Académique Documents
Professionnel Documents
Culture Documents
Abstracts submitted to the 43rd FEBS Congress, taking place in Prague, Czech Republic from 7th to 12th July 2018, and accepted by the
Congress Organizing Committee are published in this Supplement of FEBS Open Bio. Late-breaking abstracts are not included in this issue.
Indexing
Abstracts published in FEBS Open Bio Supplement for the 43rd FEBS Congress will be included individually in the Conference Proceedings
Citation Index published by Web of Science.
P.01 DNA, RNA and nucleotides P.15 Signalling through membranes and receptors
P.02 Chromosome structure and chromatin P.16 Extracellular matrix
P.03 Genome dynamics and epigenetics P.17 Glycans
P.04 The structural organization of the cell P.18 Protein – folding, dynamics, interaction and degradation
P.05 Stem cells P.19 Structural biology
P.06 Photosynthesis and plant biochemistry P.20 Systems biology
P.07 Biotechnology P.21 Synthetic biology
P.08 Host–pathogen interactions P.22 Computational biology
P.09 Biochemistry and medicine P.23 Structural bioinformatics
P.10 Cancer biology P.24 Omics technologies
P.11 Xenobiochemistry and drug metabolism P.25 Nanoworld
P.12 Gaseous molecules P.26 Chemistry of food and environment
P.13 Redox biochemistry and signalling P.27 Biochemical education
P.14 Biochemical processes at cellular membranes P.28 Miscellaneous
POSTERS
DNA, RNA and nucleotides after the swim-up test were used for IUI and the spare sperm sus-
pension in the pellet was prepared for TUNEL staining. The
staining was done according to the TUNEL staining kit proce-
P.01-001-Mon dure and the ratio of the defected DNA in the sperms were deter-
Escherichia coli single-stranded DNA binding mined by fluorescent microscopy. A positive control was done on
protein SSB binds to AlkB and promotes DNA positive slides supplied in the kit. There was no statistical differ-
repair ence between the pregnancy and no pregnancy groups in terms
R. Nigam, M. Mohan, A. Roy of liquefaction time, volume, pH, sperm concentration, motility,
Indian Institute of Technology Hyderabad, Hyderabad, India and morphology. Although the DNA damage percentage mea-
sured by TUNEL test was less in the pregnancy achieved group
Repair of alkylation damage in DNA is essential for maintaining compared to no pregnancy group, it did not reach statistically
genome integrity. E. coli DNA repair enzyme AlkB removes vari- significant levels.
ous alkyl adducts present in DNA by oxidative demethylation.
Previous studies showed that AlkB preferentially repairs single-
stranded DNA (ssDNA). However, ssDNA remains bound to P.01-003-Wed
E. coli single-stranded DNA binding protein SSB. It is unclear Novel backbone modified antisense
whether AlkB can repair alkyl adduct present in SSB-coated
oligonucleotides with improved potency
ssDNA. We have addressed this question by studying AlkB-
mediated DNA repair using SSB-bound DNA containing alkyl- effectively inactivate oncogenic miRNA-21
adduct as substrate. We have also found AlkB interacts with comparing to phosphorothioate
SSB. Yeast two-hybrid and in vitro pull-down experiments oligonucleotides
showed that SSB interacts with AlkB via structurally disordered S. Miroshnichenko, O. Patutina, E. Burakova, B. Chelobanov,
17 amino acid residues located in the disordered CTD. With A. Fokina, D. Stetsenko, V. Vlassov, M. Zenkova
reconstituted in vitro repair reaction we observed that AlkB effi- Institute of Chemical Biology and Fundamental Medicine SB RAS,
ciently remove alkyl-adducts from shorter oligonucleotide sub- Novosibirsk, Russia
strate but the efficiency of AlkB catalyzed reaction was less
compared to SSB-bound DNA substrate. Using 70-mer oligonu- Vast amount of earlier studies confirms successful application of
cleotide containing single alkyl adduct, we demonstrate that SSB- antisense oligonucleotides targeted to oncogenic miRNAs for the
AlkB interaction was dispensable for the repair of terminally suppression of various events of carcinogenesis. Despite signifi-
located methyl adducts but crucial for the faster repair of inter- cant achievements in the field of antisense technology, develop-
nally located adducts. When ssDNA was bound to truncated ment of new oligonucleotide structures and chemical
SSB lacking the interacting residues, stimulation of AlkB activity modifications to increase the effectiveness of anticancer therapy
was partially abolished suggesting functional importance of SSB- remains an urgent and promising task nowadays. In the present
AlkB interaction. work, the new effective antisense oligonucleotides containing the
newest mesyl phosphoramidate modification of internucleotide
bonds were developed and comprehensively studied. Functional-
P.01-002-Tue ity and biological potency of designed antisense oligonucleotides
The effect of sperm DNA fragmentation on the were assayed with respect to highly oncogenic miRNA-21. The
study of biological properties of the engineered nucleic acids has
pregnancy rate in Intra Uterine Cycles (IUI) shown a number of advantageous properties such as high binding
S. Isik1, S. Ege2 affinity to the target, tremendous stability in the biological media
1
Trakya University, Edirne, Turkey, 2Near East University, and RNase H-activating ability. For these properties, the devel-
Nicosia, Mersin 10, Turkey
oped oligonucleotides significantly exceed the well-known phos-
The objective of this study was to detect the DNA damage ratio phorothioate (PS) modification. In comparison with PS analogs
of sperm samples of infertile couples scheduled for intrauterine it was shown that transfection of mesyl phosphoramidate
insemination (IUI) treatment with the method of terminal oligonucleotides to the melanoma B16 cells is accompanied by
deoxynucleotidyl transferase-mediated dUTP- biotin end –label- low unspecific cytotoxicity and leads to the more significant dose-
ing (TUNEL) test and to detect DNA damage with semen analy- dependent decrease in the level of miR-21 and reduction in
sis and pregnancy rates. The study was designed as a prospective migration activity of cancer cells. Thus, a novel backbone modi-
randomized controlled follow up study for 81 infertile couples. fied antisense oligonucleotides were developed which are a wor-
Assessment of sperm DNA damage prior to their use in IUI is thy alternative to phosphorothioates and can become a new
essential for use in assisted conception. Semen samples analyzed powerful tool in the development of antisense technology. This
with WHO laboratory manual. The percentage of each sperm work is supported by Grants of Russian Science Foundation No.
with DNA fragmentation was determined using the method of 14-44-00068 and No. 17-44-07003, Grant of Russian Foundation
TUNEL. The duration was 18 months after approval from of for Basic Research No. 16-03-01055 and State funded budget
the ethics committee. Standard semen qualities are not sufficient project (VI.62.1.3, 0309-2016-0005).
for the outcome of IUI. However, sperm DNA quality has been
emphasized on its clinical significance and its relationship with
infertility. Written consent for use of the sperm for research was
obtained from the patients who were selected for the IUI treat-
ment. Questionary was performed to obtain smoking and alcohol
consumption and days of abstinence prior to production of the
semen sample. Gradient and swim-up preparation techniques
were used in IUI. The most motile sperms in the supernatant
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 107
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
108 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 109
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
suitable for sequencing was obtained only with the use of spe- complexes. Given that minor groove ligands are known to dis-
cially designed modified adapters, which did not allow the forma- rupt a key epigenetic process of DNA methylation, we aimed to
tion of adapter-dimeric structures. Sequencing was performed investigate the impact of olivomycin A and its synthetic deriva-
using the Illumina MiSeq platform. Identification of the organ- tive LCTA-1599 on the functioning of de novo murine DNA
isms was carried out by aligning the individual reads, using the methyltransferase Dnmt3a. This enzyme establishes the DNA
BLASTn program, to the NCBI Nucleotide Collection. About a methylation pattern in eukaryotic cells. Using specially designed
third of all the reads could be identified. Overall, they were fluorescently labelled oligonucleotide substrates, we determined
attributed to the 3,500 different species from 1,500 genera. About the optimal conditions for drug-DNA binding, including the con-
22% of the identified reads belonged to organisms from the centration of Mg2+ ions that allowed for both olivomycin bind-
genus Streptomyces and Bradyrhizobium, which are soil bacteria. ing and Dnmt3a functioning. The impact of olivomycin on the
Homo sapiens took about 5% of raw data. The highest-quality Dnmt3a-DNA complex formation under these conditions turned
alignment of the paired-end reads to the reference sequence of out to be minimal. The binding of olivomycin A and LCTA-1599
the human genome could be obtained using the algorithm BWA- to Dnmt3a was not observed. We then examined the inhibitory
backtrack. Sequencing of the negative control sample showed no effect of olivomycin A and LCTA-1599 on DNA methylation by
contamination at the stage of sample preparation. Library enrich- Dnmt3a. Employing the efficient method for in vitro quantifica-
ment application by DNA hybridization made it possible to sig- tion of DNA methylation that was recently developed in our lab-
nificantly increase the concentration of individual fragments oratory, we have shown that both drugs can substantially inhibit
containing target loci for phylogenetic studies. the methylation reaction with IC50 values of 6 1 lM and
7.1 0.7 lM, respectively. Other minor groove ligands such as
dimeric bisbenzimidazoles were found to have similar IC50 values.
P.01-011-Tue The inhibitory effect of olivomycin A and LCTA-1599 may be
Defining the promoter sequence determinants attributed to the disruption of movement of Dnmt3a catalytic
for NAD+ -initiation by RNA polymerase loop toward DNA, which is necessary for the enzymatic activity
O. Ruiz-Larrabeiti, D. Pinkas, N. Panova, L. Krasny of Dnmt3a and occurs via the DNA minor groove. Our results
Institute of Microbiology, The Czech Academy of Sciences, point at an epigenetic contribution to the mechanism of anti-
Prague, Czech Republic cancer effect of aureolic acid family antibiotics. This work was
supported by the RFBR grant 16-04-01087A.
The status of the 50 -end of RNA affects its stability, localization
and translation efficiency, and it is considered as an epitranscrip-
tomic regulatory element. A breakthrough discovery of the ubiq- P.01-013-Mon
uitous NAD+ redox cofactor attached to the 50 -end of RNA in Gre-like factors serve as lineage-specific
prokaryotic and eukaryotic cells revealed the existence of a new
transcriptional regulators in Deinococcus
cap-like RNA modification. The biological significance of the
NAD+-cap and its consequences for the RNA and the cell are species
unclear, but a number of mechanistic aspects of the NAD+-cap- A. Agapov1,2, K. Azimov1,2, D. Esyunina1, A. Kulbachinskiy1,2
1
ping and its requirements are starting to be defined. It has been Institute of Molecular Genetics Russian Academy of Sciences,
showed that NAD+ can be used by RNA polymerase (RNAP) Moscow, Russia, 2Molecular Biology Department, Biological
as a non-canonical transcription initiation (di)nucleotide (NCIN) Faculty, Moscow State University, Moscow, Russia
at promoters encoding +1A (+1 is the transcription start site). Transcription is a major step in the regulation of gene expression
The efficiency NAD+-capping likely depends on promoter in bacteria. Among various proteins that interact with RNA
sequence. Here we present our study where we systematically polymerase (RNAP) and modulate its activity, a prominent
investigated and defined the promoter sequence determinants for group of factors can bind in the secondary channel of RNAP
transcription initiation with NAD+. To achieve this, we created and affect catalysis by direct interactions with the active centre.
a series of chimeric promoters composed of the sequences of pro- While GreA is a universal bacterial factor that stimulates RNA
moters that encode +1A and display different NAD+-initiating cleavage, which is important for transcriptional proofreading, the
efficiencies, and used them as templates for in vitro transcription functions of its homologues may greatly differ. The famously
with Escherichia coli RNAP. These experiments identified a pro- stress-resistant bacterium Deinococcus radiodurans encodes three
moter region and its consensus sequence that maximizes utiliza- factors from the Gre-family: GreA, Gfh1 and Gfh2. We previ-
tion of NAD+ as the transcription initiating substrate. The ously found that the Gfh factors from D. radiodurans strongly
results presented here thus constitute a step forward in the char- enhance site-specific pausing and intrinsic transcription termina-
acterization of this newly found prokaryotic RNA-capping phe- tion by RNAP. The pause-stimulatory activity of Gfh is greatly
nomenon. This work is supported by grant No. 17-03419S, from enhanced by manganese ions, which are accumulated in D. radio-
the Czech Science Foundation. durans cells under stress conditions. Uniquely, another bacterium
from the Deinococcus genus, D. peraridilitoris encodes four differ-
ent Gfh factors. In this work, we analyzed their activities in vari-
P.01-012-Wed ous in vitro transcription assays. The results show that one of the
Epigenetic aspect of function of a promising four proteins significantly inhibits transcription initiation, pro-
anticancer drug olivomycin A longs transcriptional pausing and suppresses the RNA cleavage
A. Sergeev1, A. Tevyashova2,3, E. Gromova1 activity of RNAP. At the same time, the other three Gfh factors
1
Moscow State University, Moscow, Russia, 2Gause Institute of reveal much more moderate if any effects on different steps of
New Antibiotics, Moscow, Russia, 3Moscow University of transcription. We conclude that Gfh proteins serve as important
Chemical Technology of Russia, Moscow, Russia transcriptional regulators in Deinococcus species and may poten-
tially play a role in stress resistance, but the functions of their
Olivomycin A is a promising anticancer agent that belongs to a
species-specific paralogues remain enigmatic. We speculate that in
family of aureolic acid antibiotics. However, the mechanism of
the case of D. peraridilitoris the Gfh proteins, though being
its action is not completely understood. The drug binds to the
homologous to transcription factors, may have additional func-
DNA minor groove in GC-rich regions as Mg2+-containing
tions in the cell that we will try to uncover in our future
110 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
studies. This work was in part supported by Russian Science transversions, C?T transitions followed by C?A transversions.
Foundation (grant 17-14-01393) and Russian Foundation for We established the optimal pH, Fe(II) and aKG concentrations
Basic Research (grant 18-34-00905). for enzymatic reaction. Our data proved that the protonated
form of HPA is preferentially repaired by EcAlkB, though the
reaction is stereoselective. Moreover, the number of reaction
P.01-014-Tue cycles carried out by an AlkB molecule remains limited and
Investigation of the argonaute protein from reached 38 4 enzymatic cycles before its total inactivation.
Synechococcus elongatus Molecular modeling of the AlkB/HPA complex demonstrated
A. Olina, A. Kulbachinskiy, D. Esyunina that the R stereoisomer in the equatorial conformation of the
1
Institute of Molecular Genetics Russian Academy of Sciences, HPA hydroxyl group is strongly preferred, while the S one seems
Moscow, Russia to be susceptible to AlkB-directed oxidative hydroxylation only
when HPA adopts the syn conformation around the glycosidic
Argonaute proteins are key players in the diverse RNA interfer- bond. In addition to the biochemical activity assays, substrate
ence pathways in eukaryotes. Eukaryotic argonautes bind short binding to the protein was monitored by differential scanning flu-
RNAs and use them as guides for target RNA recognition and orimetry allowing the identification of the active protein form
cleavage. Argonaute proteins, often with unusual domain archi- with cofactor and cosubstrate bound and monitoring substrate
tectures, are also found in many bacterial and archaeal species binding.
but their functions in prokaryotes remain largely obscure. Recent
studies revealed that prokaryotic argonautes can bind short
RNA or DNA molecules and act as endonucleases to protect the P.01-016-Mon
cells against foreign genetic elements. However, their cellular The amount of humans with high or low
roles and the molecular mechanisms of action are poorly under-
ribosomal repeat copy number in population
stood. In this work, we studied argonaute protein SynAgo from
the blue-green algae Synechococcus elongatus. We used purified decreases with age
SynAgo protein for in vitro experiments and demonstrated that it V. Sergeeva, E. Malinovskaya, E. Ershova, N. Lyapunova,
is able to bind short DNA guides and cleave target DNA sub- V. Izevskaya, S. Kutsev, N. Veiko, S. K. Kostyuk
strates of various structures. We further constructed S. elongatus Research Centre for Medical Genetics, Moscow, Russia
strain with tagged-argonaute expression and used it for purifica- Ribosomal RNA genes (rDNA) encode the rRNA species that
tion of SynAgo and associated nucleic acids. It was found that in form the ribosomes. The diploid human genome contains several
S. elongatus cells SynAgo is bound with short DNAs correspond- hundred copies of 43-kb rDNA units tandemly arrayed on five
ing to the whole genomic sequence. Similarly, SynAgo also asso- acrocentric chromosomes. Dysregulation of rRNA biogenesis has
ciates with short DNAs when expressed in the heterologic been implicated in some human diseases. One of the factors
Escherichia coli system suggesting that no additional factors are affecting rRNA biogenesis is the ribosomal genes copy number
required for short DNA processing. Thus, our experiments sug- (CN). Some authors have shown that the rDNA CN in human
gest that SynAgo is a DNA guided DNA nuclease that target genome reduces with age. However, other authors deny this phe-
various genomic sequences to perform its functions. To reveal nomenon. It can be explained with high degree of rDNA damage
these functions, we constructed a set of S. elongatus strains with in aged cells and low efficiency of qPCR. We compared two
various mutations in the argonaute gene and are now performing methods: qPCR and nonradioactive quantitative hybridization
in vivo experiments aimed at understanding the roles of SynAgo (NQH) in quantitative analysis of human rDNA. We have shown
in genomic defense and cell physiology. This work was supported that NQH is less sensitive to severe DNA damage than qPCR
in part by the Russian Science Foundation (grant 16-14-10377). and thus is more appropriate for damaged rDNA detection. We
have shown earlier that rDNA CN is higher in patients with
schizophrenia compared to healthy people of the same age. We
P.01-015-Wed assessed blood leukocyte rDNA CN of 250 subjects (group I, 17
1,N6-a-hydroxypropanoadenine and 3,N4-a- -71 years) in comparison to 126 subjects (group II, 72–91 years)
hydroxypropanocytosine - acrolein adducts to using NQH. The leukocyte rDNA CN showed no significant dif-
adenine and cytosine, are substrates for AlkB ference in the median for group I and group II (median 419 vs
dioxygenase 396, P = 0.05). However, the dispersion of CN for group I was
much higher than for group II (200 -711 vs 252–541). In 80% of
M. Dylewska, J. Pozna nski, J. T. Kusmierek, E. Grzesiuk,
group II subjects CN of rDNA had a very narrow dispersion –
A. Maciejewska
from 300 to 450. Thus, the amount of humans with high or low
Institute of Biochemistry and Biophysics, Polish Academy of
rDNA CN decreases with age. This work was supported by
Science, Warsaw, Poland
RFBR grant No. 17-29-06017 ofi_m.
1,N6-a-hydroxypropanoadenine (HPA) and 3,N4- a-hydroxypro-
panocytosine (HPC) are six-membered adducts formed in reac-
tion of adenine and cytosine with acrolein (ACR). ACR is a P.01-017-Tue
mutagenic agent originated from different sources including Ferritin2 RNA interference inhibits the
cigarette smoke, exhaust fumes and overcooking. It is also gener- formation of iron granules in honey bees
ated endogenously during oxidative stress as a by-product of
(Apis mellifera)
lipid peroxidation. E. coli AlkB dioxygenase (EcAlkB) is DNA
C. Hsu
repair enzyme that remove alkyl lesions from bases via an oxida-
Chang Gung University; Chang Gung Memorial Hospital,
tive mechanism restoring native DNA structure. It belongs to the
Taoyuan, Taiwan
superfamily of 2-a-ketoglutarate (aKG) and Fe(II) dependent
dioxygenases. AlkB is induced within E. coli system of adaptive Iron granules containing superparamagnetic magnetite act as mag-
response to alkylating agents (Ada response). Our in vivo data netoreceptor for magnetoreception in honey bees. Biomineralization
show that HPA and HPC show mutagenic properties and, gener- of iron granules occurs in the iron deposition vesicles of tropho-
ated in plasmids, causes (respectively) A?C and A?T cytes and requires the participation of actin, myosin, ferritin2,
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 111
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
and ATP synthase. If the formation of iron granules can be sup- Silencing of NRP1 significantly suppressed lung cancer cell
pressed, it can help to understand the mechanism of magnetore- migration, metastasis, and tumor angiogenesis. Thus, develop-
ception in honey bees. Whether ferritin RNA interference can ment of specific aptamers against NRP1 would be a promising
inhibit the formation of iron granules is unknown. In this study, therapy for NSCLC patients. Here, we screened a DNA aptamer
double-stranded RNA of ferritin2 was injected into hemolymph library against NRP1 protein by using a nitrocellulose filter sys-
from the abdomen of newly emerged worker bees to knock down tematic evolution of ligands by exponential enrichment (SELEX).
ferritin2. The results showed that ferritin2 RNA interference Three NRP1-binding aptamers, AP5, AP15, and AP37, were iso-
declined the mRNA and protein expression of ferritin2 and the lated. We further screened a customized aptamer microarray, on
formation and accumulation of 7.5 nm iron particles leading to which a short aptamer library was made based on the sequences
immature iron granules. Superparamagnetic magnetite is formed of AP5, AP15, and AP37, and we got several short aptamers
in the center of mature iron granules. These findings can be used with high NRP1-binding affinity and specificity. We found that
to further study the magnetoreception of honey bees. NRP1-specific aptamers decreased the migration ability of the
highly invasive NSCLC cells, CL1-5, compared with that in cells
treated with scramble-aptamers. Moreover, tube formation ability
P.01-018-Wed of HUVEC cells were also reduced while treating with NRP1-
DNA aptamers against procalcitonin as specific aptamers. These results showed that NRP1-specific apta-
emerging diagnostic molecules for sepsis mers decrease the migration ability of tumor cells and angiogene-
Y. Chen1, Y. Yeh1, L. Chau2, T. Hong1 sis. This study revealed that NRP1-specific aptamers might be
1
National Cheng Kung University, Tainan, Taiwan, 2National developed a useful therapeutic strategy to lung cancer in the
Chung Cheng University, Chiayi, Taiwan future.
112 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 113
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
bioinformatics approach to assess the effect of adenosine modifi- support of Italian association for cancer research (IG2013, pro-
cations on mRNA abundance and protein production in bacteria. ject code 143, to Prof. L. Xodo) and the grant of the President
For every gene we collected published available data about its of the Russian Federation for young Russian scientists (MK-
mRNA and protein levels in different conditions, absolute pro- 2474.2018.3, to Dr. A. Tikhomirov).
tein number, translation efficiency, and adenosine modifications.
We correlated mRNA and protein abundances for genes in each
growth condition and compared mean correlation coefficients P.01-026-Tue
among all conditions between modified and unmodified genes. C-reactive protein and renin-angiotensin
Modified genes have statistically significant lower correlation (P- system gene polymorphisms in patients with
value Mann-Whitney test <0.01) between mRNA and protein coronary artery stenosis
abundances. Using ribosome profiling data we analyzed the
K. B. Bogatyreva, M. M. Azova, Z. K. Shugushev,
impact of modifications on intermediate stages of cell informa-
A. V. Aghajanyan, L. V. Tskhovrebova, A. Ait Aissa,
tion flow: from mRNA to ribosomes and from ribosomes to pro-
M. L. Blagonravov, S. P. Syatkin
teins. Modifications affect ribosome occupancy in such a way
Peoples’ Friendship University of Russia (RUDN University), 6
that translation efficiency only slightly correlates with mRNA
Miklukho-Maklaya St., Moscow, Russia
level. Ribosome occupancy within modification sites is slightly
lower than in other parts of the genes. We didn’t find any differ- The latest studies in the field of molecular cardiology demon-
ence in ribosome distribution upstream or downstream of modifi- strate the important role of inflammation and renin-angiotensin
cation sites. Was created the model that predicts protein system (RAS) in the pathogenesis of atherosclerosis. Gene poly-
abundance based on expression level, translation rate, and adeno- morphisms of their components have been described to be associ-
sine modification status. The model explains up to 63% of the ated with the development of coronary artery disease. Our work
variation in protein abundance, the model based on decision trees was aimed at studying the polymorphisms of the C-reactive pro-
shows that m6A abundance has 15% contribution to protein tein (CRP), angiotensin II receptor type 1 (AGTR1), and angio-
level prediction. So, our calculations show statistically significant tensin (AGT) genes in patients with coronary artery stenosis. 65
impact m6A modifications on information flow from mRNA to patients and 59 healthy individuals participated in the study. All
protein level. This work was supported by RSF grant No. 14-24- of them were Russians. Total DNA was extracted from the blood
00159. and genotyped using the Real-time PCR. The CRP C1444T,
AGT Thr174Met, AGT Met235Thr, and AGTR1 A1166C poly-
morphisms were analyzed. The Chi-square test and Fisher’s exact
P.01-025-Mon test were used to estimate differences between groups. It was
G-quadruplex RNA in Kirsten ras transcript as found that in patients the frequencies of most of minor alleles
promising target for anticancer therapy were significantly higher (AGT 174T 40.7% vs. 11%; AGTR1
A. Tikhomirov1,2, G. Miglietta3, S. Cogoi3, A. Shchekotikhin1,2, 1166C 39.2% vs. 19.5%; CRP 1444T 42.2% vs. 24.7%), which
L. Xodo3 was accompanied by growth of the frequency of homozygous
1
Gause Institute of New Antibiotics, Moscow, Russia, 2D. I. rare genotypes. Comparison of patients with multifocal
Mendeleev University of Chemical Technology of Russia, Moscow, atherosclerosis (n = 20) with other patients (n = 45) revealed that
Russia, 3Department of Medical and Biological Sciences, in this group the genotypic distributions of RAS genes were dif-
University of Udine, Udine, Italy ferent due to a significant increase in the frequency of heterozy-
gotes (AGT Thr174Met 15.8% vs. 4.3%; AGT Met235Thr 21%
Quadruplex-forming motifs of promoters of some oncogenes are vs. 10.9%; AGTR1 A1166C 52.6% vs. 15.2%). The obtained
considered as prospective targets for cancer chemotherapy. The results may be of interest for personalized medicine because
human Kirsten ras (KRAS) transcript is capable to form several heterozygosity for the given polymorphisms may predispose to
putative G-quadruplexes (G4s) as predicted by methods of the multifocal atherosclerosis. To confirm this idea it is necessary
bioinformatics. Among the three expected G4 motifs only one to continue research in larger samples. The publication was pre-
was recognised by BG4, an antibody specific for G-quadru- pared with the support of the “RUDN University Program 5–
plexes that confirms the initial assumption. Previously, it was 100”.
demonstrated that heteroarene-fused anthraquinones have a
strong affinity and selectivity to telomeric and Harley ras G-
quadruplexes. Therefore, we investigated an ability of series P.01-027-Wed
ligands on heteroareneanthraquinones scaffolds to bind to G4s The expression of a nested alternative open
in the 50 -UTR of mRNA and suppress the KRAS oncogene in reading frame may be a novel mechanism for
pancreatic cancer cells. Found, that 4,11-bis(2-aminoethylamino)
anthra[2,3-b]furan-5,10-dione (ATFD) stabilizes G4 in the intronless gene expression control
KRAS transcript (DTM up to 32 °C). Moreover, synthesis and T. V. Komarova1,2, E. V. Sheshukova1,2, N. M. Ershova1,2,
evaluation of biotinylated ATFD by means of biotin-streptavi- Y. L. Dorokhov1,2
1
din pulldown assay revealed that ATFD binds to RNA G4s in Vavilov Institute of General Genetics, Russian Academy of
the KRAS transcript under low-abundance cellular conditions. Sciences, Moscow, Russia, 2Lomonosov Moscow State University,
Dual-luciferase assays showed that ATFD repress translation in Moscow, Russia
a dose-dependent manner. The new G4-ligand efficiently pene- The regulation of gene expression in animal and plant tissues
trate into the Panc-1 cancer cells, suppressing protein p21KRAS at different stages of development and after exposure to exter-
to <10% of the control followed by apoptosis together with a nal stimuli occurs at the transcriptional, post-transcriptional
dramatic reduction of cell growth at submicromolar concentra- and post-translational levels. Recently, in the Solanaceae family
tions. As the result one may conclude, a suppression of the of plants, we found an intronless gene for the Kunitz protease
KRAS oncogene via stabilization of G4s in the 50 -UTR of inhibitor-like protein (KPILP) with low mRNA levels in the
mRNA by low molecular weight ligands can be considered as a leaves of an intact plant. However, these levels sharply
new strategy in G4-oriented anticancer therapy. Acknowledg- increased after a 72-h incubation in the dark and under biotic
ment: this work has been carried out in part with the financial stresses. To identify the mechanism for the regulation of
114 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
Solanaceae KPILPs, we identified and examined the function Science Centre, Poland, grant no.: UMO-2014/13/B/NZ1/00043
of the KPILP nested alternative open reading frame (aORF) (to B.W.).
that controls the expression of maternal mRNA. Here, we
investigated the regulation of KPILP under stress conditions at
the level of transcription. We isolated a portion of Nicotiana P.01-029-Tue
benthamiana chromosomal DNA upstream of the KPILP cod- Eukaryotic ribosome discriminates 3’ context
ing sequence (proKPILP) and showed that proKPILP directed and determines efficiency of stop codon
the expression of the E. coli UidA reporter gene encoding b-D- readthrough
glucuronidase (GUS) in plant. A bioinformatic analysis
E. Sokolova, T. Egorova, A. Schuvalov, E. Alkalaeva
revealed potential light-responsive elements in the proKPILP
Engelhardt Institute of Molecular Biology, the Russian Academy
sequence. To understand if these elements influence proKPILP
of Sciences, Moscow, Russia
activity, agroinjected plants were isolated for 3 days in com-
plete darkness. Then, the level of GUS and corresponding Nucleotide context, surrounding stop codons, significantly affects
mRNA content were assessed in the leaves and compared with the efficiency of translation termination. To study the mechanism
the constitutive CaMV 35S promoter. The results showed that, of its influence, we assessed properties of several 3’ contexts,
as in the case of the 35S promoter, darkness had a negligible unfavorable for translation termination, in the reconstituted
effect on the activity of the proKPILP. We concluded that the mammalian translation system. Using pairs of stop codons
stress-dependent content of the KPILP mRNA in the leaves is divided by hexanucleotides we revealed, that the ribosome recog-
determined mainly by the aORF-mediated mechanism of nizes leaky and strong 3’ stop codon contexts by itself. Stop
maternal mRNA control. Thus, Solanaceae KPILP is an codon readthrough depends only on the presence of suppressor /
intronless matryoshka gene and is the first example of a gene near-cognate tRNA and leaky 3’ context. We estimated also the
expression regulation mechanism that is probably characteristic impact of 3’ contexts on translation termination activity of
for intronless genes. This study was performed with financial eRF1. All tested contexts demonstrated approximately equal
support from the Russian Foundation for Basic Research (pro- translation termination efficiency in the presence of release fac-
ject No. 17-29-08012). tors. However, comparison of basal readthrough levels showed
the discriminate response among different weak context. It can
reflect much more sophisticated mechanism of balancing between
P.01-028-Mon readthrough and termination, orchestrated by nucleotide sur-
Transcriptome analysis reveals influence of rounding of a stop codon that could be surmised on the first
RNases from toxin-antitoxin systems on gene glance. The work was supported by the Russian Science Founda-
tion (grant No. 14-14-00487) and by the Program of Fundamen-
expression in Staphylococcus aureus
tal Research for State Academies for the years 2013 to 2020 (No.
B. Wladyka1, A. Sabat2, M. Bukowski1, M. Hydzik1, K. Hyz1,
1201363822).
K. Chlebicka1, V. Akkerboom2, A. Friedrich2, E. Bonar1
1
Jagiellonian University, Krakow, Poland, 2University Medical
Center Groningen, Groningen, Netherlands P.01-031-Mon
Toxin-antitoxin (TA) systems are widespread in bacteria. A typi- 2’-Modified RNA aptamers specific to
cal TA system consists of a toxin, which is a stable protein, often photoprotein obelin as a platform for new
with enzymatic activity, and an antitoxin, constituting a labile bioluminescent biosensors
component with inhibitory properties towards the toxin. In case
A. Davydova1, M. Vorobyeva1, V. Krasitskaya1,2,3, P. Vorobjev1,
of plasmid-encoded TA systems a role in the maintenance of
A. Tupikin1, M. Kabilov1, L. Frank2,3, A. Venyaminova1
mobile genetic elements is attributed to these entities. However, 1
Institute of Chemical Biology and Fundamental Medicine,
localization of TA genes in chromosomal parts of bacterial gen-
Siberian Branch, Russian Academy of Sciences, Novosibirsk,
omes implies the existence of other functions. Staphylococcus
Russia, 2Institute of Biophysics Siberian Branch of Russian
aureus is a carrier of chromosomally-encoded MazEF TA system
Academy of Sciences, Krasnoyarsk, Russia, 3Siberian Federal
and less frequently of PemIK-Sa1, which is present in a number
University, Krasnoyarsk, Russia
of staphylococcal plasmids. Toxins from these systems are
sequence-specific RNases towards UACAU and UAUU, respec- Aptamers are in vitro selected DNA or RNA molecules that can
tively. We hypothesize that the toxins, released from TA com- specifically bind a wide variety of targets from small molecules to
plexes upon activation, may degrade transcripts, and thus proteins and living cells. Aptamers represent a high-potential
influence gene expression. To challenge this hypothesis we intro- alternative to monoclonal antibodies due to an opportunity to
duced plasmids carrying toxins’ genes under inducible promoter generate an aptamer to almost any desired target, longer shelf-
to S. aureus null mutants for the respective TA systems. Upon life, cost-effective chemical synthesis, stability in a wide range of
induction of toxins’ expression, RNA was isolated and RNA-seq conditions, and low toxicity and immunogenicity. These unique
was performed using Illumina platform. Interestingly, over- properties determined aptamer applications as prospective thera-
expression of MazF toxin resulted in rather moderate changes in peutic agents as well as biosensors for diagnostic purposes. In
gene expression. The number of genes with over two-fold differ- this study we proposed a new design of bioluminescent aptasen-
ence was below twenty, however slightly increased over the time. sor made of two different aptamer modules: one is responsible
Conversely, in case of PemK-Sa1 toxin over 160 gene transcripts for specific binding of analyte, the other provides bioluminescent
were affected. This clearly indicates that the length of the recog- reporting. We suggested that aptamers to Ca2+-regulated photo-
nized target sequence correlates negatively with the number of protein obelin could be used as universal reporting modules for
altered transcripts. Moreover, among transcripts down-regulated bifunctional aptasensors. Due to the high quantum yield and
by PemK-Sa1 was RNAIII, which is an important molecule in low-level background, obelin established itself as a promising
the accessory gene regulator system, which suggests the existence reporter molecule. We performed in vitro selection of 2’-F-modi-
of a possible regulatory cascade/coupling. The presented data fied RNA aptamers against His-tagged obelin immobilized on
demonstrate that TA RNase toxins may modulate gene expres- Ni-NTA sepharose beads. After high-throughput sequencing and
sion in bacteria. The study was supported by the National bioinformatical analysis, we obtained a series of individual
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 115
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
116 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
P.01-035-Tue Real-Time PCR analysis. The stage of liver fibrosis was identified
with the elastography method (FibroScan, Echosens, France).
6S RNA as a transcription regulator in
Liver fibrosis stages 0, 1, 3, and 4 were detected in 6 (12.7%), 3
Rhodobacter sphaeroides (6.4%), 2 (4.3%), and 36 (76.6%) patients respectively. We did
D. Elkina1, L. Weber2, O. Burenina3, E. Kubareva1, not find the patients with liver fibrosis stage 2. It was found that
R. Hartmann4, G. Klug2 the frequency of allele A among patients was 38.3% which is sig-
1
A.N. Belozersky Institute of Physico-Chemical Biology MSU, nificantly higher than in the control group (12.5%;
Moscow, Russia, 2Institute of Microbiology and Molecular P = 0.005768). The frequency of heterozygotes CA was not sig-
Biology, Justus-Liebig-University, Giessen, Germany, 3Skolkovo nificantly different between the group of patients and the control
Institute of Science and Technology, Moscow, Russia, 4Institute of group (38% and 25% respectively, P = 0.4425). Homozygous
Pharmaceutical Chemistry, Philipps University, Marburg, Germany AA genotypes were revealed only in patients with liver fibrosis,
E. coli 6S RNA was one of the first discovered non-coding the frequency was 19.1%. Thus, we suggest that the presence of
RNAs. It forms a complex with the housekeeping RNA poly- allele A of the COL1A1_1 gene, particularly in the homozygous
merase (RNAP) holoenzyme. The important finding was that state, may be one of the genetic factors involved in the develop-
RNAP utilizes 6S RNA as a template for the transcription of ment of liver fibrosis in patients abusing alcohol. The publication
short product RNAs (pRNAs). We analyzed the expression and was prepared with the support of the “RUDN University Pro-
function of 6S RNA in a-proteobacterium R. sphaeroides that is gram 5–100”.
a common model organism to study regulated formation of
photosynthetic complexes and the response to (photo)oxidative
stresses. R. sphaeroides 6S RNA was shown to have a unique
P.01-038-Tue
expression pattern: it peaks at the end of exponential growth The role of human Gle1 isoforms in translation
and strongly decreases during prolonged stationary phase. termination
According to our Northern blot analyses, the levels of pRNAs T. Egorova1, A. Shuvalov1, B. Eliseev2, L. Yeramala2,
are the highest in mid-to-late exponential phase and largely N. Melnikova1, A. Dmitriev1, P. Kolosov3, C. Schaffitzel4,
decrease toward prolonged stationary phase. This trend is also E. Alkalaeva1
1
seen in the pRNA-Seq data; 12 to 16- meric pRNA are Engelhardt Institute of Molecular Biology, the Russian Academy
assumed to be long enough to remain bound to 6S RNA and of Sciences 119991, Moscow, Russia, 2European Molecular
to rearrange its structure which triggers RNAP release. It is Biology Laboratory, Grenoble Outstation 71 Avenue des Martyrs
shown that E. coli and B. subtilis cells lacking 6S RNA worse 38042, Grenoble, France, 3Institute of Higher Nervous Activity and
or better survive at some stress conditions. For R. sphaeroides Neurophysiology, The Russian Academy of Sciences 117485,
we found the retarded growth phenotype of the 6S RNA dele- Moscow, Russia, 4School of Biochemistry, University of Bristol,
tion strains under high salt conditions, which can be explained BS8 1TD, Bristol, United Kingdom
by the dysregulated expression of SspA salt stress protein. The
Gle1 is a conservative essential and multifunctional protein
spatial vicinity of ssrS and sspA genes may also point to an
involved in mRNA export from the nucleus into the cytosol.
interesting mechanistic scenario: newly transcribed 6S RNAs
Yeast Gle1 also participates in initiation and termination of
may bind to the same RNAP by which they were synthesized
translation. Mutations of human Gle1 (hGle1) cause fetal
to reduce the frequency of incidences where an RNAP (after
motoneuron diseases (LCCS1 and LAAHD) and amyotrophic
transcription of 6S RNA) reinitiates transcription at the nearby
lateral sclerosis development. In human cells two isoforms
promoter that drives sspA expression. This work was supported
(hGle1B and hGle1A) of this protein are formed as a result of
by the Russian Science Foundation (project No. 14-24-00061).
alternative splicing. hGle1B is involved in mRNA export from
the nucleus, and hGle1A is involved in stress granule (Sg) forma-
P.01-037-Mon tion. In the present work, we studied the role of hGle1 isoforms
in translation termination using a reconstituted mammalian
The polymorphism of the COL1A1_1 gene as a
translation system. We show that hGle1B binds to eRF1 and the
risk factor of liver fibrosis development in eRF1-eRF3 complex (but not to eRF3) and activates translation
patients abusing alcohol termination. Conversely, hGle1A slightly inhibits translation ter-
M. Bayarsaikhan, E. V. Tarasenko, I. V. Garmash, mination. It effectively binds to pre-termination complexes
G. I. Myandina, O. S. Arisheva, A. S. Ivanov, E. M. Zheludova, (preTCs) and competes with eRF1 for binding to preTCs. We
M. M. Azova, S. P. Syatkin evaluated the levels of hGle1 isoform expression in Hek293 cells
Peoples’ Friendship University of Russia (RUDN University) 6 after different types of stress stimulation. Heat shock induced an
Miklukho-Maklaya St., Moscow, Russia increase of the mRNA expression of both hGle1 isoforms.
Sodium arsenite treatment decreased the amount of hGle1A
The main causes of liver fibrosis are hepatitis B and C, alcohol
mRNA. We conclude that the two isoforms of hGle1 play differ-
abuse, and genetic factors. Liver fibrosis is characterized by a sig-
ent roles in translation termination: hGle1B enhances the effi-
nificantly increased extracellular matrix which includes several
ciency of translation termination, while hGle1A binds preTCs
types of collagen molecules. The most common is type I collagen.
and interferes with termination, possibly leading to Sg formation.
Mutations changing the structure of collagen fibers can lead to
The work was supported by the Russian Science Foundation
serious functional defects in connective tissue and the liver fibro-
(grant No. 14-14-00487).
sis development. The aim of the present study was to investigate
the association between the COL1 A1_1 C/A (rs1107946) poly-
morphism and liver fibrosis (F0-F4) in patients abusing alcohol.
47 patients (36 men and 11 women; the period of alcohol con-
sumption was 15.6 9.5 years) and 30 healthy individuals were
examined. Patients with hereditary liver diseases or infected by
hepatitis B and C viruses were excluded from the study. The
polymorphism of the COL1A1_1 gene was determined using the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 117
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
P.01-039-Wed species, cultivated and wild. The obtained genes encoded pro-
teins, which contain conserved zinc finger and HMG-like
Targeting ligand decorated exosome as a
YABBY domains and nuclear localization signals characteristic
carrier of miRNA21 antisense oligonucleotide for YABBY transcription factors, and also, the conserved motifs
for brain cancer therapy specific for the YAB1/YAB3 subfamily. The YABBY1 and
G. Kim, M. Lee, M. Kim, J. Oh, Y. Lee YABBY3 expression patterns were determined in the leaves and
Hanyang University, Seoul, South Korea flowers of S. lycopersicum, S. chmielewskii, S. peruvianum, S. hab-
Extracellular vesicles (EVs) are membrane-derived vesicles that rochaites, three C. annuum accessions, C. chinense, C. frutescens,
secreted by various types of cells and involved in cell-cell signal- and C. pubescens. In analyzed tomato species, the possible nega-
ing. Exosome is the smallest subtype among EVs with size of 30 tive correlation between YABBY1 and YABBY3 co-expression
to 100 nm in diameter. Since exosomes were small and native to level and the size of lateral organs was observed. The proposed
the animals, they have several advantages as a drug delivery car- evolutionary history suggests the more recent origin of YABBY3
rier. Rabies Virus Glycoprotein (RVG) is a small peptide that compared to YABBY1, as well as the emergence of these paralo-
specifically bind to the acetylcholine receptor. It already has been gous genes from the common ancestor before Dicots divergence
reported that RVG decorated exosome can target brain by sys- on Rosids and Asterids. The study was supported by the Russian
temic injection. To generate a stable cell line that can secrete Science Foundation grant 16-16-10022.
RVG-exosomes (RVG-exos), plasmid DNA encoding fusion
protein of RVG and Lamp2b was transfected into 293T cell.
P.01-041-Tue
RVG-exos and Unmodified-exosomes (Un-exos) were isolated
and evaluated as a gene carrier. The result of SEM and Dynamic Novel 2’-F-RNA aptamers specific to protein
Light Scattering shows round shaped particles with a size of markers of glycemia – a basis for
30~100 nm. The miRNA21 antisense oligonucleotide (ASO) was bioluminescent aptasensors
loaded into the exosomes by electroporation. After transfection M. Vorobyeva1, A. Davydova1, E. Shatunova1,2, P. Vorobjev1,2,
into C6 cell, cellular uptake of ASO loaded exosomes were A. Tupikin1, M. Kabilov1, E. Bashmakova1,3,4, V. Krasitskaya1,3,
measured by flow cytometer. In vivo therapeutic effect of RVG- L. Frank3,4, A. Venyaminova1
1
exo-ASO was tested in an intracranial tumor rat model. Rats Institute of Chemical Biology and Fundamental Medicine SB
were bored 2 mm hole on the skull and C6 cells were implanted RAS, Novosibirsk, Russia, 2Novosibirsk State University,
into the brain. A week later, ASO loaded RVG-exo and Un-exo Novosibirsk, Russia, 3Institute of Biophysics SB RAS, Federal
were administrated by tail vein injection. The result of Nissl Research Center “Krasnoyarsk Science Center SB RAS”,
staining of the brain section showed that delivery of RVG-exo- Krasnoyarsk, Russia, 4Siberian Federal University, Krasnoyarsk,
ASO had anti-cancer effect on brain tumor. As programmed cell Russia
death 4 (PDCD4) and phosphatase and tensin homologue
Nucleic acid aptamers are now generally considered as the most
(PTEN) are pro-apoptotic gene that has known to be inhibited
prominent alternative to monoclonal antibodies thanks to their
by miRNA21, the induction of them may be involved in the
high target affinity and specificity, ease of chemical synthesis and
tumor volume in the animals. Immunohistochemistry (IHC)
a possibility to generate an aptamer with desired properties by
showed that the delivery of RVG-exo-ASO induced PTEN and
in vitro evolution on a lab bench. In particular, aptamers are
PDCD4 more efficiently than that of Un-exo-ASO group, show-
becoming increasingly popular as recognizing elements for
ing anti-tumor effect of the ASO delivery. In conclusion, RVG-
biosensing platforms. In this study, we generated 2’-F-modified
exo delivered the ASO into the rat brain effectively and may be
RNA aptamers specific to protein markers of blood glucose level
useful as a therapeutic agent for the brain tumor.
– glycated and non-glycated human hemoglobin and serum albu-
min. All target proteins were covalently immobilized on magnetic
P.01-040-Mon beads. To select aptamers capable to bind only the glycated pro-
tein, each cycle comprised an incubation of the RNA pool with
Differential expression profiling of the YABBY1
the non-glycated target (the negative selection step) followed by
and YABBY3 genes in Solanum and Capsicum the incubation with the glycated one. In the case of the aptamers
species affine to the total hemoglobin or albumin, only the incubation
M. Filyushin1, M. Slugina1, O. Pyshnaya2, E. Kochieva1, with the ‘normal’ protein was applied. The enriched RNA pools
A. Shchennikova1 were subjected to high-throughput sequencing and bioinformatic
1
Institute of Bioengineering, Research Center of Biotechnology of analysis. For each set of candidate aptamers we designed their
the Russian Academy of Sciences, Moscow, Russia, 2Federal truncated forms on the basis of RNAfold secondary structure
Scientific Vegetable Center, Moscow, Russia prediction. Truncated aptamers were then chemically synthesized
The plant-specific bifunctional YABBY transcription factors have and screened for their affinity and specificity to the correspond-
an important role in driving the evolution of the leaf and gynoe- ing targets. The best aptamers to the total/glycated hemoglobin
cium. YABBY genes are involved in the initiation, growth and or albumin were then integrated as recognizing elements of biolu-
structural organization of almost all aboveground lateral organs, minescent aptasensors with the use of Ca2+-dependent photopro-
vegetative or reproductive, defining their adaxial-abaxial asymme- tein obelin as a reporter. The investigation of their sensitivity and
try, and in the control of shoot apical meristem organization and specificity revealed the optimal design of aptasensors. Novel
activity. The present study was focused on the identification and aptamers and aptasensors developed in this work could be fur-
comparative characterization of YABBY1 and YABBY3 genes in ther used for the assessment and monitoring of the short-term
species of tomato (Solanum section Lycopersicon) and pepper (the percentage of glycated albumin) and long-term (the percent-
(Capsicum), their polymorphism, expression profiling in lateral age action of glycated hemoglobin) blood glucose control. The
organs, and evaluation of possible correlations between the work is supported by Russian Science Foundation (Grant No.
expression pattern and the leaves and flowers size. The complete 16-14-10296).
YABBY1 and YABBY3 genomic sequences were identified in 13
accessions of 11 tomato species and 6 accessions of 4 Capsicum
118 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 119
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
P.01-045-Wed P.01-047-Tue
Selection of DNA aptamers to odorous Benzothiazole derivatives for staining DNA
substances using Capture-SELEX protocol structures: selectivity for G4/duplex junctions
N. Komarova, A. Kuznetsov A. Turaev1, A. Aralov2, V. Tsvetkov1, A. Varizhuk1,
Scientific-Manufacturing Complex Technological Centre, G. Pozmogova1
1
Zelenograd, Moscow, Russia SRI of Physical-chemical Medicine, Moscow, Russia, 2Institute of
Bioorganic Chemistry, Moscow, Russia
Food industry requires analysis of different odorous compounds
to test product freshness, to discriminate between different prod- Non-canonical nucleic acid structures (ncDNA), such as G-quad-
ucts and to control the stages of foods and beverages manufac- ruplexes (G4), are currently attracting much attention because of
turing process. Aptamers, which are short ssDNA or RNA their diverse biological roles. Express methods for ncDNA visual-
fragments displaying affinity to the target molecule, can be used ization based on specific light-up probes are gaining popularity.
for developing new odorant detection methods. Aptamers to a We synthesized new benzothiazole derivatives – analogs of a
certain target can be selected from the randomized sequences known dye thiazole yellow – and evaluated their applicability as
library using systematic evolution of ligands by exponential light-up probes for discriminating between ssDNA, dsDNA and
enrichment (SELEX). In this research new aptamers with affinity ncDNA. Several new dyes exhibited specificity for G4s. Fluores-
to guaiacol, ethylguaiacol and homofuraneol were obtained. cence quantum yields of the leading new dye in complexes with
These substances contribute to coffee, wine and beer flavors. G4s from oncogene promoters were comparable to those of a
Capture-SELEX protocol was used for aptamer selection because “gold standard” light-up probe thioflavin T. Interestingly, both
it suits well for selection of aptamers to small organic molecules. thiazole yellow and the new dyes demonstrated remarkable fluo-
Mix of few targets was used for selecting the aptamers from 98 rescence increase upon complexation with a double-module DNA
nt ssDNA bank having 60 nt randomized region. After 12 rounds structure composed of G4 and duplex fragments. Molecular
of selection, the aptamers were selected to each target separately modeling (docking) results suggest that the pronounced sensitiv-
in the final 13th round followed by cloning and Sanger sequenc- ity of the probes for the double-module structure is accounted
ing of 96 clones for each target. 7-8 aptamer candidates were for by stacking interactions with nucleic bases from the G4/du-
selected to each target. A novel isothermal DNA amplification plex junction. We assumed that several benzothiazole-based
based technique was used for affinity measurements. New apta- ligands might recognize the ncDNA-dsDNA junctions. The
mers for guaiacol, ethylguaiacol and homofuraneol are capable assumption is being verified using an expanded set of DNA struc-
to detect at least 0.5 lM of the target in solution. This work was tures. The results have implications for visualizing ncDNA in
supported by the Russian Science Foundation (project #16-19- duplex media. This work was supported by Russian Science
10697). Foundation [14-25-00013].
P.01-046-Mon P.01-048-Wed
Single nucleotide loops stabilize Synthesis and Degradation of Ms1 in
intramolecular parallel G4 DNA Mycobacterium smegmatis
D. Kaluzhny, A. Beniaminov, O. Mamaeva, A. Shchyolkina
M. Janouskova1, M. Sikov a1, J. Pospısil1, P. Palenıkova1,
Engelhardt Institute of Molecular Biology, Russian Academy of P. Bartl , J. Hnilicova , L. Krasny1
2 1
1
Sciences, Moscow, Russia Department of Microbial Genetics and Gene Expression, Institute
of Microbiology, Czech Academy of Sciences, Praha, Czech
Conformation of intramolecular G-quadruplex(G4) DNA
Republic, 2Faculty of Nuclear Sciences and Physical Engineering,
depends on multiple factors including nucleotide sequence.
Czech Technical University in Prague, Praha, Czech Republic
Apparently the length of the loops inputs certain constrains on
the resulting G4 DNA fold and affects its thermodynamic stabil- Adaptation of microorganisms is necessary for their survival in
ity. To shed some light on this issue, we report here on effect of changing environment. In this process, regulatory roles are
shortening of each of three loops on the resulting folding of the played by small non-coding RNAs (sRNAs). Ms1 is an abundant
G4 DNA and its conformational stability. Telomeric DNA sRNA (rivalling in amounts those of rRNA) found in Mycobac-
repeats d(TTAGGG)4 have been chosen as a starting model of terium smegmatis and it has homologs in all mycobacteria includ-
G4 structure. We considered all variants of potential quadruplex ing Mycobacterium turberculosis. Ms1 forms a complex with the
sequences in which one, two or all three TTA loops were RNA polymerase (RNAP) core and it is a pleiotropic regulator
replaced by a single thymine nucleotide. Circular dichroism, gel of gene expression, enhancing survival of the cell under various
migration, chemical probing with DMS and ZnP1 (a porphyrin types of stress. Ms1 is highly expressed and stable in stationary
derivative sensitive to G4 conformation) were applied to monitor phase and it is rapidly degraded when the cell is shifted into
conformational changes occurred upon shortening each loop to nutrient-rich medium. The accumulation of Ms1 in the cell
single nucleotide. We demonstrate that all variants of sequences depends both on its synthesis and degradation but the specific
form intramolecular structures. Circular dichroism data of all mechanisms involved are unknown. Here, we identify and charac-
DNA sequences confirm the formation of G4 structures of vari- terize the Ms1 promoter, the dynamics of Ms1 expression, and
able conformations. DNA sequences containing a single short reveal the presence of a transcription factor involved in regula-
loop formed a mixed quadruplex structure. More than two single tion of its expression. Further, we identify an RNase, Polynu-
thymine loops in quadruplex sequence lead to formation of par- cleotide phosphorylase (PNPase) to interact with Ms1. With
allel G4 structure with significant increase in thermodynamic sta- recombinant PNPase we demonstrate that it is able to degrade
bility. Our findings assist in understanding how nucleotide Ms1 in vitro and identify Ms1 secondary structures that affect its
sequence governs the conformation and stability of the G4 DNA. stability. RNAseq data show that PNPase is expressed ~10x more
This study was financially supported by the Russian Science in exponential than in stationary phase, inversely correlating with
Foundation (project 16-14-10396). the accumulation dynamics of Ms1. In summary, we provide a
comprehensive characterization of how the intracellular level of
Ms1 is controlled, paving the way to potential future designs
120 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
altering its expression in the case of pathogenic species. This (PQS). Primer extension method allowed to detect the stops cor-
work is supported by grant 946216 from the Grant Agency of responding the beginning of PQS sequences. We compared sev-
Charles University. eral polymerases and solution conditions in synthesis of PQS
containing sequences. Replacing of K+ ions by Cs+ ions in pri-
mer-extension reaction resulted in synthesis of full length DNA
P.01-049-Mon product as was observed by denaturing PAAG. Accordingly, the
In children with autism spectrum disorders, an presence of K+ and not Cs+ ions led to inhibition of PCR when
increase in cell-free DNA content aggravates amplifying PQS containing DNA. The obtained results allow to
amplify effectively G-rich DNA sequences by PCR and provide a
oxidative stress with decreasing activity of the
new method for detection of stable cation-dependent alternative
antioxidant system DNA structures. This work was supported by the Program of
S. Kanonirova1, Y. Chudakova1, E. Ershova1, L. Porokhovnik1, fundamental research for state academies for 2013–2020 years
N. Veiko1, S. Nikitina2, N. Simashkova2, N. Korovina3, (№ 01201363818).
O. Dolgikh1, G. Shmarina1, S. Kostyuk1
1
Research Centre of Medical Genetics (RCMG), Moscow, Russia,
2
Mental Health Research Center, Moscow, Russia, 3Scientific and P.01-051-Wed
Practical Center of Pediatric psychoneurology, Moscow, Russia Changes in the transcriptome of coelomocytes
Autism spectrum disorders (ASD) affect ~1% of the world’s pop- from sea star Asterias rubens under endotoxic
ulation and are characterized by impaired social interaction, com- stress
munication, and behavior. We report here a new piece of V. Bardashev1, A. Bezmenova2, M. Logacheva3, A. Penin3,4,
evidence for elevated cell-free DNA (cfDNA) in ASD. Children V. Pozarskaya3, T. Neretina5, D. Kuprash1,6
with ASD (n = 112) aged 4–12 were tested with ABC, SRS-2, 1
Biological Faculty, M.V. Lomonosov Moscow State University,
SCQ, AMSE scales and found to meet the F84.0, 84.1, 84.5 Moscow, Russia, 2Skolkovo Institute of Science and Technology,
ICD-10 and DSM-IV-TR criteria. The controls were healthy chil- Moscow, Russia, 3A.N. Belozersky Institute Of Physico-Chemical
dren (n = 64) aged 4–12. We found that plasma cfDNA in ASD Biology, M.V. Lomonosov Moscow State University, Moscow,
group was 2–4 times higher, while nuclease activity was 1.8-times Russia, 4Extreme Biology Laboratory, Institute of Fundamental
lower than in controls (P < 0.01). The more severe disease, the Medicine and Biology, Kazan Federal University, Kazan, Russia,
higher was the cfDNA content in ASD children’s plasma. Oxida- 5
White Sea Biological Station, Biological Faculty, M.V.
tive stress (OS) followed the autism progress. We supposed that Lomonosov Moscow State University, Moscow, Russia,
the surplus amount of cfDNA acts as a pathogenetic factor that 6
Engelhardt Institute of Molecular Biology, Russian Academy of
aggravates OS and potentiates apoptosis. Lymphocytes isolated Sciences, Moscow, Russia
from controls (n = 22) and ASD cases (n = 36) were exposed
in vitro to cfDNA obtained from ASD cases at 50 ng/mL for 1– Echinoderms occupy a critical phylogenetic position in deuteros-
3 h. In control cells, the exposure to cfDNA induced a transient tomes evolution. Coelomocytes are mobile cells present in the vis-
burst of reactive oxygen species (ROS) by a factor of 2–2.5 ceral coelom of the sea star and considered to be critically
against the baseline, however, it was accompanied by a 2-fold important for the host defense. We obtained 140.9 million of Illu-
increase in the expression of NRF2 and SOD1 transcription fac- mina MiSeq pair reads of RNA from sea star coelomocytes and
tors, which normalized the ROS level. In ASD cells, the exposure assembled them into 139 thousand contigs with N50 = 1911.
also provoked a 2–3-fold increase in ROS content, but no activa- After removing redundant contigs, the final assembly of 13002
tion of the antioxidant system followed: NRF2 expression was contigs (unigenes) with N50 = 2913 was obtained. Comparison to
3.5–4 times and 2–3 times lower, and SOD1 expression was 2–2.5 similarly assembled transcriptomes of A. rubens oocytes and S.
times and 1.6–2 times lower than in healthy cells, respectively, purpuratus coelomocytes demonstrated about 80% of common
after 3 and 24 h of the start of exposure (P < 0.01). Our findings transcripts while 20% were cell type specific. Protein domain
could help to reveal novel prognostic markers of ASD, and search identified a number of unigenes that may be related to
develop new therapies. The study was supported by RFBR grant evolution of the immune system. TIR domains and their flanking
#17-04-01587_A. sequences defined 5 putative Toll-like receptors and 2 homologs
of the adapter molecule MyD88. Unigenes encoding NACHT
and DD domains suggested 8 potential NOD-like receptors
P.01-050-Tue expressed in coelomocytes. 17 unigenes encoded polypeptides
Primer-extension stops at G4 DNA in with full length SRCR domain and were annotated as putative
oncogene promotors scavenger receptors. We found homologs of complement compo-
nents B, C2 and C3, peroxidase and NO-synthase, cathepsins,
G. Chashchina1,2, A. Beniaminov1, D. Kaluzhny1
1 granzymes, homologs of RAG-1/RAG-2 recombinase, transcrip-
Engelhardt Institute of Molecular Biology, Russian Academy of
tional factor NFkB, as well as intracellular signaling and extra-
Sciences, Moscow, Russia, 2Moscow Institute of Physics and
cellular recognition domains of several cytokine receptors.
technology, Dolgoprudny, Moscow Region, Russia
Differential gene expression in response to stress and endotoxin
A number of oncogene promoters in the human genome contain was analyzed in silico, followed by analysis of selected candidate
G-rich nucleotide sequences that are able to form G-quadruplex genes by quantitative real-time PCR. Of 14 genes activated by at
(G4) structure. Folding of some of them was demonstrated least 5 fold, 6, including the top 2, appear to encode transcription
in vitro and in vivo. Such G4 structures are considered potential factors of leucine zipper and zinc finger families domains related
molecular targets in treatment of cancers. Amplification of G-rich to stress responses and cell activation. The top 2 stress-induced
DNA sequences often considered problematic and potential G4 unigenes (annotated as early growth response protein 1-B-like
structures are referred a possible reason. As so, polymerase stops isoform X1 and fos-related antigen 1 isoform X1) also showed a
could be a convenient method for detecting G4 structures in the significant increase of expression in response to E. coli in vivo.
genomic DNA. In this work, we used double stranded DNA
fragments of four oncogene promoters (C-MYC, C-KIT, KRAS
and NRAS) which contained the potential quadruplex structure
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 121
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
P.01-052-Mon P.01-054-Wed
Polyplex using tumor-specific aptamers, Wy-5a A promising biomarker for diagnosis of breast
and AS1411 for targeting delivery of cancer: miRNA
therapeutic gene to prostate cancer € G€
N. S. Yilmaz1, O. ulbahar1, B. Sen1, G. Çetinkaya2, E. Tezel2
1
J. Lee, M. Lee, Y. Kim, J. Oh, Y. Lee Gazi University Medical Faculty, Department of Medical
Hanyang University, Seoul, South Korea Biochemistry, Ankara, Turkey, 2Gazi University Medical Faculty,
Department of General Surgery, Ankara, Turkey
Prostate cancer (PCa) is one of the most frequently diagnosed
cancers among western male population. So far, treatment of An important part of breast cancer studies focused on the discov-
PCa mostly relies on chemotherapy and radiation therapy, which ery of biomarkers that will enable early diagnosis, because it can
have high toxic to normal tissues and side effects. Therefore, new be completely cured with early diagnosis, MicroRNAs (miRNAs)
treatments for PCa have been developed to make up for non-spe- are accepted as potential biomarkers for the early detection of
cific therapy. Aptamers are short single-stranded oligonucleotides breast cancer. MiRNAs posttranscriptionally regulating gene
that can bind defined targets with high affinity and specificity. It expression are known to affect tumor formation, growth and
is derived from Cell-SELEX (systematic evolution of ligands by spread. We aimed to determine whether there is a statistically sig-
exponential enrichment). Compared to antibodies, it has attrac- nificant difference in serum cancer-specific miRNA levels among
tive characteristics such as low immunogenicity and higher stabil- the patients diagnosed with breast cancer and healthy controls.
ity. In this study, tumor specific aptamers, Wy-5a and AS1411, The study included 40 breast cancer patients admitted to Gazi
were applied on targeting delivery of nucleic acids. Wy-5a and University Hospital and had not received any treatment yet, and
AS1411 bind to membrane protein related to progression of PCa 35 healthy women as a control group. 86 different miRNAs iso-
and nucleolin highly expressed on most cancer cell surfaces, lated from serum samples. Isolated miRNAs were transformed
respectively. Wy-5a and AS1411 were conjugated to polyethyle- into cDNA using a reverse transcription kit, then pre-amplifica-
neimine (25 kDa, PEI25k) using maleimide polyethylene glycol tion reaction and exonuclease reaction is done. After PCR, analy-
succinimidyl ester (Mal-PEG-NHS, Mw 2000) as a linker. PEI- sis of the obtained RT-PCR results (Ct values) was calculated.
PEG-Wy-5a and PEI-PEG-AS1411 formed stable complexes with For the intergroup comparisons Student T-test was used. Com-
plasmid DNA (pDNA). The transfection assays showed that pared with the control group, in patients with breast cancer the
PEI-PEG-Wy-5a and PEI-PEG-AS1411 had higher transfection levels of let-7a-5p, miR-125a-5p, miR-148a-3p, miR-21-5p, miR-
efficiencies to PC3 prostate cancer cells than their counter-parts 100-5p, miR-150-5p, miR-27b-3p, let-7d-5p, miR-15b-5p, miR-
without aptamers. In addition, the cytotoxicity of PEI-PEG-Wy- 125b-5p, miR-126-3p were found to be statistically significantly
5A and PEI-PEG-AS1411 was lower than PEI25k. Therefore, increased (P < 0.05); while the levels of miR-205-5p, miR-181a-
PEI-PEG-Wy-5A and PEI-PEG-AS1411 may be useful for thera- 5p, miR-301a-3p, miR-127-5p, miR-215-5p, miR-193a-5p, miR-
peutic gene delivery targeting to PCa. 372-3p, miR-149-5p, miR-181c-5p decreased (P < 0.05). MiRNAs
which have a statistically significant difference between the two
groups in the study have potential for early diagnosis of breast
P.01-053-Tue cancer. However, further studies are needed so that serum miR-
Combination therapy of adiponectin gene and NAs can be validated as a noninvasive biomarker early in the
RAGE binding peptide for acute lung injury diagnosis of breast cancer.
C. Piao1, J. Oh1, Y. Lee1, J. H. Park2, M. Lee1
1
Hanyang University, Seoul, South Korea, 2Inje University, Seoul, P.01-055-Mon
South Korea
Detection of b-thalassemia Cd 39 mutation by
Acute lung injury (ALI) is a severe lung inflammatory disease, using piezoelectric biosensor for non-invasive
induced by various causes including infections, traumas, and ische-
prenatal diagnosis
mia-reperfusion damages. Previously, it was reported that RAGE
U. K€okbas, K. Kartlasmıs, E. D€
undar Yenilmez, A. Tuli,
binding peptide (RBP) reduced inflammation response in the ALI
L. Kayrın
animal models by inhibition of RAGE-mediated signal pathway.
Department of Biochemistry, Faculty of Medicine, Çukurova
Interestingly, RBP has high positive amino acids, suggesting that it
University, ADANA, Turkey
may bind plasmid DNA (pDNA) and be useful as a carrier of
pDNA. In the current study, RBP was characterized as a carrier of b-Thalassemia is one of the most monogenic autosomal recessive
the adiponectin (APN) gene as well as therapeutic peptide. Our disorder characterized by defective production of the b-chain of
previous report showed that APN gene had an anti-inflammatory hemoglobin. Definition of the b-globin genotype is necessary for
effect and protected the endothelial cells in the ALI animal models. genetic counselling in the carriers, and for predicting prognosis
Therefore, the delivery of the APN gene with RBP may synergistic and management options in the patients with thalassemia. DNA-
effect in the treatment of ALI. A gel retardation assay and a hep- based prenatal diagnosis of b-thalassemias routinely relies on
arin competition assay confirmed that RBP formed stable com- polymerase chain reaction (PCR) and gel electrophoresis. The
plexes with the APN plasmid (pAPN). In vitro transfection assays aim of this study is to develop a new procedure, a DNA-based
showed that the RBP/pDNA complex had higher transfection effi- piezoelectric biosensor, for the detection of b-thalassemia Cd 39
ciency than naked pDNA in the L2 lung epithelial cells. In addi- mutation on the fetus’ cell free DNA from maternal blood, the
tion, the RBP/pDNA complex higher gene delivery efficiency than common b-thalassemia mutation type in Turkey. Cell free fetal
naked DNA in the LPS induced ALI animal models. Furthermore, DNA taken from maternal whole blood. Bioactive layer was con-
the RBP/pAPN complex had higher anti-inflammatory effect in stituted by binding 2-Hydroxymethacrylate Methacryloami-
the ALI animal models, reducing the pro-inflammatory cytokine doscysteine (HEMA-MAC) nanopolymers on the electrode’s
levels. The results suggested that RBP may be useful as a carrier of surface. Single oligonucleotide probes specific for Cd 39 mutation
pDNA into the lungs as well as an anti-inflammatory therapeutic of b-thalassemia were attached to the nanopolymer. The mea-
peptide. Therefore, the RBP/pAPN complex may have a synergis- surements were executed by piezoelectric resonance frequency
tic effect for the treatment of ALI. which is caused by binding of the cell free fetal DNA in media
122 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
with the single oligonucleotide probe on the electrode surface. (acid phosphatase 4) have been identified to cause hypoplastic
The results were confirmed by the conventional molecular AI. In this study, we identified novel heterozygous compound
method as ARMS. The piezoelectric resonance frequencies ACP4 mutations in a hypoplastic AI family using whole exome
obtained by hybridization of the cell free fetal DNA on bioactive sequencing and Sanger sequencing: c.262C>A (p.Arg88Ser) and
layer were found 221 15, 279 8, and 318 9 Hz for the c.419C>T (p.Pro140Leu). Western blot revealed that the mutant
samples of normal b-globin, heterozygote, and homozygote of proteins were expressed in a greatly reduced level compared to
Cd 39 mutation, respectively. The developed biosensor serves as the wild type protein. To further explicate the effect of the
a specific result to Cd 39 mutation. It could accurately discrimi- altered protein sequence on ACP4 homodimerization, Co-IP was
nate between normal and Cd 39 mutation samples. Because of done. Moreover, to compare AP activity levels of wild type and
low costs, fast results, specificity and high detection/information mutant forms, acid phosphatase assay was performed. The aim
effectiveness as compared with conventional prenatal diagnosis of this study was to elucidate how the compound heterozygous
methods, we can be offered this technique as an alternative to ACP4 mutations, located in extracellular part of ACP4 protein,
conventional molecular methods. affect protein structure and function of ACP4.
P.01-056-Tue P.01-058-Mon
Preferential binding of p53 and BRCA1 Effects of paricalcitol on hydrogen peroxide-
proteins to naturally occurring G-quadruplexes induced oxidative damage in human umbilical
forming sequences vein endothelial cells through vitamin D
J. Cerven1, N. Mikyskova2, A. Niziolova2, M. Bartas2, receptor signaling
V. Brazda3, P. Pecinka1 M. Akıs1,2, M. Kant1, Z. Y€ uce3, M. Dizdaroglu4, H. Islekel1,5
1
Faculty of Science, Institute of Environmental Technologies, 1
Department of Medical Biochemistry, Faculty of Medicine, Dokuz
University of Ostrava, Ostrava, Czech Republic, 2Faculty of Eylul University, Izmir, Turkey, 2Department of Medical
Science, Department of Biology and Ecology, University of Biochemistry, Faculty of Medicine, Balikesir University, Balikesir,
Ostrava, Ostrava, Czech Republic, 3Institute of Biophysics, Turkey, 3Department of Medical Biology and Genetic, Faculty of
Academy of Sciences of The Czech Republic, Brno, Czech Medicine, Dokuz Eylul University, Izmir, Turkey, 4Biomolecular
Republic Measurement Division, National Institute of Standards and
Aim of our work was to study preferential binding of BRCA1 Technology, Gaithersburg, Maryland, United States of America,
5
and p53 proteins to different forms of G-quadruplexes found in Department of Molecular Medicine, Graduate School of Health
promotor regions of p21 and ESR1 genes. Based on enhanced Sciences, Dokuz Eylul University, Izmir, Turkey
occurrence of sequences, that are able to form G-quadruplexes in Oxidative DNA damage is caused by the imbalance between
promotor regions of large scale of genes including genes that are reactive oxygen species (ROS) and the antioxidants in cells.
involved in cell cycle regulation, our hypothesis is, that formation Accumulation of DNA damage might lead to a variety of muta-
of G-quadruplexes can be a part of regulation of gene expression tions and consequently to genomic instability. Paricalcitol is a
and cell cycle progression/arrest. For both p53 and BRCA1 pro- synthetic vitamin D analog that shows similar physiological
teins binding to Non-B DNA motifs have been proven to be one properties with vitamin D, but has less hypercalcemic effects.
of their modes of action. P53 protein is involved in regulation of Paricalcitol exerts most of its pleiotropic actions via binding to
expression of p21 and BRCA1 is involved in ESR1 regulation vitamin D receptor (VDR). The relation between vitamin D and
pathway. Understanding mechanisms of regulation is important non-calcemic analogs such as paricalcitol with oxidative DNA
for discovery of new anti-cancer therapeutic methods, for exam- damage is noteworthy due to the high prevalence of vitamin defi-
ple possibility of using G-quadruplex stabilisation drugs. Using ciency and the importance of genomic instability. The aim of this
bioinformatic tools we have found sequences able to form G- study is to investigate the dose-dependent effects of paricalcitol
quadruplexes in promotor regions of human p21 and ESR1 on the formation of ROS, levels of oxidatively induced DNA
genes. Sequences of maximally 45 bp, with four repetitions of at damage and expressions of DNA repair enzyme APE1 and VDR
least 3 guanines separated by loops of maximally 15 bp were in a model of hydrogen peroxide (H2O2)-generated oxidative
searched. We have found 4 sequences able to form G-quadru- stress in human umbilical vein endothelial cells (HUVEC). Three
plexes in p21 promoter and 3 sequences in ESR1 promoter. doses (0.1–10–100 nM) of paricalcitol and 100 nM of ZK159222
Using less strict criteria dozens of potential sequences were antagonist to evaluate the role of VDR were used in the study.
found. We have PCR amplified these promotor regions from The generation of ROS was assessed fluorometrically using the
HCT16 cell lines. Using B4G antibody we have tested ability of probe dichlorofluorescein diacetate (DCFH-DA). APE1 and
these sequences to form G-quadruplex structures in different con- VDR gene expressions were analyzed by qPCR. Treatment of
ditions and tested preferential binding of p53 and BRCA1 pro- 300 lM H2O2 significantly increased ROS as compared to con-
teins to these sequences using EMSA and immunochemical trol (P < 0.001). Paricalcitol pretreatments attenuated excessive
methods. ROS in HUVECs exposed to H2O2 (P < 0.05). Moreover, this
effect was performed by VDR-mediated signaling. Treatment of
HUVEC with H2O2 significantly decreased APE1 and VDR
P.01-057-Wed mRNA expressions (P < 0.05). But, preincubation with different
Functional analysis of novel compound doses of paricalcitol did not have a significant effect on the
heterozygous ACP4 mutations causing expressions of the APE1 and VDR. This study demonstrated that
hypoplastic amelogenesis imperfecta paricalcitol might protect endothelial cells from H2O2-generated
Y. J. Kim, J. Kang, J. W. Kim oxidative stress. For further work, the levels of damaged DNA
School of Dentistry Seoul National University, Seoul, South Korea nucleosides will be analyzed using liquid chromatography-tandem
mass spectrometry.
Amelogenesis imperfecta (AI) is a rare hereditary condition
affecting tooth enamel. The affected enamel can be hypomineral-
ized and/or hypoplastic. Recently, recessive mutations in ACP4
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 123
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
P.01-062-Tue
P.01-060-Wed mRNA 5’ UTR modulates positive effect of
miRNA and cytokine expression in active incorporated m5C and N1 mΨ nucleotides on
Behcet’s patients mRNA expression
N. Eyerci1, E. Diyarbakir2 A. Anisimova1,2, K. Akulich1,2, T. Abakumova3, T. Zatsepin3,
1
Kafkas University, Faculty of Medicine, Department of Medical V. Gladyshev2,4, S. Dmitriev2,5,6
Biology, Kars, Turkey, 2Ataturk University, Faculty of Medicine, 1
School of Bioengineering and Bioinformatics, Lomonosov Moscow
ERZURUM, Turkey State University, Moscow, Russia, 2Belozersky Institute of
Behcet’s disease is a chronic multisystem inflammatory disorder Physico-Chemical Biology, Lomonosov Moscow State University,
characterized mainly by recurrent oral ulcers, ocular involvement, Moscow, Russia, 3Skolkovo Institute of Science and Technology,
genital ulcers, and skin lesions, presenting with remissions and Skolkovo, Moscow Region, Russia, 4Division of Genetics,
exacerbations. Environmental and genetic factors are thought to Department of Medicine, Brigham and Women’s Hospital,
contribute to its onset and development. Although the etiology Harvard Medical School, Boston, United States of America,
5
of Behcet’s disease is not yet clear, new immunogenetic findings Department of Biochemistry, Biological Faculty, Lomonosov
provide clues in the pathogenesis. miRNAs have created a new Moscow State University, Moscow, Russia, 6Engelhardt Institute
paradigm for gene regulation and pathways involved in the of Molecular Biology, Russian Academy of Sciences, Moscow,
pathogenesis of autoimmune diseases. The goal of this study was Russia
to investigate the expression of miRNAs in patients with active mRNA transfection is considered to be a promising approach for
Behcet’s disease (BD). Peripheral blood mononuclear cells gene delivery due to minimal hazards of genome modification
(PBMCs) were obtained from BD patients with exacerbation and and no dependence on cell proliferation status. It may be success-
from healthy controls. Expression level miRNAs were studied fully applied for stem cell reprogramming, vaccination and sub-
using Dynamic Array System. The levels of cytokines mRNAs stitutional therapy. However, in vitro transcribed mRNAs
124 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
activate the innate immune response, complicating transfection of above this temperature. This work was supported the project
primary cells. Incorporation of modified nucleotides into a tran- SYMBIT reg. number: CZ.02.1.01/0.0/0.0/15 003/0000477
script can both decrease the immune response and improve
mRNA stability. However, published data on the effects of
nucleotide modifications on protein production are controversial. P.01-065-Tue
Here, we examined the effects of two modified nucleosides, N1- Identification of RNA secondary structure in
methylpseudouridine and 5-methylcytidine, within a luciferase Mycoplasma gallisepticum living cells
transcript, as well as three 5’ cap variants, on reporter mRNA T. Semashko, D. Evsyutina, G. Fisunov, V. Govorun
expression in several in vitro and in vivo systems. We compared Federal Research and Clinical Center of Physical-Chemical
efficiency and kinetics of regular and modified transcript transla- Medicine, Moscow, Russia
tion in cell extracts, immortalized cultured cells, and mouse pri-
mary hepatocytes and fibroblasts. All mRNAs bearing m7G-cap, Mycoplasma gallisepticum is a representative of the class Molli-
both with and without m5C and N1 mΨ, efficiently produced cutes. It is characterized by lack of cell wall and many metabolic
luciferase for up to 10–12 h in living cells and for 1–2 h in cell- pathways, reduced genome size and loss of many of the known
free systems. The effects of mRNA modifications on translation regulators. Due to this, genome-wide profiling of RNA secondary
efficiency differed for the systems used. For modified mRNAs, structure may be a useful tool for studying the transcriptome of
we documented a delay in product appearance, indicating a the cell and its regulation. To determine the RNA secondary
decrease in elongation rate that was especially pronounced in the structure in vivo, the method of chemical probing with dimethyl
case of m5C containing transcripts. By using reporter mRNAs sulphate (DMS) with further detection of modification sites by
with various 5’ untranslated regions, we showed that the modi- high-throughput sequencing was chosen. We optimized condi-
fied nucleotides within mRNA leader negatively affect translation tions for modification of M. gallisepticum RNA by DMS. Then
initiation, presumably due to interference with ribosomal scan- we developed a method of obtaining strand-specific cDNA
ning. Thus, 5’ leaders with poor CU content may be preferred library for further high-throughput sequencing. Samples of inter-
for therapeutic applications. The work was supported by the est were sequenced and analyzed using a number of bioinformat-
grant of the Russian Federation government № 14.W03.31.0012. ics tools. Quality of identified RNA secondary structures was
checked by comparing our data with the predicted conservative
structure of RNase P. Genome-wide RNA secondary structure
P.01-063-Wed map at nucleotide resolution with quantitative structural infor-
Various G-quadruplex structures of the human mation was created. Differences in structure for genes and anti-
sense RNA and for coding sequence, intergenic regions, 5’UTR
telomere sequence studied by circular
and 3’UTR were determined. This work was supported by Rus-
dichroism and Raman spectroscopies sian Science Foundation 14–24–00159 “Systems research of mini-
y1, I. Kejnovska1, P. Mojzes2, M. Vorlıckova1
J. Palack mal cell on a Mycoplasma gallisepticum model”.
1
IBP CAS, v.v.i., Brno, Czech Republic, 2Institute of Physics,
Faculty of Mathematics and Physics, Charles University in
Prague, Prague, Czech Republic P.01-066-Wed
In this work, we combine conventional CD measurements with Mutation m14441T>C in ND6 gene of
Raman spectroscopy (RS) to probe solution structures adopted mitochondrial DNA inhibits antioxidant
by the 22-mer DNA sequence of AG3(TTAG3)3 (wild type, response in fibroblasts of patients with Leigh-
referred to as wt hereinafter) derived from the human telomere like syndrome
repeat region as well as its abasic modification, in which adenine
M. S. Abramova1,2, M. S. Konkova1, A. A. Kalyanov1,
in one loop at the position 19 (ap19) or three loop adenines at
E. A. Nikolaeva1,3, N. N. Veiko1, E. M. Malinovskaya1,
the positions 7, 13 and 19 (ap7,13,19) were removed. As shown
L. V. Kameneva1, V. A. Sergeeva1, P. G. Tsygankova1,
by us, CD spectra unambiguously differentiate between quadru-
S. V. Kostyuk1
plex structures adopted by wt, ap19 and ap7,13,19 in the pres- 1
Research Centre of Medical Genetics (RCMG), Moscow, Russia,
ence of different stabilizing cations (Na+ or K+), at different 2
Pirogov Russian National Research Medical University
ionic strengths and temperatures. Here we aim to provide addi-
(RNRMU), Moscow, Russia, 3I.M. Sechenov First Moscow State
tional and complementary information to the earlier CD studies
Medical University, Moscow, Russia
by means of RS. As demonstrated by several researchers over the
last five decades, conventional non-resonant RS is highly sensi- Mitochondria have the key role in energy production. Mutations
tive to DNA structure and its conformational transitions induced in mitochondrial DNA (mtDNA) can lead to hereditary mito-
by various physicochemical parameters. We have already shown chondrial disorders connected to disturbances in oxidative phos-
that RS can be used to monitor transition from antiparallel to phorylation and metabolic pathways. Dying cells emit DNA that
parallel G-quadruplex folding induced by increasing oligonu- becomes part of the oxidized cell-free DNA (cfDNAoxy) pool.
cleotide concentration. In the present study, Raman spectra of CfDNAoxy as well as irradiation dose of 10 cGy can induce
wt, ap19 and ap7,13,19 quadruplexes stabilized by Na+ and K+ oxidative stress in healthy donors’ fibroblasts by increasing ROS
cations at rather high oligonucleotide concentrations (~ 1 mM) production, activating NRF2-signaling pathway. NRF2 activa-
have been compared with those measured in the absence of stabi- tion is an adaptive response to endogenous and exogenous oxida-
lizing cations. The greatest difference was observed between the tive stress and it leads to resistance of cells towards exposure to
Raman spectra of ap7,13,19 and those of the other two damaging and toxic agents. The significance of mutations
sequences, wt and ap19, reflecting the fact that ap7,13,19 forms a m14441T>C in ND6 and m.3945C>A in ND1 in mtDNA of two
predominantly parallel quadruplex, whereas wt and ap19 seem to patients with Leigh-like syndrome (in both cases heteroplasmy
adopt mixture of parallel/antiparallel alignment. Specific Raman was 100%) on the mitochondrial function under cfDNAoxy
markers also reflect further increase in the extent of parallel fold- treatment or irradiation with 10 cGy was assessed. It was shown
ing of ap7,13,19 with increasing temperature (max. at ~50°C), as that mitochondrial potential is decreased 60–80% in fibroblasts
well as thermal denaturation of all three quadruplex structures with mutation in the ND6 gene compared to healthy donors. It
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 125
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
decreases 2–4 times (P < 0.01) when cells are treated with P.01-068-Tue
cfDNAoxy or irradiated with 10 cGy. In fibroblasts of healthy
Guanine quadruplexes in the oct4 gene
donors and in case of mutation in ND1 gene mitochondrial
potential increases when cells are treated with cfDNAoxy or irra- promoter
D. Renciuk, I. Kejnovska, D. Subert, M. Brazdova,
diated with 10 cGy. In fibroblasts of healthy donors and in case
of mutation in ND1 gene expression of NRF2 is decreased when M. Vorlıckova
cells are treated with cfDNAoxy or irradiated with 10 cGy. Cells Institute of Biophysics, Academy of Sciences of the Czech
with mutation in ND1 gene, however have a 50% decrease in Republic, Brno, Czech Republic
NRF2 level. Mutation in m14441T>C causes a decrease in NRF2 Oct4 (POU5f1) gene codes the key regulator of cellular pluripo-
levels 5–8-fold and NRF2 protein 3–4-fold (P < 0.01). Thus, tency. Recently, we have described a guanine quadruplex motif
m14441T>C in ND6 of mtDNA significantly influences signaling in close proximity to the oct4 transcription start site (TSS) and
pathways involved in the response of cells to oxidative stress. we confirmed its influence on the regulation of the oct4 gene
This work was supported by RFBR grant No. 16-04-01099_A. expression. Guanine quadruplexes (G4) are non-canonical sec-
ondary structures of nucleic acids, based on several stacked
square-shaped planar tetrads; each formed by four Hoogsteen-
P.01-067-Mon hydrogen bonded guanines, and stabilized by monovalent cations
Single-stranded DNA aptamers to in central cavity. Our recent bioinformatics analysis revealed
Mycobacterium tuberculosis RNA polymerase seven other potential quadruplex-forming motifs (PQS) within
I. Petushkov1, Z. Morichaud2, B. Konstantin2, A. Kulbachinskiy1 the -5000 to 500 bp range around the mouse oct4 TSS, some of
1 them located within the highly conserved regulatory regions of
Institute of Molecular Genetics, Russian Academy of Sciences,
Moscow, Russia, 2IRIM, CNRS, Univ Montpellier, Montpellier, the oct-4 gene, including the proximal and distal enhancers. We
France characterized these PQS in terms of overall topology and thermal
stability by circular dichroism and UV absorption spectroscopy
Mycobacterium tuberculosis is a human pathogen with a highly
and gel methods. Out of the eight selected sequences, six form
regulated transcription machinery which is composed of the
monomolecular, i.e. relevant G4 with melting temperature higher
RNA polymerase core enzyme (RNAP), the major sigma subunit
than 50°C, two exceeding 80°C, under physiological salt concen-
(sigmaA) and 12 alternative sigma subunits. RbpA is an essential
trations. Interestingly, we observed that the folding of the
transcription regulator, which is found only in Actinobacteria
quadruplexes ranged from pure antiparallel G4 through hybrid
and is absent in other species including Escherichia coli. Alterna-
forms to pure parallel ones; as shown before, each quadruplex
tive sigma subunits and RbpA are implicated in stress response
type might have significantly distinct interacting partners and this
and antibiotics resistance. The mechanisms of promoter recogni-
variability might be broadened by the variability of the loops.
tion by alternative forms of RNAP holoenzyme, and the role of
Our results indicate potential biological regulatory role of some
RbpA in this process remain only partially understood, in part
of the quadruplexes and offer solid base for consecutive in cell
due to the low growth rate and high pathogenicity of M. tubercu-
studies involving functional assays as well as detection of G4
losis. To uncover the mechanisms of specific transcription initia-
within the cells. This work was supported by the CSF (17-
tion we used an in vitro transcription system with purified RNAP
19170Y) and by the project SYMBIT reg. nr. CZ.02.1.01/0.0/0.0/
and transcription factors of M. tuberculosis in combination with
15 003/0000477 financed by the ERDF.
analysis of specific single-stranded DNA aptamers obtained by
in vitro selection. We obtained ssDNA aptamers to RNAPs con-
taining either sigmaA or sigmaB subunits in the presence and in P.01-069-Wed
the absence of the RbpA factor. We showed that the aptamers
Oxidized cell-free DNA causes a short-term
are specifically recognized by RNAP, with apparent dissociation
constants of the aptamer-holoenzyme complexes being in the activation of TLR9-signaling pathway in MSCs
nanomolar range. We identified extended -10-like promoter A. A. Kalyanov1, M. S. Konkova1, V. A. Sergeeva1,
elements in aptamers to both sigmaA or sigmaB holoenzyme E. M. Malinovskaya1, E. S. Ershova1, N. N. Veiko1,
forms. Some aptamers did not contain known promoter elements L. V. Kameneva1, M. S. Abramova1,2, A. D. Filev1,
suggesting that additional as yet unidentified elements may con- S. V. Kostyuk1
1
tribute to promoter recognition by mycobacterial RNAP. Finally, Research Centre of Medical Genetics (RCMG), Moscow, Russia,
2
we demonstrated that the aptamers sense RNAP interactions Pirogov Russian National Research Medical University
with the sigma subunits or RbpA, and RbpA can strongly stimu- (RNRMU), Moscow, Russia
late the binding of some of them to the RNAP holoenzyme. Low-dose ionizing radiation (LDIR) causes cell death of a frac-
Thus, the aptamers may provide a useful tool for analysis of tion of population in mesenchymal stem cells in vitro, which
RNAP interactions with promoters, transcription factors and leads to release of oxidized cell-free DNA (cfDNA). Oxidized
antibiotics. The work was supported in part by the Russian cfDNA can cause adaptive response to LDIR in MSC. We have
Foundation for Basic Research (grant 17-54-150009) and PRC shown that oxidized cfDNA like LDIR induces ROS production,
Russie CNRS/RFBR. DNA oxidation, DNA damage, cell-cycle arrest, activation of
reparation processes, antioxidant response and apoptosis inhibi-
tion in MSCs. Could TLR9-signaling pathway play a significant
role in protection of MSCs from apoptosis? To answer this ques-
tion, cells were treated with oxidized DNA (300 mM H2O2/Fe2+/
EDTA) in concentration 50 ng/mL. The level of oxidation mar-
ker 8-oxodG in the obtained DNA was measured with ESI-MS/
MS and was ~ 400 8-oxodG per 106 nucleotides. This oxidized
DNA causes a 3–4-fold increase in TLR9 mRNA level within an
hour after addition. Three hours later the mRNA level returns to
control levels. Protein TLR9 levels increase 2–3-fold 1 h after
start of incubation and decrease to control levels 3 h after.
126 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
Unoxidized DNA doesn’t affect TLR9 mRNA and protein levels. P.01-071-Tue
Gene expression levels of adaptors and effectors of TLR9-signal-
Catalytically active Argonaute proteins from
ing pathway change in response to addition of oxidized DNA:
after 1 h mRNA levels of MYD88, TIRAP, HSPD1, MAP2K3 mesophilic bacteria
increase 3- 4 -fold, levels of TICAM2, SARM1, TOLLIP, HRAS, D. Yudin1, S. Ryazansky1, A. Kulbachinskiy1, A. Aravin2,
HSPA1A and MAPK8IP3 increase 2–3-fold, there is a 1.5-fold A. Kuzmenko1
1
increase in the level of expression of PELI1 and RIPK2. Expres- Institute of Molecular Genetics, Russian Academy of Sciences,
sion of genes EIF2AK2, PPARA increases 3- 4-fold, genes Moscow, Russia, 2Division of Biology and Biological Engineering,
TRAF6, FADD, IRAK1 and IRAK2 2-fold. 3 h after start of California Institute of Technology, Pasadena, CA, United States of
incubation levels of these genes return to control. Blocking America
TLR9-signaling pathway with ODN 2088 (TCCTGGCGGGG Argonaute proteins are an integral part of eukaryotic RNA inter-
AAGT) and chloroquine in combination with oxidized DNA did ference machinery. They bind small noncoding RNAs and utilize
not lead to increase in the expression of these genes. Thus, oxi- them for guided cleavage of complementary RNA targets or indi-
dized cfDNA causes a short-term activation of TLR9-signaling rect gene silencing by recruiting additional factors. Argonaute
pathway in MSCs. This work was supported by RFBR grant proteins are also encoded in many bacterial and archaeal gen-
No. 16-04-01099 _ A. omes (pAgos). pAgos from thermophilic bacteria were initially
studied to gain structural insight into eukaryotic RNA interfer-
ence. They were later shown to cleave DNA substrates in a
P.01-070-Mon guided manner employing small RNAs or DNAs, which appear
Folding rules of intercalated cytosine to be generated autonomously by pAgos. Thus, pAgos might be
tetraplexes based on genome-derived C-rich considered as means of prokaryotic defense against invasive
DNA fragments genetic elements. Here we characterize pAgos from non-cultivable
Z. Dvorakov akova, I. Kejnovska,
a, D. Renciuk, P. Skol or pathogenic mesophilic bacteria. Candidate proteins were
K. Bedn arov
a, M. Vorlıckova selected through bioinformatic screening of genomic databases.
Institute of Biophysics, Academy of Sciences of the Czech Corresponding pAgo genes were chemically synthesized and used
Republic, Kr alovopolsk
a 135, CZ - 612 00, Brno, Czech Republic for expression in a heterologous system. Upon expression in
E. coli these proteins have been shown to associate with short
Intercalated cytosine tetraplex (i-motif, iM) is a four stranded (14–25 nt) 5’-phosphorylated DNA molecules. Such short DNA
DNA structure which is adopted by cytosine-rich regions in loading relies on the catalytic activity of pAgos and is abolished
DNA at slightly acidic pH. This structure is formed by two par- in catalytically dead protein variants, which bear amino acid sub-
allel duplexes bound by hemi-protonated cytosine base pairs stitutions in the DEDX catalytic tetrad. Further in vitro assays
(C∙C+), which are mutually intercalated in antiparallel fashion. have shown that purified pAgos cleave various DNA substrates
iM structure formation and stability depends on: 1) the number in a guide-dependent manner. They display high activity at tem-
and lengths of cytosine stretches, 2) number and sequence of peratures ranging from 30 to 45 °C, with the efficiency of cleav-
non-C nucleotides between cytosine stretches forming iM loops age being greatly affected by ionic strength, supplied divalent
and 3) pH value. Using electronic circular dichroism (CD), UV cations and guide molecules. This suggests that all studied pAgos
absorption spectroscopy and electrophoretic methods, we have act as DNA-dependent DNA nucleases which may subsequently
examined iM formation and stability of the four C3TAA repeats be used as means of targeted genome editing in eukaryotic organ-
of the human telomere DNA sequence and of its modified ana- isms. This work was supported in part by the Grant of the Min-
logues with substituted cytosines at selected positions. The istry of Education and Science of Russian Federation
obtained results have enabled us to formulate simple rules for the 14.W03.31.0007.
manner of iM folding: At least four alternating C∙C+ pairs are
needed to form intramolecular iM structure; one or two missing
terminal Cs in different repeats decrease the iM thermal stability, P.01-072-Wed
but the intramolecular folding is preserved. Loss of the middle C The role of Drosophila CPEB protein Orb2 in
in the repeat hinders the intramolecular iM folding and instead a
regulation of localized translation
bimolecular iM is formed. The bimolecular iM consists of two
R. Gilmutdinov1, E. Kozlov1, K. Yakovlev1, P. Schedl1,2,
iM parts. In this case, even mere three C∙C+ pairs in one part,
Y. Shidlovskii1,3
probably stacked on the intact six C∙C+ pairs of the second part, 1
Institute of Gene Biology, Russian Academy of Sciences, Moscow,
suffice to maintain bimolecular iM stable. To test the robustness
119334, Russia, 2Department of Molecular Biology, Princeton
of these rules they were applied to C-rich sequences derived from
University, Princeton, United States of America, 3I.M. Sechenov
the promoter of mouse oct4 gene that have irregular numbers of
First Moscow State Medical University, Moscow, 119048, Russia
cytosines in the block, as well as, variable primary sequence and
length of the loop. The behaviour of iMs formed by such a com- mRNA localization is a phenomenon important for development
plex sequences seem to be more complicated and cannot be fully and functioning of polarized cells. Multiple localized mRNAs
followed by these simple rules. This work was supported by the carry motifs at their 3’ UTRs called cytoplasmic polyadenylation
CSF (17-19170Y) and by the project SYMBIT reg. nr. elements (CPEs). These elements are recognized by a highly con-
CZ.02.1.01/0.0/0.0/15 003/0000477 financed by the ERDF. served family of RNA binding proteins, cytoplasmic polyadenyla-
tion element binding proteins (CPEBs). The CPEBs belong to
regulatory switch proteins that have dual functions either to
repress or to activate translation. Orb2 is one of the CPEB pro-
teins in Drosophila. Previous studies have revealed its functions
in asymmetric cell division, viability, motor function, learning,
and memory in Drosophila. Recent experiments indicated that
Orb2 regulates its own expression in at least two different con-
texts, in the nervous system in the processes of learning and
memory formation and in the testis in the spermatogenesis. To
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 127
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
128 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS DNA, RNA and nucleotides
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 129
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
DNA, RNA and nucleotides POSTERS
stability of transcription complexes and thus facilitate their dis- presented in the environmental samples. Diagnostics of Clavibac-
lodging by DNA repair and replication factors. We obtained a ter michiganensis ssp. sepedonicus, the causal agent of quarantine
series of mutations of this class in Escherichia coli RNAP and disease called as ring rot of potato is particularly difficult. In the
analyzed their transcription properties in vitro. We found that latent infection Cms bacteria occurs at low concentration in the
most of the mutations destabilize promoter complexes of RNAP. potato tissue and often without causing symptoms on plants. In
At the same time, they do not greatly affect the rate of transcrip- addition the gram-positive bacteria it is characterized by the pres-
tion elongation, although certain amino acid substitutions signifi- ence of a cell wall with exopolysaccharide coat, which more or
cantly stimulate site-specific transcriptional pauses. Interestingly, less surrounds the surface of the tested bacterial cells and can
none of the mutations changed the ability of RNAP to transcribe make the identification difficult. One of the most important fac-
DNA templates containing various types of damaged nucleotides, tors enabling effective molecular diagnostics of Cms bacteria is
including AP-sites, oxidized and alkylated bases, and thymine- the selection of an effective method of DNA isolation that allows
thymine dimers. Furthermore, the mutant RNAPs did not reveal to obtain a high sensitivity of the detection method. Therefore,
increased sensitivity to the Mfd protein that couples transcription the aim of the presented research was to develop and evaluate
to DNA repair. Thus, we propose that the stringent phenotypes the effectiveness of the DNA isolation method used in the diag-
of the mutations are likely explained by their effects on transcrip- nosis of Cms strains with different degree of mucous and evalua-
tion complex stability. Since most of the mutated residues flank tion of the influence of bacterial mucous on the sensitivity of the
the DNA binding channel of RNAP, these effects may likely PCR assay. The studies were carried out using different mucoid
result from changes in RNAP-DNA contacts at various steps of Cms strains. In the first step, the bacterial suspensions were stan-
RNA synthesis. This work was supported in part by the Russian dardized spectrophotometrically and after obtaining the bacterial
Science Foundation (grant 17-14-01393). colonies growing on the microbiological medium, the working
suspensions 108 cfu/mL (colony forming units per ml) for each
strain were prepared. Than the suspensions series with a concen-
P.01-079-Mon tration from 106 to 100 cfu/mL were prepared and used to isola-
Regulation of the activity of ribosomal RNA tion of bacterial DNA. Because of the high sensitivity of the
promoters by region 3.2 of the RNA classical molecular diagnosis methods on the environmental con-
polymerase sigma subunit and DksA taminants, the efficiency of the developed isolation method was
evaluated using the classical PCR assay.
D. Pupov, I. Petushkov, D. Esyunina, A. Kulbachinskiy
Institute of Molecular Genetics, Russian Academy of Sciences,
Moscow, Russia P.01-081-Wed
Ribosomal RNA promoters are the most active bacterial promot- Heat shock proteins as a part of response to
ers during exponential phase of growth but their activity is stress in Chironomidae midges
silenced during stationary phase. The rRNA promoters form O. Kozlova1, Z. Abramova1, O. Gusev1,2, T. Kikawada3,4
intrinsically unstable complexes with RNA polymerase, which is 1
Kazan Federal University, Kazan, Russia, 2RIKEN, Yokohama,
required for efficient transcription initiation from these promot- Japan, 3National Institute of Agriculture and Food Research
ers. In Escherichia coli, regulation of the rRNA synthesis was Organization, Tsukuba, Japan, 4The University of Tokyo, Chiba,
shown to depend on the action of transcription factor DksA, Japan
which binds RNA polymerase in the secondary channel and
together with ppGpp dramatically decreases the stability of Chironomids (non-biting midges) are among the most stress-resis-
rRNA promoter complexes. We demonstrate that the low stabil- tant insects in the world that makes them an interesting model
ity of rRNA promoter complexes depends on region 3.2 of the for studying various aspects of response to different kinds of abi-
RNA polymerase sigma subunit that directly interacts with the otic stress. Acting like molecular chaperons, heat shock proteins
template DNA strand in the open promoter complex. Mutations play an important role in protection of proteins and the whole
in region 3.2 significantly increase promoter complex stability, cell from adverse abiotic factors, and besides, they are quite con-
thus making transcription initiation less sensitive to the action of servative in structure and sequence, especially among closely
DksA and ppGpp both in vitro and in vivo. Not only the stability related organisms. In this study we compare the number and
of promoter complexes is increased but also the apparent affinity activity of HSP-coding genes in larvae of several Chironomidae
of DksA is decreased, suggesting the existence of an allosteric species from different habitats, being exposed to desiccation, heat
pathway connecting sigma region 3.2 with the binding site of shock and other kinds of severe stress. Methodology of our study
DksA. Together, our data reveal an unexpected negative role of is based on high-throughput DNA and RNA sequencing, de-novo
the sigma subunit in promoter complex formation. The unstable genome assembly and further accurate, transcriptome-based pre-
complexes thus become a target for highly sensitive regulation diction of protein-coding regions, therefore the obtained results
during bacterial stress response. This work was supported in part present reliable platform for understanding molecular evolution
by the Russian Science Foundation (16-14-10377) and Russian of stress-resistance in insects. Detailed analysis of expansion and
Foundation for Basic Research (17-54-150009). contraction of HSP-coding genes shows that high ability to with-
stand stress is not always necessary linked to changes in number
of such genes in a genome, while sometimes accommodation to
P.01-080-Tue abiotic stress goes on the way of regulation. Together with
Evaluation of DNA isolation to the sensitivity revealing common functional patterns of response to abiotic
stress in different species (i.e. upregulation of HSP20, HSP70 and
of molecular diagnostics of Cms bacteria
HSP90-coding genes), we also detect some species-specific stress-
K. Salamo nska, W. Przewodowski, D. Szarek, A. Przewodowska,
induced genes, which, in many cases, do not have any ortholo-
W. Stochła
gous genes in other species. This work was supported by Russian
Plant Breeding and Acclimatization Institute - National Research
Science Foundation grant No. 14-44-00022P.
Institute, Bonin, Poland
Molecular diagnostics of bacteria isolated from an environment
is often difficult because of different factors and contaminants
130 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Chromosome structure and chromatin
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 131
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Chromosome structure and chromatin POSTERS
P.02-004-Mon P.02-006-Wed
Chromosome number in Ixodes tick cell lines Analysis of protein complexes binding at
K. Kotsarenko, P. Vechtova, J. Lieskovska, J. Sterba termini of 50–250-kb forum domains in human
Faculty of Science, University of South Bohemia, Ceske
Budejovice, Czech Republic
cells
O. Kretova, I. Slovohotov, N. Tchurikov
The tendency to gain or lose chromosomes is typical for the indi- Engelhardt Institute of Molecular Biology Russian Academy of
vidual cells within a tick cell lines, similarly to cell lines of Sciences, Moscow, Russia
another origin. It was shown that I. scapularis cell lines IDE1, 2,
We developed a new approach for the genome-wide profiling of
8, and 12, as well as ISE5 and 18, had a diploid number of chro-
double-strand breaks (DSBs) with 1-bp precision. Using this
mosomes, while coincidently some cell subpopulation had the
approach we detected large chromosomal domains (50–250 kb),
tendency to be aneuploid or tetraploid. The karyotype of I. rici-
denoted forum domains, that are delimited by hot spots of DSBs,
nus cell lines for a long time remained unstudied. The aim of our
and contain genes that are expressed much more coordinately
work was to analyze and compare the karyotypes of I. scapularis
than in random domains of the same size (-z->4, P-
and I. ricinus tick cell lines. Methods. Mitotic chromosomes of
value < 0.0001). The data suggested a strong link between chro-
cell lines ISE6, 18, IRE11 and IRE/CTVM19, 20 were prepared
mosomal breakage and transcription. Previously we detected that
using colchicine treatment and methanol-acetic acid fixation.
PARP1 and HNRNPA2B1 specifically bind at hot spots of DSBs
Flow cytometry analysis of tick cells was performed after ethanol
in human cells at termini of forum domains. It was supposed that
fixation and propidium iodide staining. In the ISE6 line, cells
PARP1 binding might be connected with the suggested mecha-
were predominantly diploid (87.4%) with the modal chromosome
nism of coordinated expression of genes located in domains
number 22, 12.6% of cell population were aneuploid. In the
delimited by hot spots of DSBs. It is known that PARP1 behaves
ISE18 line, most of the cells were tetraploid (75.0%), while
as a strong regulator of chromatin structure and transcription
18.3% and 6.7% of cells were octoploid and aneuploid, respec-
and is even capable of somatic cell reprogramming. To elucidate
tively. IRE11 cells with the modal chromosome number 22 were
whether PARP1 recognizes forum domains termini and recruits
diploid (65.5%), with 24.6% being tetraploid, 7.8% being
different proteins that are also involved in the coordinated
132 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Chromosome structure and chromatin
regulation of genes in a forum domain we used the isolation of promoter interaction. Expression of the Abd-B gene in the
such complexes using genome-wide preparation representing the appropriate parasegment-specific pattern is controlled by cis-reg-
forum domains termini that were immobilize on paramagnetic ulatory domains: iab-5 to iab-9. Each of these regulatory
particles. Our data indicated that the complexes possessing domains is bracketed by boundary elements. In particular
PARP1, HSP90, HSP70, TBP5, EF1A1 and some other proteins the Fab-7 boundary is located between the iab-6 and iab-7 do-
bind specifically with forum domains termini. The data are mains and display two important functions: to block crosstalk
important for understanding the mechanisms of coordinated between the regulatory domains and to facilitate specific distance
expression of genes in human chromosomes. The study was sup- interactions between the iab-6 enhancer and the promoter in
ported by the grant from Russian Foundation for Basic Research order to proper activate Abd-B in PS11. The 1.6 kb Fab-
(No. 18-04-00680). Computer analysis of the proteomics data 7 boundary includes four chromatin-specific nuclease-hypersensi-
was performed under support by the Program of fundamental tive sites: HS*, HS1, HS2 and HS3. HS3 is Polycomb Responsi-
research for states academics for 2013–2010 years (No. 0103- ble Element (PRE) and responsible for repression of premature
2014-0005). activation of the iab-7 enhancer. Using attP-mediated replace-
ment strategy we found that only distal 240 bp part of the HS1
region (dHS1) in combination with PRE can form functional ele-
P.02-007-Mon ment. The dHS1 or HS3 alone had no activity, while dHS1 in
PARP1 modulates nucleosome structure in the combination with five sites of the architectural protein Pita
absence of NAD+ restored both boundary functions. The Pita sites alone functioned
N. Maluchenko1, D. Sultanov1, N. Gerasimova1, A. Feofanov2, as an insulator that block crosstalk between the iab-6 and iab-7
V. Studitsky3 domain / Abd-B promoter. Probably that dHS1 functions as
1
Lomonosov Moscow State University, Moscow, Russia, the Abd-B promoter tethering element that facilitates the specific
2
Lomonosov Moscow State University; Shemyakin-Ovchinnikov enhancer-promoter interaction across the insulator. It was shown
Institute of Bioorganic Chemistry, Moscow, Russia, 3Fox Chase previously that dHS1 is bound by Late Boundary Complex
Cancer Center, Philadelphia, United States of America (LBC) that is essential for boundary functions. ChIP in embryos
showed that the Clamp protein, component of LBC, bound to
The mechanisms that guarantee access of transcription factors, dHS1 only in combination with Pita sites or HS3. These results
polymerases and other effectors to genomic DNA, which is are in agreement with a model that Pita or HS3 facilitate recruit-
tightly packed into chromatin remain poorly understood. Human ing of protein complex to dHS1 that can fulfill all boundary
poly-ADP-ribose polymerase 1 (PARP1) is a protein sensor of functions. The work was supported by the Russian Science Foun-
DNA breaks, transcriptional regulator and an enzyme responsi- dation grant N 14-24-00166.
ble for poly-ADP-ribosylation of numerous proteins, including
histones and chromatin-associated proteins. To study the interac-
tion of PARP1 with chromatin, we used three mononucleosomal P.02-009-Wed
templates, each containing two fluorescent labels placed into dif- Antibody analysis supports the G4/IM-synaptic
ferent parts of nucleosomal DNA: either near the entrance/exit
complex structures
of DNA in/from nucleosome or near the nucleosomal dyad. Each
V. Severov, N. Barinov, V. Tsvetkov, A. Varizhuk, D. Klinov,
nucleosomal template contained a short DNA fragment extend-
G. Pozmogova
ing from the nucleosomes and supporting PARP1 binding.
SRI of Physical-chemical Medicine, Moscow, Russia
spFRET microscopy analysis of these nucleosomes in solution
revealed two subpopulations: compact high-FRET nucleosomes G-quadruplexes (G4s) are noncanonical nucleic acid structures
(major subpopulation) and low-FRET nucleosomes containing consisting of planar guanine tetrad arrangements. They play
partially uncoiled DNA (minor subpopulation). Binding of important roles in regulation of key cellular processes, such as
PARP1 to nucleosomes in the absence of NAD+ results in for- DNA recombination, replication, transcription and others. Wher-
mation of a uniform population of complexes that have an inter- ever there is a G-quadruplex forming sequence in one strand the
mediate FRET efficiency, indicating global structural complimentary strand contains a C-rich sequence capable of
reorganization of the entire nucleosomal DNA. Addition of forming an I-motif (IM). Although structures of G4s and IMs
NAD+ results in dissociation of PARP1 from the nucleosomes themselves are well-characterized, little is known about their
and the recovery of the nucleosomal structure. The data suggest behavior in dsDNA. In our previous work we reported AFM
that PARP1 can increase accessibility of nucleosomal DNA for studies of DNA duplexes containing (G3T)nG3 sequences
external probes by reversibly reorganizing nucleosomes. This (n = 15) in the middle regions. In the case of n ≥ 3, the middle
newly discovered nucleosome structure-modulating activity of regions in theory could fold into G4/IM; while in the case of
PARP1 is independent of its catalytic function and likely plays n = 1, 2, no intrastrand noncanonical structures are possible.
an important role in PARP1-dependent DNA repair. This study Surprisingly enough, AFM scanning of the duplexes revealed
was supported by RFBR grant 17-54-33045. intermolecular G4/IM-synaptic complexes in all cases (the images
contained cruciforms and higher order structures). No signs of
such complexes were visible in AFM images of the control
P.02-008-Tue duplex that lacks the (G3T)nG3 middle region. These data con-
Mapping of functional elements in Fab-7 firm our hypotheses about several ways of self-association of
boundary involved in regulation of Drosophila non-canonical structures (Varizhuk et al. Biochemistry-Moscow
2017; Protopopova et al. PCCP 2018). Here we used anti-G4-
hox gene Abd-B
DNA antibody (clone 1H6) to prove existence of G4 structure in
M. Sabirov, N. Postika, O. Maksimenko, P. Georgiev,
our synaptic complex. AFM images show interaction of the anti-
O. Kyrchanova
body with the junctions of cruciforms and higher order struc-
Institute of Gene Biology, RAS, Moscow, Russia
tures. These results confirm possible structures of the G4/IM-
The regulatory region of the Drosophila Hox gene Abd-B is one synaptic complexes elucidated by molecular modeling. Not every
of the best models to study mechanisms and proteins involved in DNA-cruciform was recognized by antibody molecules due to
organization and regulation of the specific distance enhancer- possible covering of G4 in the synaptic complex structure.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 133
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Chromosome structure and chromatin POSTERS
Collectively, our findings demonstrate that G-rich DNA sites can on promoters of target genes. Target regions was characterized
form G4/IM-synaptic complexes - potentially important factors by depletion of active marks and enrichment with repression
of chromatin remodeling, recombination, replication, and tran- marks. On the other hand depletion of Kaiso also led to alter-
scription processes. This work was supported by Russian Science ation in epigenetic environment of target gene promoters. We
Foundation [14-25-00013]. hypothesize that in cellular processes, such as an inflammation,
where hyperosmotic conditions are observed, deSUMOylation of
Kaiso may lead to stable inactivation of target genes by generat-
P.02-010-Mon ing repressive chromatin structures, while Kaiso knockout will
The structural and functional analysis of the have different consequences. Thus, we propose a novel biological
orc6 protein – Drosophila model of the Meyer- role for Kaiso in the regulation of homeostasis. The work was
Gorlin syndrome supported by RNF 14-14-01202
M. Balasov1, K. Akhmetova1, G. Zhu2, I. Chesnokov1
1
University of Alabama at Birmingham, Birmingham, United P.02-012-Wed
States of America, 2HKUST, Hong Kong, Hong Kong
Multiple roles for insulator complex LBC in
The origin recognition complex (ORC) is important for DNA regulating chromosome architecture in
replication in eukaryotic cells. ORC is also involved in other cell
Drosophila
functions. The smallest ORC subunit, Orc6, has an active role in
A. S. Kurbidaeva1,2,3,4, Y. V. Shidlovskii3,5, P. Schedl3,4
both DNA replication and cytokinesis. The structural and func- 1
Department of Molecular Biology, Princeton University,
tional analyses revealed that Orc6 in eukaryotes has a homology
Princeton, United States of America, 2Russian Academy of
with transcription factor TFIIB. Orc6 has a DNA binding activ-
Sciences, Moscow, Russia, 3Institute of Gene Biology, Russian
ity on its own and is important for DNA binding of whole six-
Academy of Sciences, Moscow, 119334 Russia, 4Department of
subunit ORC. We propose that Orc6 may position the whole
Molecular Biology, Princeton University, Princeton, NJ 08540,
complex at the origins of DNA replication similar to the role of
USA, 5I.M. Sechenov First Moscow State Medical University,
TFIIB in positioning transcription pre-initiation complex at the
Moscow, 119048 Russia
promoter. The cytokinesis function of Drosophila Orc6 is
achieved through its interaction with the septin protein Pnut, Chromatin structure influences gene expression on a local level
which together with Sep1 and Sep2 forms a septin complex, a by regulating expression of individual genes, and on a global
highly conserved polymerizing protein assembly that is critical level by changing the activity of large gene domains or entire
for cytokinesis in many species. Septins are weak GTPases that chromosomes. Examples of global regulation include repression
can assemble into large filaments. The binding of Orc6 to Pnut of the fly homeotic gene complexes by PcG silencing and the
stimulates septin complex filament formation. Mutational analy- upregulation X-chromosome gene expression by MSL complexes.
sis revealed that both N-terminal and C-terminal domains of Like the formation of local chromatin domains, these global
Orc6 are important for this activity of the protein. The C-termi- mechanisms depend upon chromosome architectural elements.
nus of Orc6 contains a highly conserved motif important for the MSL complexes exploit the pre-existing X-chromosome topologi-
interaction of the protein with the core ORC. A mutation found cal organization. This organization depends special elements
in patients with Meier-Gorlin Syndrome (MGS) maps to the called Chromatin Entry Sites (CES) that are distributed along
same segment in Orc6, destabilizing the interaction of Drosophila the X-chromosome. CES are boundaries of topological domains.
and human Orc6 with the core Orc1-5 sub-complex. We have Likewise, a large network of contacts between Polycomb
shown that mutations in this region result in reduced DNA repli- response elements (PREs) in different PcG target loci has been
cation. In order to study the effects of MGS mutation in animal discovered in genome-wide studies. Thus, the MSL and PcG
model system we introduced MGS mutation in Orc6 and estab- chromatin modifying complexes would appear to share a similar
lished Drosophila model of the disease. Obtained flies are unable dependence upon chromosome architecture and architectural ele-
to fly and display planar cell polarity defects. The availability of ments. We find that a large complex, LBC, is responsible for the
the Drosophila model of MGS will allow thorough studies reveal- architectural functions of CES and PREs. The LBC has previ-
ing crucial and conserved mechanisms of organism development ously been shown to be required for the architectural functions
and disease pathogenesis. of insulators. The LBC binds to CES and to PREs. Mutations
which disrupt LBC binding in vitro abrogate PcG/CES func-
tions in vivo. We propose that the architectural functions of LBC
P.02-011-Tue provide a scaffold for the efficient recruitment and subsequent
Role of Kaiso in homeostasis regulation spreading of the MSL and PcG complexes, facilitating the epige-
S. Zhenilo, D. Kaplun, E. Prokhortchouk netic regulation of transcription. This work was supported by
Institute of Bioengineering, Research Center of Biotechnology of Russian Scientific Foundation grant 16-14-10346 and Russian
the Russian Academy of Sciences, Moscow, Russia Foundation for Basic Research grant 16-34-00509 mol_a.
Kaiso is a member of the BTB/POZ zinc finger family, which is
involved in cancer progression, cell cycle regulation, apoptosis
and WNT signaling. It regulates methyl-dependent repression of
gene transcription by recruiting corepressors N-CoR and SMRT.
We showed early that transcriptional properties of Kaiso may
depend on its posttranslational modification- SUMOylation, that
is changed with the hyperosmotic stress. SUMOylation reverts
Kaiso from repressors to activators. Here, we demonstrated how
Kaiso modification influences on setting and maintenance of epi-
genetic marks. By genomic DNA editing, we generated cell line
with Kaiso that cannot be modified by SUMO. The presence of
non-modified Kaiso in cells results in changes of epigenetic marks
134 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Chromosome structure and chromatin
P.02-013-Mon P.02-015-Wed
Changes in dynamics and kinetics of Biochemical and structural analysis of Msh4/
chromatin binding of proteins from the Msh5 protein complex involved in meiotic
replication machinery during licensing for DNA crossover formation
replication M. Firlej, J. Weir
S. Uzunova Friedrich Miescher Laboratory of the Max Planck Society,
Institute of Molecular Biology, Sofia, Bulgaria T€
ubingen, Germany
DNA replication is a process that is strictly controlled. One of its Meiosis is a form of cell division that occurs in sexually repro-
most important stages is the licensing of the genetic material for ducing organisms and results in the production of haploid game-
initiation of DNA synthesis, when every origin of replication is tes from diploid parental cells. A key process in meiosis is
“tagged”, so that a single activation event can take place per reciprocal homologous recombination, also known as crossing-
round of the cell division cycle. Some of the key proteins of the over. This process not only promotes genome diversification but
replication apparatus in the eukaryotic cells are Claspin, Tim and is also essential to ensure faithful segregation of chromosomes.
Tipin, which form a triple complex, that controlled the activity While it has been established that crossovers are required for
of the MCM helicase, and And1, that stabilizes the DNA poly- error-free gametogenesis, little is known about their formation
merase a / primase complex to the Replisome. The evaluation of and regulation. Crossovers are derived from programmed dou-
the kinetic changes of chromatin binding of these harmonization ble-strand DNA breaks, but most breaks do not result in cross-
factors during early and late G1-phase, where the chromatin is overs. It has been previously shown that the meiosis-specific
licensed for replication and the formation of the Initiation com- ZMM proteins (Zip, Msh, Mer) promote crossover formation in
plex takes place, can elucidate the intimate mechanisms of con- budding yeast, with homologs of these proteins found in many
trol, that aim to execute the dynamic program of DNA synthesis. other eukaryotes. In the following poster, I focus on the investi-
In our study, by means of modern microscopic techniques, we gation of the role of two ZMM proteins Msh4 and Msh5. These
study the kinetic changes of chromatin binding of key, GFP- two proteins form a DNA binding complex, which recognizes
tagged, replication proteins - MCM6, PolA2, Claspin and And1 double Holliday junctions in an ATP dependent manner and fur-
during the early and late G1-phase of the cell cycle. ther slides onto the homologous arms of a four-way junction.
The Msh4/Msh5 heterodimer potentially stabilizes strand inva-
sion during meiotic homologous recombination. In order to
P.02-014-Tue investigate the function of the complex in meiotic recombination
Drosophila repression protein Insv is involved process I produced and purified the Saccharomyces cerevisiae
in activity of the Fab7 boundary from the Msh4/Msh5 complex from insect cells, and then I explored their
potential interactions with different proteins and different DNA
bithorax complex substrates. Interactions were analyzed in vitro, with a view to the
A. Fedotova1, A. Bonchuk2, P. Schedl3, P. Georgiev4 high-resolution structural analysis of large complexes. Under-
1
Institute of gene biology of the Russian academy of sciences, standing the mechanisms underlying the regulation of the cross-
Moscow, Russia, 2the Institute of Gene Biology of the Russian overs formation will further our understanding of the critical
Academy of Sciences, Moscow, Russia, 3Princeton University, process of meiosis and its regulation.
Princeton, United States of America, 4the Institute of Gene
Biology of the Russian Academy of Sciences, Moscow, Russia
Insensitive protein (Insv) was first describe as a nuclear corepres- P.02-016-Mon
sor for the Notch transcription factor Suppressor of Hairless (Su Control of DNA double-strand break formation
(H)). Insv lacks domains of known biochemical function but con- in meiosis
tains a single BEN domain and negatively regulate some E(spl) D. Rousova, J. Weir
complex genes in nervous system of Drosophila. The genome- Friedrich Miescher Laboratory of the Max Planck Society,
wide mapping of Insv binding sites in embryo have shown that T€
ubingen, Germany
most Insv sites overlap with known fly architectural proteins,
including CP190 and CTCF. In particular, Insv binding sites Meiosis is a key step in sexual reproduction, which leads to the for-
were found in well-studied boundaries Fab-7 and Fab-8 in the mation of haploid gametes from a diploid cell. During genome
reduction, the homologous chromosomes are segregated into
bithorax complex. Using in vitro approaches and the yeast two-
daughter cells, therefore they have to be physically linked via
hybrid assay, we found that Insv directly interacts with CP190.
By the series of deletion, we mapped a region in the N-terminus homologous recombination. The linkage is enabled by repair of
of Insv [135–150] that interacts with the BTB domain of CP190. programmed double-stranded DNA breaks (DSBs) using the
homologous chromosome instead of the sister chromatid. Forma-
To determine whether Insv is boundary factor, we used muta-
tions in the Fab-7 boundary that weakly inactivate its function. tion of DSB has to be strictly controlled - too little breaks would
Combination of the null insv (insv23B) mutation with the Fab-7 not be sufficient to hold homologs together, too many breaks
mutation dramatically disrupted boundary function. Expression would lead to chromosome destabilization. It is known that DSB
formation in S. cerevisiae involves regulation by phosphorylation
of the wild type Insv protein rescued boundary activity in insv23B;
Fab-7 mutant flies. The Insv mutant lacking CP190-interacting and the assembly and disassembly of the Hop1-Red1-Mek1- and
domain also completely restored the boundary activity. While Rec114-Mer2-Mei4-complexes together with the nucleosome bind-
our results suggest that the CP190-interacting domain is not ing protein Spp1. However, mechanistic detail of these processes
are lacking. We use an in vitro reconstitution approach combining
essential for Insv function in Fab-7, we found that it is important
for recruiting Insv to many sites in polytene chromosomes. Taken biochemistry and structural biology to provide insight into the
together our results suggest that Insv is a new architectural/insu- mechanisms underlying DSB control; both in time and in space.
Unravelling DSB control would represent a significant step for-
lator protein. The work is supported by Russian Science Founda-
tion (grant 14-24-00166). ward in our understanding of break positioning, genome stability
and, ultimately, the critical process of meiosis as a whole.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 135
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Chromosome structure and chromatin POSTERS
136 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Genome dynamics and epigenetics
P.02-020-Tue the hESC lines. Transition to the na€ıve state was accompanied
by a loss of XIST expression, loss of Xi H3K27me3 enrichment
A whole-genome map of chromatin-bound
and a switch in Xi replication synchronously with active X,
RNAs except for two regions. This pattern of Xi reactivation was
A. Gavrilov1, A. Zharikova2, A. Galitsyna1,2,3, S. Razin1, observed in all cells in two hESC lines. However, these cell lines
A. Mironov2,3 were unable to undergo classical XCI upon spontaneous differen-
1
Institute of Gene Biology, Russian Academy of Sciences, Moscow, tiation. We conclude that na€ıve culture conditions do not resolve
Russia, 2Department of Bioengineering and Bioinformatics, the variability in XCI status in female human ESC lines and
Lomonosov Moscow State University, Moscow, Russia, 3Institute establish an irreversible heterogeneous pattern of X reactivation.
for Information Transmission Problems, Russian Academy of
Sciences, Moscow, Russia
The recent studies show that the major part of the eukaryotic P.03-002-Tue
genome is transcribed to produce a broad range of RNAs, The role of SCD1 in thyroid hormone
including both protein-coding and noncoding RNAs (ncRNAs). dependent control of epigenetic modification
ncRNAs participate in various biological processes from regulat- in cardiomyocytes
ing enzymatic activities to sustaining genome imprinting and A. Olichwier, A. Filip, M. Wolosiewicz, P. Dobrzyn
nuclear body biogenesis, but the mechanism of action is still Laboratory of Molecular Medical Biochemistry, Nencki Institute
unclear for most ncRNAs. Several studies reveal the role of of Experimental Biology, Polish Academy of Sciences, Warsaw,
ncRNAs in establishing the spatial organization of chromosomes. Poland
The further progress in studying ncRNAs and their functions in
chromatin will depend on the availability of the genome wide The main factors of epigenetic regulation are enzymes involved
spectrum of RNA associations with chromosomes, the RNA- in alteration of chromatin structure, i.e. DNA methyltransferases
DNA interactome. An objective of our work is to develop a new (DNMTs), histone methyltransferases (e.g. lysine-specific
wholegenome method to simultaneously identify the sites of asso- demethylase 1 [LSD1]), and histone deacetylases (HDACs).
ciations with chromosomes for all RNA molecules present in the Moreover, epigenetic modifications which might be induced by
cell nucleus. The gist of the method, designated Red-C (RNA thyroid hormones (TH) action are involved in pathogenesis of
ends on DNA capture), is adaptermediated RNADNA ligation several cardiovascular diseases. TH through nuclear TH receptors
in fixed nuclei followed by the analysis of the chimeric RNA- can modulate the expression of various genes involved in epige-
DNA molecules using high-throughput sequencing. In pilot netic control (e.g. DNMT1). Moreover, TH through regulation
experiments we applied Red-C protocol to uncover RNA-DNA of protein kinase A (PKA) can rapidly change chromatin methy-
interactome of the cultured human erythroleukemia cells (line lation level and structure. Furthermore, one of the main enzyme
K562). We studied interaction of various RNA biotypes that regulates lipid metabolism, stearoyl-CoA desaturase 1
(lincRNA, antisense RNA, snoRNA, snRNA, piRNA, eRNAs, (SCD1), which gene expression is under TH control, affects the
protein-coding RNA, miRNA, etc.) with different DNA elements level of DNA methylation in 3T3 adipocytes. Therefore, the aim
such as promoters, enhancers, insulators, gene bodies, transcrip- of the presented study was to investigate the role of SCD1 and
tion factor binding sites, as well as with different types of active TH in regulation of epigenetic modifications in the heart. To
and repressed chromatin that can be distinguished based on dis- induce hyperthyroidism SCD1+/+ and SCD1-/- mice were
tribution of epigenetic marks. We also compared the obtained injected with triiodothyronine (T3). Performed analyses showed,
RNA-DNA contact maps with the profiles of genome partition- that SCD1 deficiency increased LSD1 and HDAC4 protein con-
ing into topologically associating domains and A/B spatial com- tent as well as PKA phosphorylation level in cardiomyocytes.
partments. The results of our study demonstrate a possible role Moreover, we observed decreased global DNA methylation level
of ncRNAs in establishing/mediating the 3D genome organiza- and DNMT1 protein level in SCD1-/- heart. Interestingly, TH
tion and in regulating gene expression. This work was supported caused increase in global DNA methylation level and LSD1 pro-
by the Russian Science Foundation (grant 18-14-00011). tein level in SCD1-/- heart when compared to WT controls. Fur-
thermore, in hyperthyroidic SCD1-/- cardiomyocytes we observed
drop in phosphorylation of PKA, what was correlated with ele-
Genome dynamics and epigenetics vated HDAC4 protein level. Therefore, obtained results empha-
size the important role of SCD1 expression in epigenetic
P.03-001-Mon modifications in the heart and suggest that SCD1 is an important
Epigenetic reprogramming by na€ıve conditions element of response to TH action in the heart. National Science
Center (Poland) grant UMO-2014/13/B/NZ4/00199.
establishes an irreversible state of X
chromosome reactivation in human ESCs
A. Panova, A. Bogomazova, M. Lagarkova, S. Kiselev
Vavilov Institute of General Genetics, Moscow, Russia
Female human pluripotent stem cells (PSCs) have variable X-
chromosome inactivation (XCI) status. One of the X chromo-
somes may either be inactive (Xi) or display some active state
markers. Long-term cultivation of PSCs may lead to an erosion
of XCI and partial X reactivation. Such heterogeneity and insta-
bility of XCI status might hamper the application of human
female PSCs for therapy or disease modeling. We attempted to
address XCI heterogeneity by reprogramming human embryonic
stem cells (hESCs) to the na€ıve state. We propagated five hESC
lines under na€ıve culture conditions. PSCs acquired na€ıve cells
characteristics although these changes were not uniform for all of
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 137
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Genome dynamics and epigenetics POSTERS
P.03-003-Wed P.03-005-Tue
Clarifying the role of DNA methylation in tree Role of stearoyl-CoA desaturase 1 in
phenotypic plasticity epigenetic control of pancreatic islet cells
M. D. SOW1, A. Le Gac1, C. Lafon-Placette1, A. Delaunay1, identity
I. Le Jan1, R. Fichot1, S. MAURY1, M. Mirouze2, S. Lanciano2, A. M. Dobosz1, J. Janikiewicz1, A. M. Borkowska2,
J. Tost3, V. Segura4, C. Chaparro5, C. Grunau5, I. Allona6, A. Dziewulska1, W. M. Kwiatek2, A. Dobrzyn1
G. Le Provost7, C. Plomion7, J. Salse8, C. Ambroise9, 1
Laboratory of Cell Signaling and Metabolic Disorders, Nencki
S. Gribkova10, S. H. Strauss11 Institute of Experimental Biology PAS, 3 Pasteur Street, 02-093
1
LBLGC, ORLEANS, France, 2IRD, UMR 232 DIADE, Warsaw, Warsaw, Poland, 2Institute of Nuclear Physics Polish
Laboratory of Plant Genome and Development, Perpignan, France, Academy of Sciences, PL-31342 Krakow, Krakow, Poland
3
Laboratory for Epigenetics and Environment, Centre National de
G enotypage, CEA-Institut de G enomique, EVRY, France, 4AGPF, Type 2 diabetes (T2D) is multifactorial disorder characterized by
INRA, UMR 588, ORLEANS, France, 5IHPE, Universit e de chronic hyperglycemia due to peripheral tissue insulin resistance
Perpignan, UMR 5244, Perpignan, France, 6Centro de and progressive loss of pancreatic islets function. Recent studies
Biotecnologıa y Gen
omica de Plantas, UPM– INIA, Campus de have shown that the loss of endocrine cells identity may be one
Montegancedo UPM, Pozuelo de Alarc on, Madrid, Spain, 7INRA, of the main reasons for development of pancreatic islets dysfunc-
UMR1202 BIOGECO, Bordeaux, France, 8INRA/UBP UMR tion in T2D. The maintenance of identity of insulin- and gluca-
1095 GDEC, Research group PPAV, Clermond-Ferrand, France, gon-secreting cells in pancreatic islets depends on dynamic
9
Laboratoire Heudiasyc, Unit e Mixte de Recherche/Centre control of transcription factors (TFs) expression. The gene
National de la Recherche Scientifique 6599, Compi egne, France, expression patterns in pancreatic islets have been influenced by
10
Laboratoire de Statistique Theorique et Appliqu
ee, Universit
e fatty acids (FAs) through epigenetic mechanisms. In the present
Pierre et Marie Curie, Paris, France, 11Department of Forest study we tested the hypothesis that stearoyl-CoA desaturase 1
Ecosystems and Society, Oregon State University, Oregon State, (SCD1), a significant control point in FAs metabolism, affects
97331, United States of America expression patterns of TFs involved in maintenance of pancreatic
islet cells identity via epigenetic modifications. The experiments
In a context of global climate change, trees as sessile and long were carried out in vivo on SCD1 knock-out (SCD1 KO) mice
lifespan organisms need to develop mechanisms enabling them to and in vitro on INS-1E b-pancreatic cell line, where lipotoxicity
adapt and to survive. These last years, epigenetic mechanisms was induced by palmitic acid treatment. Our data show that pan-
such as DNA methylation have been proposed as a valuable creatic islets of SCD1 KO mice are characterized by different
resource since they can be triggered by the environmental condi- microarchitecture and decreased protein level of TFs which are
tions in a reversible-way. However, evidences for their role in tree crucial for maintenance of b-cells identity (Pdx1, FoxO1, Isl1)
phenotypic plasticity are still lacking (Br€autigam et al., 2013; Plo- and increased of those characteristic exclusively for a-cells (gluca-
mion et al., 2016). In this context, we develop different comple- gon, Arx) comparing with wild type mice. Furthermore, we
mentary approaches: noticed that inhibition of SCD1 activity as well as silencing of
1 A correlative approach with simultaneous analysis of methy- SCD1 gene expression, lead to global DNA hypomethylation and
lome and transcriptome dynamics in the shoot apical meristem decrease in methyltransferase 1 (Dnmt1) protein level in INS-1E
(center of shoot morphogenesis) of poplar in various environ- cells. In addition, we also observed significant changes in methy-
ments (Gourcilleau et al., 2010; Lafon-Placette et al., 2013; lation pattern within promoter regions of identity TFs (Pdx1).
Bastien et al., 2015; Le Gac, 2017; Lafon-Placette et al., 2017). Obtained results suggest that SCD1 activity/expression and DNA
2 A reverse genetic approach, using RNAi clones of Populus methylation pattern alterations may lie beneath maintenance of
tremula x alba (Zhu et al., 2013; Conde et al., 2017; Le Gac pancreatic islets functional identity. This study was supported by
et al., in prep) hypo or hypermethylated and grown under NCN UMO2013/10/E/NZ3/00670 and National Centre NCBR
environmental constraints. grant STRATEGMED 3/305813/2/NCBR/2017
3 A population approach, using natural populations from
diverse geographic origins to explore microevolutive adapta-
tion to local environment and phenotypic plasticity (Project P.03-006-Wed
‘EPITREE’ ANR 2018-2021, S. Maury). Our previous data Transforming growth factor beta 1 (TGF-b1)
highlight a relationship between DNA methylation in the treatment as a new in vitro model of
shoot apical meristem biomass productivity and a possible
connection with phytohormone signaling in response to abiotic osteoarthritis
stress. New data will be also presented concerning the stability L. Allas1, Q. Rochoux1, M. Berthelot1, S. Leclercq2,
of the epigenetic modifications and their genetic diversity in K. Boumediene3, C. Bauge1
1
populations. Altogether, our data provide new insights into EA7451 BioConnecT - UniCaen, Caen, France, 2Clinique Saint-
how trees modulate their epigenomes to ensure developmental Martin, Service de Chirurgie Orthop
edique, Caen, France,
3
plasticity and adaptation in a changing environment. EA7451 BioConnecT - UniCaen, Caen, France
Osteoarthritis (OA) is a rheumatic disease characterized by carti-
lage degradation, inflammation and osteophyte formations.
Osteophytes are initiated by the process of chondrocytes hyper-
trophy. Recent studies showed that blockage of TGF-b1 signal-
ing in cartilage of OA mouse models prevents osteophyte
formations. Furthermore, it has been demonstrated in vivo, that
inhibition of Enhancer of zeste homolog 2 (EZH2), Histone 3
lysine 27 methylase, attenuates hypertrophy. Our study aimed to
evaluate the hypertrophic effect of TGF-b1 in human articular
chondrocytes and to investigate whether EZH2 inhibition would
attenuate TGF-b1-induced hypertrophy in vitro. Human primary
138 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Genome dynamics and epigenetics
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 139
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Genome dynamics and epigenetics POSTERS
infer possible roles of the residues and their interactions. Interest- P.03-011-Tue
ingly, a cluster of residues that showed the largest differences in
Different performance of PTEN in the lung
motion between the natural substrate pair of Fpg, oxoG:C, and
its least preferred substrate, oxoG:A, was found outside the tissues and in the blood of pneumoconiosis
apparent active site and DNA-binding groove, near the linker M. Ye
that connects the two domains of the protein. Additionally, we CDC, Beijing, China
have identified a conserved cross-groove bridge, a structure Silicosis is a respiratory disease due to long-term silica dust expo-
involving several residues that connect the domains non-cova- sure. Our previous study has demonstrated that silica dust medi-
lently. We have tested the importance of these structural elements ates the activation of PI3K/PTEN/AKT/MAPK/AP-1 pathway in
by making site-directed mutants in E. coli and L. lactis Fpg. The human embryo lung fibroblasts (HELFs). The purpose of this
scanning alanine mutagenesis of the near-linker region surpris- study is to identify genome-wide aberrant DNA methylation profil-
ingly resulted in the enzyme variants that had better specificity ing in lung tissue from silicosis patients. We all know that the key
towards oxoG:C. On the contrary, elimination of the cross- to epigenetic study is the selection of target organs. In the study of
groove bridge inactivated Fpg, making it unable to bind DNA, pathogenesis of many diseases, due to unable to get target organs,
and also increased the cooperativity of protein thermal unfolding, we can only choose relatively easily acquired organs and tissues to
indicative of stabilizing interactions mediated by the bridge. The do our studies. Thus the partial information of epigenetic studies
work was supported by RSF (grant 17-14-01190). are lost. This time, we were very lucky to collect lung tissue samples
from patients with pneumoconiosis and to extract DNA. This pro-
vides a gold standard for clarifying DNA methylation changes in
P.03-010-Mon the lung tissue of patients with pneumoconiosis. We performed
The effect of PTENP1 pseudogene methylation genome-scale DNA methylation profile of lung tissues from silico-
on PTENP1 and PTEN transcription in human sis patients to identify DNA methylation patterns in silicosis
cells through Illumina Human Methylation 450K Bead chip. We found
T. Kovalenko1, L. Ozolinya2, L. Patrushev1 86770 CpG sites and 79660 CpG sites significantly differed in
1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the methylation status in early-stage and advanced-stage compared
Russian Academy of Sciences, Moscow, Russia, 2Pirogov Russian with normal lung methylation data from GEO, respectively. PTEN
National Research Medical University, Moscow, Russia promoter hypermethylation might be associated with the decrease
of PTEN protein. In turn, lung tissue samples are difficult to
Genetic mutations in the tumor suppressor gene PTEN are often obtain in daily physical examination. We will test the results of the
found in human malignant cells, and such genome changes are lung tissue sample studies in the blood of patients with pneumoco-
especially characteristic of endometrial cancer. At the same time niosis. We found that there was an significant hypomethylation of
the three transcripts of PTENP1 pseudogene – sense RNA, and PTEN in the blood of patients with silicosis, and at the same time,
antisense RNAs a and b – are involved in regulation of PTEN there was an significant increasing of the mRNA of PTEN. So it is
gene expression. In our previous research, we found that 5’- a very interesting results that there was an total opposite perfor-
region of PTENP1 was methylated in normal endometrium, as mance of PTEN in the lung tissues and in the blood of silicosis.
well as in endometrial hyperplasias and carcinomas of middle-
aged and elderly women. The aim of this study was to investigate
the impact of PTENP1 methylation on PTENP1 pseudogene and P.03-012-Wed
PTEN gene expression in human cells. Genomic DNA was iso- In vivo visualization of chromatin interactions
lated from 8 cell cultures (HEK 293, MRC-5V2, MCF-7, Hep
in an ecdysone-dependent locus of D.
G2, SCOV-3, SKBR-3, COLO 375 and HeLa) using standard
methods. The methylation status of PTENP1 5’-terminal region melanogaster
was studied using the methylation-sensitive PCR. PTENP1 anti- E. V. Putlyaev1, K. V. Anisovich1,2, Y. V. Shidlovskii1,3,
sense-a and b–transcripts were detected by RT-PCR. Relative P. Schedl1,4
1
levels of PTENP1-sense and PTEN RNAs were determined by IGB RAS, Moscow, Russia, 2Lomonosov Moscow State
RT-qPCR (Litvak method). It was shown that PTENP1 was University, Moscow, Russia, 3I.M. Sechenov First Moscow State
methylated in 5 of 8 cell lines (MRC-5 V2, MCF-7, HepG2, Medical University, Moscow, Russia, 4Department of Molecular
SCOV-3 and HeLa). We found that PTENP1 antisense-a tran- Biology, Princeton University, Princeton, United States of America
scription was completely suppressed in all cell lines with PTENP1 The in vivo dynamics of chromatin interactions in polytene chro-
methylation. Nevertheless, PTENP1 antisense-b transcription mosomes of D. melanogaster under the ecdysone stimulation still
remained unchanged in these cells (except of HepG2). It was remains an unstudied question. We have designed the genetic
shown that PTENP1 methylation correlated with a decreased constructions, that will allow to replace three different regulatory
PTENP1 sense RNA expression. We also detected an elevated elements in the eiP74EF gene by means of specific recombination-
PTEN gene expression in 4 out of 5 cell lines with the PTENP1 mediated cassette exchange (RMCE). To visualize the dynamics of
methylation (MRC-5V2, HepG2, SCOV-3 and HeLa). Thus, we chromatin in this locus in vivo we are applying the technique, pre-
assume that PTENP1 methylation may act as a positive regulator viously developed by Thomas Gregor and Kerstin Bystricky. Two
of PTEN gene expression through a suppression of the PTEN specific bacterial centromere-like sequences (termed ParS) and their
transcription negative regulator (PTENP1 antisense-a RNA). specific fluorescently-labeled protein ligands (ParB) were used for
labeling the locus of interest on the chromosome. There are also
two labels available for in vivo visualization of the transcription
events: the MS2 and PP7 phage-originating stem-loop forming
sequences and their protein ligands (fluorescently labeled capsid
proteins of these phages). According to the concept of our experi-
ment, we are planning to make a series of fly lines by the RMCE.
In all fly lines the two ParS sequences will be inserted in the bound-
ary regions, while the internal regulatory element will be labeled
140 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Genome dynamics and epigenetics
with MS2-stem-loop repeats. Each of the three named regulatory positive inter- and intragenic enhancer was comparable. Surpris-
elements will be deleted in a separate fly line, so that the compar- ingly high number of PARP1 peaks occurred in areas characterized
ison of the average distances between these point on the genome by single histone marks in the following descending order:
could be evaluated by the LSCM. This relatively simple evaluation H3K4me1>H3K4me4>H3K27me3>H3K27ac. Further analysis
will give valuable information on: (1) in vivo dynamics of spatial revealed that some H3K4me1/H3K27ac/PARP1 enhancers were
interactions inside the 74EF locus in its silent and ecdysone- associated with close and actively transcribed genes and PARP1
induced states; (2) possible differences in the mode of chromatin contribution to activation of their transcription was confirmed by
interactions between the polytene and the corresponding normal PARP1 silencing. Concluding, the identification of PARP1-bound
chromosome; (3) the role of the three main regulatory elements of genomic regions, which are featured by gene regulatory histone
the eiP74EF gene in the transcriptional response to ecdysone. This marks may predict PARP1-dependent genes. This study has been
work was supported by Russian Scientific Foundation grant 16-14- supported by Polish National Science Centre grant UMO-2013/11/
10346. D/NZ2/00033.
P.03-013-Mon P.03-015-Wed
UHRF1 depletion induced cellular senescence Experimental evolution of S. cerevisiae -
by decreasing DNMT1 transcription evolutionary stability of functional modules in
S. Min1,2, S. M. Kwon1, G. Yoon1,2 yeast genome
1
Department of Biochemistry & Molecular Biology, Ajou J. Klim, D. Izak, U. Zielenkiewicz, S. Kaczanowski
University School of Medicine, Suwon, South Korea, 2Department Institute of Biochemistry and Biophysics Polish Academy of
of Biomedical Science, The Graduate School, Ajou University, Sciences, Warsaw, Poland
Suwon 16499, South Korea, Suwon, South Korea
Experimental evolution involves long-term maintenance of a large,
Global loss of DNA methylation has been implicated in chrono- reproducing population and is considered as essential tool for
logical aging and cellular senescence. In our previous study, we investigating clonal dynamics, competition, fitness and genetic
revealed that Ubiquitin-like with PHD and Ring Finger Domains basis of complex traits. The purpose of our research was to con-
1 (UHRF1)/DNA Methyltransferase 1 (DNMT1) axis is critical duct experimental evolution of selected S. cerevisiae strains, cou-
at initiation of senescent reprogramming in human diploid pled with whole genome sequencing which allow us to perform
fibroblast (HDF) model. However, the underlying mechanism detailed analysis of changes in so-called “functional modules”.
has not been investigated in detail. In this study, we found that Functional modules are groups of genes (between which genetic
UHRF1 increased DNMT at transcription level. UHRF1 interactions occur) participating in the same biological process
increased ubiquitination of p53 as E3 ligase and subsequently (e.g. transport). It might determine whether these modules have a
inhibited the transcription activity of p53, a transcriptional common evolutionary path and what is the scale of genetic interac-
repressor of DNMT1. Consistently, DNMT1 promoter regions tions in genome. Previous studies suggest that the loss of function
possess p53 binding sequence and p53 knockdown increased of one of the genes from given module often leads to the inactiva-
DNMT1 promoter activity and its. Moreover, UHRF1 knock- tion of another gene from that module, which compensates the pre-
down increased p21 by increasing transcriptional activity of p53 vious loss. Presumably, this is due to the fact that the deletion of a
and induced HDF senescence. Our results suggest that UHRF1 given gene causes module damage—so that the entire mechanism is
depletion could downregulate DNMT1 by enhancing p53 tran- malfunctioning and therefore it is preferable to remove other mod-
scriptional activity, leading to a cellular senescence. Further ular genes. For research we chose cog7D and nup133D genes
detailed study on the ubiquitination of p53 by UHRF1 is cur- mutants. Cog7 is a part of vesicular transport module whereas
rently under investigation. Nup133 is one of nuclear pore complex protein. Both of them have
many putative compensatory interactions and show growth rate
defects compared to the wild-type strain. After about 300 genera-
P.03-014-Tue tions mutant strains (and wild-type strain as a control) were sub-
Bioinformatic ChIP-Seq-based approach to jected to genome re-sequencing for identification of compensatory
identify PARP1-dependent genes in cancer mutations resulting from each primal mutation. We found that
cells majority of mutations identified in this study occurred in genes
T. Płoszaj1, J. Pietrzak2, A. Robaszkiewicz2 involved in cell aggregation and stress response. Moreover com-
1
Department of Molecular Biology, Medical University of Lodz, pensatory evolution did not promoted substantial divergence
Narutowicza 60, 90-136 Lodz, Poland, Lodz, Poland, 2Department across populations. The work is supported by the grant from the
of General Biophysics, University of Lodz, Pomorska 141/143, 90- National Science Centre of Poland, number 2014/13/B/NZ8/0471.
236 Lodz, Poland, Lodz, Poland
Chromatin immunoprecipitation followed by new generation P.03-016-Mon
sequencing (ChIP-Seq) allows tracing histone modifications and Investigation of TIMP3, FBN-EDA and FBN-EDB
association of particular proteins with chromatin. Using ChIP-Seq
gene expression levels in patients with
data and Galaxy (version 18.01.rc1) we searched for regions in the
genome, which were occupied by PARP1 as well as characterized thoracic aortic aneurysm
by histone marks typical for active gene promoters (H3K4me3, Z. M. Isik Saglam, H. Ozdemir1, E. Çoskunpinar1,
H3K27ac), active (H3K4me1, H3K27ac) and inactive enhancers M. Yanartas2, S. Pence1
1
(H3K4me3, H3K27me3). Reads were mapped to the human gen- Institute of Experimental Medicine, Istanbul University, Istanbul,
ome (b37: hg19) with Map with Bowtie for Illumina, peaks were Turkey, 2Kartal Kosuyolu Yuksek Ihtisas Training and Research
called in MACS and overlapping intervals were analyzed by using Hospital, Istanbul, Turkey
BEDTools. Only about 1% of total PARP1 was found in gene pro- Thoracic aortic aneurysms are characterized by extracellular
moters. A similar number of peaks was also observed in active and matrix breakdown associated with progressive smooth muscle cell
inactive enhancers. Furthermore, the abundance of PARP1- rarefaction. Hypertension and presence of congenital bicuspid
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 141
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Genome dynamics and epigenetics POSTERS
aortic valve (BAV) are risk factors for the disease, but a genetic P.03-019-Mon
predisposition also plays a prominent role in etiology. For many
Epigenetic regulation of inflammatory
years, it has been known that patients with Marfan syndrome
(MFS), an autosomal dominant syndrome, are predisposed to macrophage polarization by histone
thoracic aortic aneurysm. MFS results from mutations in Fib- deacetylase-dependent mechanisms in
rillin1 gene, which encodes fibrillin-1, a component of elastin- experimental atherosclerosis
associated microfibrils. The study was completed with 16 cases. A. Manea1, S. Manea1, M. Antonescu1, A. Lazar1, H. Muresian2,
Tissue samples obtained during surgery were freezed immediately M. Simionescu1
1
in liquid nitrogen and stored at -80°C until RNA isolation. Beta Institute of Cellular Biology and Pathology “Nicolae Simionescu”,
actin gene used as control for RNA quality and quantity normal- Bucharest, Romania, 2The University Hospital of Bucharest,
ization. Gene expression analysis was performed by qRT–PCR. Bucharest, Romania
Analysis of the obtained data was done with SPSS 17.0 program.
Monocyte (Mon)-derived macrophages (Mac) are versatile cells
ANOVA, Fischer and Chi square tests were used for characteris-
adapting their phenotype in response to the microenvironment in
tic demographic and other comparative data representations.
the process of atheroma formation. Mac actively orchestrate
Scatter Plot used in the representations of the expression evalua-
important inflammatory and oxidative reactions. Two major Mac
tions. Expression of FBN-1, FN-1 and TIMP-3 gene did not
populations with different major phenotypes have been described:
show any significant difference between TAA patients and
the pro-inflammatory (M1) and anti-inflammatory (M2). Histone
healthy subjects. Figure: TIMP-3, FBN-1, FN-1 Genes Fold
acetylation enzymes control epigenetic mechanisms implicated in
Change Graphs. Uncontrolled increasing of MMP activity plays
the regulation of cell phenotype. The aim of this study was to
an important role pathogenesis of acute and chronic diseases by
find approaches to mitigate atherogenic Mac functions by modu-
degradation of ECM. Difference between MMP and TIMPs
lating histone deacetylases (HDAC)-dependent pathways in
expression cause ascending aorta dissection. According to the
experimental atherosclerosis. Human THP-1 Mac and Mac
results of this study these genes were expressed at a higher level
derived from freshly isolated mouse Mon were polarized into M1
but it was not gained statistical significant results. The present
and M2-Mac. The cells were further exposed (24 h) to vehicle or
work was supported by the Research Fund of Istanbul University
structurally distinct HDAC inhibitors (SAHA, tubacin or CI-
(Project No. 28248.).
994). Human non-atherosclerotic and atherosclerotic samples and
ApoE-/- mice were used. The mice, maintained on normal (ND)
P.03-017-Tue or high-fat cholesterol-rich diet (HD), were randomized to receive
vehicle/suberoylanilide hydroxamic acid (SAHA). HDAC1 and 2
Identification of a new insulator-associated
proteins were induced in M1-Mac as compared to resting Mac
protein in Drosophila (M0) and M2-Mac. Pharmacological inhibition of HDAC
A. Mikhailova1, F. Gorbenko1, A. Srivastava2, R. K. Mishra2, reduced the gene/protein expression of inflammatory molecules
P. Georgiev1, M. Erokhin1, D. Chetverina1 (MCP-1, TNFa, TLR2/4, NOS2, CD80/86) and the activation of
1
IGB RAS, Moscow, Russia, 2CSIR-Centre for Cellular and STAT1 in M1-Mac. Immunohistochemical staining of human
Molecular Biology (CCMB), Hyderabad, India atherosclerotic lesions revealed that HDAC proteins are up-regu-
The Drosophila GAGA factor (GAF) binds to GA-repeats and lated within media and Mac-rich areas (CD68+/CD45+). The
involved in multiple nuclear processes incl. activation and silenc- augmented expression of HDAC proteins and M1-Mac markers
ing of gene expression, nucleosome organization and remodeling, correlated with the severity of atherosclerotic lesions, were found
higher order chromosome architecture and mitosis. These func- in the aortas of ApoE-/- mice. Treatment of ApoE-/- (HD) mice
tions are provided by multiple interaction partners of GAF pro- with SAHA down-regulated the markers of M1-Mac and reduced
tein. In previous study using affinity purification coupled with the extent of atherosclerotic lesions. The data suggest the phar-
high throughput mass spectrometry we have identified GAF asso- macological targeting of HDAC-dependent pathways may be an
ciated protein complexes, including proteins from LBC and effective therapeutic strategy in atherosclerosis. Work supported
CP190 complexes, including insulators. Moreover, we identified a by UEFISCDI (PN-III-P4-ID-PCE-2016-0665).
number of factors with DNA-binding potential that together with
GAF could be involved in recruitment and functional activity of
insulator complexes to chromatin. One of them is the Sry-delta, a P.03-020-Tue
protein containing a cluster of six C2H2-type Zinc finger motifs Differences in methylation of iPSCs, obtained
that are predicted to bind DNA. In current study using yeast by integration and integration -free methods,
two-hybrid (Y2H) assay we performed a search of Sry-delta are determined only by transgene silencing
direct partners. As a result, we have found that Sry-delta directly R. Sultanov1,2, Y. Zhukova2, O. Lebedeva2, A. Bogomazova2,
interacts with the BEAF-32, a known insulator DNA binding M. Lagarkova2
protein. Using deletion analysis, we precisely mapped the 1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
domains required for interactions between Sry-delta and BEAF- Russian Academy of Sciences, Moscow, Russia, 2FRCC PCM,
32. This finding suggests the existence of cooperative interactions Moscow, Russia
between different DNA-binding factors that could bind together
to provide a greater selectivity and affinity for protein complexes Induced pluripotent stem cells (iPSCs) generating from somatic
recruitment to different set of loci. The reported study was cells have the ability to self-renew, to differentiate into any type
funded by RFBR according to the research project No. 17-54- of somatic cells, and are promising tool for basic research and
45098 and by No. GAP0494. regenerative medicine. The reprogramming factors (oct 4, sox 2,
klf 4, c-myc) can be delivered to the cell either by vectors inte-
grating into the genome (lentiviruses, retroviruses) or by non-
integrative methods (e.g., plasmids, Sendai virus, synthetic
mRNAs). To evaluate the contribution of the reprogramming
method to epigenetic characteristics of iPSCs, isogenic system
should be utilized. Here we report analyses of methylation
142 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Genome dynamics and epigenetics
profiles for iPSC lines derived from fibroblasts of healthy donors P.03-022-Mon
via integrating (retro- and lentiviral transfection) and non-inte- The evolution of homeologous gene
grating (Sendai virus infection) reprogramming using an Illumina
450K Methylation BeadChip platform. The data of the GEO and
expression in a recent allotetraploid Capsella
the ArrayExpress portals, as well as our home data set of iso- bursa-pastoris
genic iPSCs, were used. Overall, iPS obtained by different meth- A. Klepikova1,2, A. Kasianov1,3, I. Kulakovskiy3,4,5,
ods, are indistinguishable in the analysis of DNA methylation. E. Gerasimov1,2,6, A. Fedotova7, E. Besedina8,
There were differences only in 8 CpG sites that were located in A. Kondrashov1,9, M. Logacheva1,2,10, A. Penin1,2,6,10
1
the exons of the MYC, KLF4 and SOX2, and OCT 4 genes and A. N. Belozersky Institute of Physico-Chemical Biology MSU,
hypermethylated in integrative viruses-derived iPSCs. Differences Moscow, Russia, 2Institute for Information Transmission Problems
are reproduced both in isogenic lines, and in the general set of (the Kharkevich Institute), Moscow, Russia, 3N.I. Vavilov
samples. We assumed that these CpGs reflect the methylation of Institute of General Genetics of RAS, Moscow, Russia,
4
the exogenic reprogramming genes. We validated our data by Engelhardt Institute of Molecular Biology, Russian Academy of
bisulfite sequencing of exogenic and endogenic KLF and C-myc. Sciences, Moscow, Russia, 5Skolkovo Institute of Science and
It turned out that exogenic inserts are fully methylated in IPSCs Technology, Moscow, Russia, 6Faculty of Biology, Lomonosov
of good quality, while in imperfect clones hypomethylation Moscow State University, Moscow, Russia, 7A. N. Belozersky
occurs. Thus, we can determine the quality of IPSCs using Institute of Physico-Chemical Biology, Moscow, Russia, 8Faculty
methylation data of just several CpGs. At the same time, negligi- of Bioengineering and Bioinformatics, Lomonosov Moscow State
ble differences in the methylation of iPSCs established by differ- University, Moscow, Russia, 9Department of Ecology and
ent methods make it possible to combine data on IPSCs obtained Evolution, University of Michigan, Ann Arbor, United States of
by various methods into large datasets for further analyses. The America, 10Extreme Biology Laboratory, Institute of Fundamental
work was supported by RSF 14-15-00930. Medicine and Biology, Kazan, Russia
Polyploidization and subsequent sub-, neofunctionalization or loss
of duplicated genes is one of the major mechanisms of plant gen-
P.03-021-Wed ome evolution. Capsella bursa-pastoris (shepherd’s purse) is a
Phenotype does not necessarily follow recently emerged allotetraploid and a promising model object for
genotype: Identification of an incompletely the studies of the early consequences of polyploidization. Its
penetrant novel POLR1D variant as a likely sequenced genome and close relativeness to classic model object
cause of Treacher Collins syndrome Arabidopsis thaliana make C. bursa-pastoris especially convenient.
H. Sah1,2, B. Sanlidag3, E. Manara4, K. Terali5, S. Paolacci4, We have identified 16,798 pairs of homeologous genes in two sub-
G. Mocan6, S. G. Temel7, E. Dirik3, M. Bertelli4, genomes of C. bursa-pastoris. We have studied differential expres-
M. C. Ergoren6 sion of homeologs in ten samples covering the main parts of plant.
1
Near East University, Health Science Institute, Department of 44% of homeologous pairs were differential expressed in at least
Molecular Medicine, Nicosia, Cyprus, 2Near East University, one sample. Generally, one gene from pair was downregulated in
Medical Faculty, Department of Medical Biology, Nicosia, Cyprus, one or more samples or upregulated in the same manner, but in
3
Near east university, faculty of medicine, department of pediatrics, rare cases homeologs showed discordant expression. In such pairs
Nicosia, Cyprus, 4MAGI Euregio, Bolzano, Italy, 5Near East homeolog from one genome was downregulated in some samples
University, Faculty of Medicine, Department of Medical gene and upregulated in other. This pattern of differential expres-
Biochemistry, Nicosia, Cyprus, 6Near East University, Faculty of sion is a possible reflection of ongoing processes of subfunctional-
Medicine, Department of Medical Biology, Nicosia, Cyprus, ization. Time-series gene expression data are a powerful tool for
7
Uludag University, Faculty of Medicine, Department of Medical the analysis of the dynamic biological processes and can provide a
Genetics, Bursa, Turkey detailed view to the differences in homeolog’s behavior. Transition
to flowering is a well-studied on A. thaliana process and can serve
Inherited in an autosomal-dominant manner, Treacher Collins as a model system for gene subfunctionalization analysis. Time ser-
syndrome (TCS; MIM 154500) is a disorder of craniofacial devel- ies of gene expression in Capsella meristems during floral transition
opment that occurs with an estimated incidence of 1:50,000 live were used for homeolog expression analysis. Differentially
births. TCS is characterized by a combination of hypoplasia of expressed pairs of genes were studied for the evidences of post-
the facial bones, bilateral downward slanting of the palpebral fis- polyploidization changes.
sures, colobomas of the lower eyelids, cleft palate, malformation
of the external ears, atresia of the external auditory canals, and
bilateral conductive hearing loss. 8% of individuals with TCS P.03-023-Tue
have a pathogenic variant within two genes (POLR1C and Characterization and in silico modelling of bi-
POLR1D), each of which codes for a subunit of RNA poly- allelic POLR3A mutations as a cause of
merase I. The current study presents the case of a two-year-old
male patient with clinical manifestations suggestive of TCS, such
Wiedemann–Rautenstrauch syndrome
M. Betmezoglu1,2, K. Terali3, E. Manara4, G. Mocan1,
as malformed external ear, underdeveloped chin and serious res-
S. G. Temel5, M. Bertelli4, M. C. Ergoren1
piratory problems. Sequence analysis of genomic DNA from the 1
Near East University, Medical Faculty, Department of Medical
patient and his parents revealed that the patient inherited the
Biology, Nicosia, Cyprus, 2Near East University, Health Science
novel POLR1D c.299Tin silico modeling, we predict that the sub-
Institute, Department of Molecular Medicine, Nicosia, Cyprus,
stitution of the evolutionarily conserved leucine with a proline in 3
Near East University, Faculty of Medicine, Department of
the POLR1D product (AC19) destabilizes the larger of the two
Medical Biochemistry, Nicosia, Cyprus, 4MAGI Euregio, Bolzano,
a-helices normally involved in extensive intersubunit contacts.
Italy, 5Uludag University, Faculty of Medicine, Department of
Overall, we identified a pathogenic new variant within POLR1D
Medical Genetics, Bursa, Turkey
that leads to TCS in the patient but displays incomplete pene-
trance in the parent carrying the same variation. Wiedemann–Rautenstrauch syndrome (WRS; OMIM: 264090),
neonatal progeroid syndrome, is a rare autosomal recessive
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 143
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Genome dynamics and epigenetics POSTERS
disorder with fewer than fifty-five reported cases to date. Clinical P.03-025-Mon
characteristics include prenatal and severe postnatal growth retar-
Targeting single-strand DNA breaks for DNA
dation, unusual facial features, dental anomalies, lipoatrophy and
sparse scalp hair. There is no specific disease-causing gene for repair
WRS, although the possible association of WRS with bi-allelic N. Gerasimova1, N. Maluchenko1, D. Sultanov1, M. Valieva1,
POLR3A variants (c.1909+18G>A, c.2617C>T) has recently been E. Kotova2, A. Feofanov1,3, V. Studitsky2
1
shown in a single patient. The current study presents the case of a Lomonosov Moscow State University, Moscow, Russia, 2Cancer
four-year-old female with clinical manifestations suggestive of Epigenetics Program, Fox Chase Cancer Center, Philadelphia,
WRS, such as premature aging phenotype, bilateral ponto-mesen- United States of America, 3Shemyakin-Ovchinnikov Institute of
cephaly, osteopenia, lipoatrophy in the caudal areas, and alopecia Bioorganic Chemistry, RAS, Moscow, Russia
areata. Two novel compound heterozygous POLR3A variants Single-strand DNA breaks (SSBs) is the prevalent form of
(c.3337-11T>C, c.3568C>T [p.Q1190*]), which have been inherited endogenous DNA damage in the living cells. They are frequently
from both parents, were found to be associated with WRS through caused by reactive oxygen species, which directly disintegrate
sequencing. Based on in silico analyses, the c.3337-11T>C variant backbone sugars and often produce damaged 3’ and 5’ termini
was predicted to cause abnormal gene splicing, while the truncating surrounding the break. SSB repair (SSBR) often engages proteins
p.Q1190* variant was predicted to reduce the catalytic activity of dedicated to base excision repair (BER) and is considered as its
RNA polymerase III and/or impair the interactions between the sub-pathway. In higher eukaryotes one of the major proteins
POL3RA product (C160) and its interacting partners, such as sev- detecting such breaks and orchestrating SSBR is poly(ADP-
eral other components of the enzyme and assembly factors/class ribose) polymerase 1 (PARP-1). Elongating RNA polymerase II
III transcription factors. Absence of the POLR3A gene may con- (RNAP II) also participates in detection of these lesions. SSBs in
tribute to disrupted interactions of POLR3A with DNA and have template strand (TS-SSBs) of a transcribed DNA cause arrest of
downstream effects on transcription factors important for neuro- some RNAPs with efficiencies varying significantly depending on
logic and cellular function. To conclude, the proband’s genotype the 5’ and 3’ backbone end chemistries at the break and the pres-
suggests alternative phenotypic roles for POLR3A variants and ence or absence of adjacent abasic sites. In contrast, SSBs in
may expand the clinical spectrum. Further studies on the func- non-transcribed strand (NT-SSBs) are shown to inhibit RNAPs
tional significance of both POLR3A variants as well as possible only during transcription through a nucleosome. The effect of
more patients diagnosed with WRS are required to confirm the TS-SSBs on transcript elongation by E. coli RNAP and RNAP
increasingly recognized association between POLR3A and WRS. II was not yet investigated in details. In the present work we
established an in vitro experimental system for investigation of
transcription of histone-free and nucleosomal DNA in presence
P.03-024-Wed of randomly or uniquely introduced TS-SSBs. The effect of TS-
Dynamic changes in the replication complex SSBs on elongation by E. coli RNAP was studied using the sys-
during normal and perturbed replication tem. Damaged templates were transcribed in vitro by E. coli
A. Atemin1, A. Ivanova2, M. Nedelcheva-Veleva3 RNAP, and efficiency of elongation through the lesion and
1 enzyme stalling were evaluated. The system can be adapted for
Institute of Molecular Biology, Bulgarian Academy of Science,
Sofia, Bulgaria, 2Institute of Molecular Biology, Sofia, Bulgaria, transcription by RNAP II and will be used to study the role of
3
Institute of Molecular Biology, BAS, Sofia, Bulgaria DNA repair machinery during ongoing transcription and to
reveal how PARP-1 affects elongation on the damaged templates.
The mechanisms of DNA synthesis are closely inspected through-
This work was supported by RFBR grant No. 17-54-33045 and
out almost the entire cell cycle. The licensing of chromatin, the
The Russian Federation Presidential Grant for Young Scientists
initiation, elongation and termination of replication are subjected
No MK-2377.2017.4 (Agreement of the Ministry of Education of
to specific control by different mechanisms. During unperturbed
the Russian Federation No 14.W01.17.2377-MK).
replication they monitor for balanced distribution of exact DNA
copy number in prodigy cells. When replication is compromised,
the S-phase checkpoint is activated in order to stabilize the repli- P.03-026-Tue
cation complex and to provoke the repair mechanisms for dam-
Simultaneous but not separated
age processing. In order to obtain a complex picture of the entire
process of DNA replication, the specific characteristics of the overexpression of the XPC and HR23B genes
dynamics of interactions in-between the replisome proteins must increases resistance to genotoxic stress
be obtained and evaluated. That is why, by means of highly sen- I. Velegzhaninov1,2, A. Rybak1, E. Belykh1, Y. Pylina1,
sitive and precisely aligned microscopy system, we study the D. Shadrin1
1
changes in the dynamics of different replication-associated pro- Institute of Biology of Komi Science Centre of Ural Branch of
teins throughout replication. Our live-cell imaging approach per- RAS, Syktyvkar, Russia, 2Polytechnical Institute of Vyatka State
mits precise detection of process fluctuations that are in a range University, Kirov, Russia
of microseconds. It aims to obtain quick and user-friendly model The massive amount of data accumulated at this point on the
for genome instability diagnostics and drug-testing. genes functions and the mechanisms in which they are involved
made possible selecting targets for the regulation of the proper-
ties of cells and organisms. In particular, it enabled us to control
cell resistance to ionizing radiation and other genotoxic agents.
Approaches utilizing CRISPR/dCas9 system allowed us to regu-
late several genes simultaneously in their natural chromosomal
context. In present work we overexpressed XPC and HR23B
genes, products of which are functionally interdependent and act
as DNA-damage recognition complex, using VP64-p65-Rta
(VPR) activator fused to nuclease-null Cas9. We showed for the
first time that HEK293T cells with the overexpression of both
144 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Genome dynamics and epigenetics
genes were more than 20% more resistant to radiation in doses execute the dynamic program of the replication machinery. In
of 1, 2, 3, 4 and 6 Gy than control cells, transfected with our work we focused on the study of the control over a key pro-
sgRNAs plasmids without the activator. Same results were tein from the human Replication complex – And1. It is necessary
obtained when cells were treated with 100 lM paraquat. At the for the stable association of Pol a to chromatin. It also links
same time, the overexpression of HR23B alone did not lead to GINS to Pol a and thus harmonizes both DNA helicase and
changes in the stress-resistance of the cells. Moreover, the resis- polymerase. As And1 has a fundamental role in replication, it is
tance to ionizing radiation was reduced by the overexpression of of major importance to determine its behavioural alterations that
the XPC gene alone which is also known from studies that used follow the cell cycle progression. All significant changes of its
various in vitro and in vivo models. Survival analysis was con- kinetics can be interrelated to the dynamic program of the repli-
ducted using the fluorometric microculture cytotoxicity assay in cation process. That is why, by means cutting-edge live-cell imag-
24 biological replicates for each data point. We assumed that the ing approaches, we studied the pattern of And1 protein levels
simultaneous overexpression of some functionally interdependent throughout the cell cycle and in the context of the dynamics of
stress-response genes (for example, coding subunits of a single DNA replication.
complex or units of a single cascade) that did not disrupt the
control of the cell cycle, or apoptosis, or other malignancy pro-
tection systems, could present a promising approach for increas- P.03-029-Tue
ing stress resistance of cells, tissues and organisms. The study Monitoring of castration-resistant prostate
was supported by a Grant of the President of the Russian Feder- cancer patients using liquid biopsy-based DNA
ation (MK-2929.2017.4). methylation biomarkers
K. Daniunaite1,2, A. Bakavicius2,3, A. Ulys2, R. Maleckaite1,
P.03-027-Wed M. Bavirsa1, A. Zalimas1,2, J. Lazutka1, F. Jankevicius2,
S. Jarmalaite1,2
Yeast protein Nhp6A binds to short GC-rich 1
Institute of Biosciences, Life Sciences Center, Vilnius University,
genes Vilnius, Lithuania, 2National Cancer Institute, Vilnius, Lithuania,
E. Gerasimov1, N. Gerasimova1, V. Studitsky1,2 3
Faculty of Medicine, Vilnius University, Vilnius, Lithuania
1
Lomonosov Moscow State University, Moscow, Russia, 2Cancer
Epigenetics Program, Fox Chase Cancer Center, Philadelphia, Prostate cancer (PCa) is the most prevalent non-cutaneous onco-
United States of America logic disease in elderly men and has high mortality rates due to
the development of castration-resistant PCa (CRPC). Despite sig-
Nhp6A is a small non-histone chromosomal yeast protein that nificant improvements in CRPC management, reliable molecular
nonspecifically binds and bends DNA. This protein is present at biomarkers are needed for well-timed personalized treatment
many promoters and transcribed regions of genome and is decisions and for monitoring disease progression. Utilizing epi-
involved in regulation of transcription. Recently it was shown genome-wide technologies, our group identified several novel
that Nhp6A participates in destabilization of the nucleosomal PCa-specific epigenetic biomarkers, PRKCB, CCDC181, and
structure in vitro that may explain its preferential localization at ADAMTS12, whose frequent methylation in tumors (N = 151)
gene promoters and other regulatory loci. The protein is also correlated with their downregulated expression (P < 0.001). We
found along the entire length of 176 genes, including the open further analyzed the suitability of this biomarker panel for liquid
reading frame. Its function in the coding regions remains biopsy-based CRPC monitoring. Methylation of the 3 genes was
unknown. Aiming to discover the mechanism of action of detectable in urine of patients diagnosed with CRPC (N = 133)
Nhp6A, we have further studied this group of genes. We calcu- as well as with localized PCa (N = 54). In urine collected from
lated length distribution and GC-content of the genes including CRPC patients before the first-line therapy with abiraterone acet-
nearby areas and compared with an average across the yeast gen- ate (AA), higher methylation levels of ADAMTS12 and GSTP1
ome. We demonstrated that Nhp6A predominantly binds to the (P < 0.050) were observed in cases that experienced short-term
coding regions of short GC-rich genes. The observed interaction disease progression. Pairwise analysis revealed decreased methyla-
is not associated directly with the high content of GC-pairs in tion levels of the genes when positive response to AA was
these DNA loci; therefore a specific, possibly regulatory mecha- observed for >6 months. In summary, our study led to identifica-
nism involving Nhp6A for this group could be proposed. Since a tion of a set of DNA methylation-based biomarkers that could
part of Nhp6A-associated genes retain some features of the be utilized for noninvasive monitoring of CRPC progression and
ancestral bacterial genome, we suggest that this genomic distribu- prediction of treatment response.
tion of Nhp6 is related to the mechanisms of regulation or main-
tenance of gene transcription that appeared early in the course of
evolution. This work was supported by the Russian Science
Foundation (project no. 14-24-00031).
P.03-028-Mon
Dynamic control of the replication protein
And1
A. Ivanova, A. Atemin, S. Uzunova, S. Stoynov, M. Nedelcheva-
Veleva
Institute of Molecular Biology, Sofia, Bulgaria
One of the most important tasks of the living cell is to duplicate
its genome and propagate. When there is an obstacle for correct
replication, the genome stability is altered and genetic disorders
or cancer can appear. That is why many proteins from the Repli-
cation complex are under tight control in order to precisely
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 145
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
The structural organization of the cell POSTERS
P.03-030-Wed P.03-031-Mon
The effect of the risk allele A of rs200395694 A novel role of spliceosomal proteins in
associated with SLE in Swedish patients on intercellular communication of glioblastoma
MEF2D gene regulation and alternative cells
splicing M. Pavlyukov1, V. Shender1, H. Yu2, S. Bastola2, N. Antipova1,
S. Abramov1,2, S. Kozyrev2, F. H. Farias2,3, J. Dahlqvist2, M. Shakhparonov1, I. Nakano2
I
D. Leonard4, M. Wilbe5,6, A. Alexsson4, G. R. Pielberg2, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
H. Hansson-Hamlin7, A. G€ oran6, K. Tandre4, M. L. Eloranta4, Moscow, Russia, 2University of Alabama at Birmingham,
L. E. R€ onnblom4, K. Lindblad-Toh2,8 Birmingham, United States of America
1
Institute of Fundamental Medicine and Biology, Kazan Federal Alternative pre-mRNA splicing dramatically increases protein
University, Kazan, Russia, 2Science for Life Laboratory, diversity within the cell. Besides being a critical mechanism for
Department of Medical Biochemistry and Microbiology, Uppsala development and regulation of cell-type-specific functions, splic-
University, Uppsala, Sweden, 3The Genome Institute, Washington ing is also involved in multiple pathologies, including cancer.
University School of Medicine,, St. Louis, Missouri, United States Dysregulations of splicing were shown to play a major role both
of America, 4Science for Life Laboratory, Department of Medical in malignant transformation and cancer progression. It is cur-
Sciences, Section of Rheumatology, Uppsala University, Uppsala, rently thought that spliceosomal proteins perform their functions
Sweden, 5Science for Life Laboratory, Department of Immunology, only within the cell. However, our study provides the first evi-
Genetics and Pathology, Uppsala University, Uppsala, Sweden, dence that various spliceosomal components may play an impor-
6
Department of Animal Breeding and Genetics, Swedish University tant role in intercellular communication. By analyzing blood and
of Agricultural Sciences (SLU), Uppsala, Sweden, 7Department of ascites samples from glioblastoma and ovarian cancer patients we
Clinical Sciences, Swedish University of Agricultural Sciences found that cancer cells can specifically secrete RNA and protein
(SLU), Uppsala, Sweden, 8Broad Institute of Massachusetts, components of spliceosomes after the courses of chemo- and
Cambridge, United States of America radiotherapy. Further experiments revealed that spliceosomal
Systemic lupus erythematosus (SLE) is a chronic inflammatory proteins are exported by apoptotic cancer cells within exosome-
autoimmune disease with diverse clinical manifestations and com- like extracellular vesicles (EVs). These EVs induce pre-mRNA
plex etiology. The common associated genetic variants do not fully splicing changes in recipient glioblastoma cells and promote pro-
explain the disease heritability, which may suggest the contribution liferation, migration, therapy resistance and aggressive mesenchy-
from rare and low frequency variants, undetectable in GWAS. We mal phenotype of glioblastoma patient-derived neurospheres both
identified a novel rare variant rs200395694 located in the MEF2D in vitro and in vivo. Mechanistically, we identified RBM11 as a
gene encoding for the myocyte-specific enhancer factor 2D tran- representative splicing factor that is upregulated in tumors after
scription factor as being associated with SLE in Swedish patients therapy and shed in EVs upon induction of apoptosis. Once
(504 SLE patients and 839 healthy controls, P = 0.014, CI = 1.1– internalized in recipient cells, exogenous RBM11 switch splicing
10), and studied the potential effect of the risk allele A on gene of MDM4 and CyclinD1 towards the expression of more onco-
regulation and splicing. The SNP shows an effect on protein-DNA genic isoforms. Clinically, the elevated expression of these iso-
binding and luciferase expression when analyzed by EMSA and forms in glioblastoma is associated with worse patient prognosis.
luciferase reporter assay thus supporting the presence of an active Taken together, these findings reveal a novel role of spliceosomal
cell-specific enhancer. The total expression of the MEF2D iso- proteins in intercellular communication between apoptotic and
forms was studied by quantitative RT-PCR using RNA isolated surviving tumor cells and suggest that targeting of EV-mediated
from cell lines. Both endogenous a1 and a2 transcripts were splicing changes may be exploited to decrease tumor aggressive-
detected in human peripheral blood mononuclear cells (PBMC), ness that would eventually improve cancer patient outcome.
monocytic cells THP-1, myelogenous leukemia cells K562 and B
cell line Daudi, but only the major a1 transcript was detected in The structural organization of the cell
Jurkat T cell line, HeLa cervical cancer cells and HEK293 human
embryonic kidney cells. The effect of the SNP on alternative splic-
ing was studied using minigene constructs with different genotypes P.04-001-Mon
transfected into 4 cell lines. No allelic difference was detected for Defects in ER-Golgi morphology and disturbed
the a1 isoform transcribed from the minigene. The alternative iso- locations of selected actin nucleators in
form a2 generated from the minigene with the rare A allele was developing spermatids of MYO6-deficient mice
significantly repressed in all tested cells. In summary, we identified P. Zakrzewski1, A. Suwi nska1, V. Chumak2, M. J. Redowicz2,
a novel rare regulatory variant associated with SLE in Swedish F. Buss3, M. Lenartowska1
patients. The risk allele exerts an effect on a putative enhancer and 1
Nicolaus Copernicus University in Torun, Torun, Poland, 2Nencki
also inhibits the splicing of the alternative MEF2D isoform. Institute of Experimental Biology, Polish Academy of Sciences,
Warsaw, Poland, 3University of Cambridge, Cambridge, United
Kingdom
Myosin VI (MYO6) is an actin-based motor protein that has
been implicated in a wide range of processes including endocyto-
sis, maintenance of Golgi morphology and regulation of actin
dynamics. In Drosophila, MYO6 plays a key role in the late
phase of spermatogenesis and mutant male flies lacking MYO6
are sterile. The fundamental mechanisms that control spermio-
genesis are conserved between evolutionally distinct animal spe-
cies, thus we hypothesised that MYO6 may also have a potential
role during this developmental process in mammals. To test this
hypothesis, we examined MYO6 expression and localisation and
146 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS The structural organization of the cell
found that two splice variants – with a small insert and no insert P.04-003-Wed
– are expressed in developing spermatids in mice. We further
Regulation of expression efficiency of genes
found that these MYO6 isoforms are associated with key actin
structures involved in sperm maturation. We next examined encoding cytoskeletal proteins in sperm of
developing spermatids in MYO6 KO mice (Snell’s waltzer; sv/sv mice after long-term modeling microgravity
mice), whose fertility is also reduced. Our ultrastructural analysis I. Ogneva1,2, M. Usik1,2
1
has revealed morphological disruptions, especially of the Golgi I.M. Sechenov First MSMU, Moscow, Russia, 2SSC RF IBMP
complex, the endoplasmic reticulum (ER) as well as the acro- RAS, Moscow, Russia
some. These results suggest that MYO6 may play a role in actin Weightlessness conditions or its modeling on the Earth lead to a
organisation and dynamics crucial for sperm development not change of the genes expression level in the different cells, but
only in Drosophila but also in mice. We further compared the how this regulation is carried out is still unclear. Therefore, the
localisation of marker proteins for the Golgi complex and the aim of this work was to determine the transcription levels of
ER and of a number of actin-nucleating proteins in developing genes encoding cytoskeletal proteins (by qPCR), the total methy-
spermatids of wild type and MYO6 KO mice. Our results show lation level of the genome (by EpiJET DNA Methylation Kit,
significant changes in the distribution of all the marker proteins Thermo Scientific), the 5hmC content (by dot blotting with speci-
tested in the absence of MYO6. In summary, our findings suggest fic antibodies, Abcam), the content of DNA-methylase/demethy-
for the first time that MYO6 may play a role in actin organisa- lase, acetylase/deacetylase of histones (by Western-blotting) in
tion and maintenance of Golgi and ER morphology during mice sperm. Modeling of microgravity effects was carried out by
spermiogenesis in mammals. 30-day antiorthostatic suspension (group 30HS, n = 7), simulated
an early period of readaptation by 12-hour recovery after suspen-
sion (group 30HS+12 h, n = 7) and formed the corresponding
P.04-002-Tue
control group (group C, n = 7). The relative mRNA content of
In cellulo nanocrystallization of the nucleoid in the genes encoding the actin cytoskeleton proteins did not change
bacteria and spores or increased after 30-day suspension, but after 12-hour recovery
Y. Krupyanskiy1, N. Loyko1,2, K. Tereshkina1, V. Kovalenko1, it increased significantly compared to the control group. At the
E. Tereshkin1, O. Sokolova1, A. Popov3 same time, the total methylation level of the sperm genome in
1
Semenov ICP RAS, Moscow 119991, Russia, 2FRC Fundamentals the 30HS group did not differ from the control one, but in the
of biotechnology, RAS, Moscow, Russia, 3ESRF, Grenoble Cedex 30HS+12 h group it decreased by 23% (P < 0.05), while the con-
9, France tent of 5hmC did not change. Also, the DNA methylases content
Survival of living organisms in constantly changing environmen- (Dnmt1 and Dnmt3a), DNA demethylases (TET1, TET2, TET3)
tal conditions is possible due to universal hereditary strategies of did not change. However, the HAT1 acetyltransferase content in
adaptation to various types of stress, based on structural, bio- the 30HS+12 h group significantly increased by 10% relative to
chemical and genetic rearrangements. One of the strategies imple- the control level (P < 0.05) in the absence of changes in the
mented in bacterial cells is related to the protection of the HDAC1 deacetylase content. One can propose a hypothesis that
nucleoid from unfavourable environmental conditions by binding an increase of the transcription efficiency of the cytoskeletal
of DNA to specific histone-like proteins, the main one being the genes under recovery after modeling of weightlessness is associated
protein Dps (DNA binding protein from starved cells), and con- with a decrease genome methylation level, which in turn is due to
densation of DNA with DPS in nanocrystalline complex which an increase of the histone acetylation. The study was supported by
was recently discovered in gram-negative bacteria. A series of program of the fundamental research SSC RF – IBMP RAS and
diffraction experiments to study structural response to stress or program of RAS presidium “Molecular and cell biology”.
stress-induced biocrystallization in bacteria E. coli, spore -form-
ing bacteria Bacillus cereus, in cells and spores of mycelial fungus
P.04-004-Mon
Umbelopsis ramanniana was performed for the first time. Zones
of increased intensity at resolution 90 A and 44 A indicate an Mitochondrial cristae changes as reflected by
ordered organization (most likely nanocrystalline) of bacterial 3D super-resolution dSTORM microscopy
nucleoid in E. coli cells. For the starving bacteria Bacillus cereus P. Jezek, A. Dlaskova, H. Engstova, L. Plecita Hlavata,
peak at a resolution of 45 A, apparently, also associated with the cek
A. Kahancova, T. Spa
presence of nanocrystalline complexes of DNA with histone-like Inst. Physiology CAS, Prague 4, Czech Republic
proteins. The spores of the fungus Umbelopsis ramanniana BKM 3D super-resolution microscopy is a powerful method for access-
F-582, as well as the spores of Bacillus cereus create ordered sets ing changes of objects that could be normally resolved only by
of DNA molecules with DNA condensing acid - soluble proteins electron microscopy. Despite the still impossible cristae resolu-
SASPs. Starving, dehydrated mycelial cells of the fungus Umbe- tion, we have previously demonstrated that mitochondrial cristae
lopsis ramanniana, form ordered structures at resolutions of 27 became widened in HepG2 cells after 72 hour hypoxic adaptation
A to 55A. The series of peaks reflects the formation of a number at 5% oxygen, with simultaneous breakdown of dimers of ATP-
of orderly protein formations with continuously changing charac- synthase leading to less dimer population within the widened cris-
teristic size of the interplanar spacings. This work was supported tae [1]. In this work, we applied the direct stochastic optical
within frameworks of the state task for ICP RAS 0082-2014- reconstruction microscopy (dSTORM) with primary Alexa-
0001. State registration # AAAA-A17-117040610310-6. Fluor-647-conjugated antibodies and thus monitored certain mor-
phological changes, such as in cristae width by dSTORM of
ATP-synthase subunit F1a. Obtained 3D images were analyzed
with the help of Ripley’s K-function modeling spatial patterns or
transferring them into distance distribution function. Resulting
histograms of distances frequency distribution provide most fre-
quent distances (MFD) between the localized single antibody
molecules. We studied the above mentioned hypoxic adaptation
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 147
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Stem cells POSTERS
of HepG2 cells, and confirmed the previously reported [1] cristae P.05-002-Tue
widening. In contrast, in fasting state of model pancreatic b-cells,
Somatic cells reprogramming and genome
INS-1E, MFD between F1a were ~80 nm at 0 and 3 mM glu-
cose, whereas decreased to 61 nm and 57 nm upon glucose-sti- editing for Stargardt disease modeling
mulated insulin secretion (GSIS) at 11 mM and 20 mM glucose, M. Lebedin1, K. Mayorova1, M. Lagarkova1, S. Kiselev2
1
respectively. Shorter F1a interdistances reflected cristae width Vavilov Institute of General Genetics, Moscow, Russia, 2Vavilov
decrease upon GSIS, since such repositioning of F1a correlated Institute of General Genetics, Russian Academy of Sciences,
to average 20 nm and 15 nm cristae width at 0 and 3 mM glu- Moscow, Russia, Moscow, Russia
cose, and 9 nm or 8 nm after GSIS (11, 20 mM glucose, respec- Degeneration of the retina occurs both in relation to age, and as
tively). Supported by GACR 17-08565S. a consequence of hereditary pathologies. A clinically similar pat-
Reference tern is often associated with different molecular pathways and
[1] Plecit
a-Hlavata L, et al., Jezek P. Hypoxic HepG2 cell adap- gene mutations. The arsenal of therapeutic approaches for these
tation decreases ATP synthase dimers and ATP production in patients is very limited. Modern advances in cellular reprogram-
inflated cristae by mitofilin down-regulation concomitant to ming and genome editing make it possible to establish a model
MICOS clustering. FASEB J., 2016 30:1941–1957. for the disease investigation and treatment. In this study we
established induced pluripotent stem cells (iPSCs) from patients
Stem cells with a clinical diagnosis of Stargardt’s disease. Mutation in the
peripherin 2 gene was found and it was shown that the mutation
does not affect the efficiency of differentiation in the pigment
P.05-001-Mon epithelium of the retina. Using the CRISPR / Cas9 system the
The role of deuterium in the biological mutation was corrected in the patient’s iPSCs. As a result, iso-
properties of human adipose derived geneic iPSC lines with a corrected mutation have been generated
mesenchymal stem cells in vitro for establishing of an in vitro model of the disease and potentially
A. Zlatska1,2, I. Zlatskyi3,4, A. Syroeshkin3 suitable for personalized therapy of Stargardt disease.
1
State Institute of Genetic and Regenerative Medicine of NAMS of
Ukraine, 67, Vyshgorodskaja st., Kiev, 04114, Kiev, Ukraine,
2 P.05-003-Wed
Biotechnology Laboratory ilaya regeneration, medical company
ilaya, 9, I. Kramskogo st., Kiev, Ukraine, 03115, Kiev, Ukraine, Immunosuppression mediated by
3
RUDN University, Moscow, Russia, 4Dumanskii Institute of mesenchymal stem cells involves cell-cell
Colloid and Water Chemistry NAS of Ukraine, Kiev, Ukraine contact mechanism
J. Suzdaltceva1, A. Gabashvili1, K. Goryunov2, Y. Rubtcov2,
Depending on the ratio of deuterium (D) and protium (H) in
V. Tkachuck2
water, its physicochemical properties change. Water, with the 1
Vavilov Institute of General Genetics, Moscow, Russia, 2Moscow
changed isotope composition D/H, has a number of unexpected
State University, Moscow, Russia
biological properties, including antitumor, antidote and metabolic
effects, but studies in this direction are insufficient. The aim of Human adipose-derived mesenchymal stem cells (hASCs) are can-
work was to study the effect of deuterium on the biological prop- didate for regenerative medicine treatments primarily due to their
erties of human adipose derived mesenchymal stem cells (ADSC) immunosuppressive properties. Various molecular mechanisms
in vitro. ADSC was prepared from lipoaspirate by enzymatic can be involved in hASC-mediated immune suppression. These
treatment. The resulting cell suspension was cultured on MEM-a, mechanisms underlying hASCs-mediated immunosuppression
prepared from a dry powder by dilution with water (MilliQ) (D/ include soluble factors and cell-cell contacts, which possibly
H = 150 ppm), deuterated water (D/H = 99 abs. at.%) and deu- depend on expression of cell adhesion molecules. To dissect cell
terium depleted water (ddw) (D/H = 10 ppm). In addition, 10% contact-dependent mechanism of hASC-dependent immune sup-
FBS, 2 mM glutamine, and 1 lg / ml FGF-2 were added to the pression in vitro co-culture system consisting of hASCs and phy-
culture medium. Cultivation was carried out in multi-gas incuba- tohemagglutinin-activated peripheral blood lymphocytes (PBL)
tors with automatic maintenance: t 37°C, absolute humidity, 5% was established. hASCs inhibited proliferation of activated PBL
of CO2 and O2. The parameters were determined: population both in trans-well and cell-cell contact culture system as assessed
doubling time (PDT), viability (using PI and FDA dyes after 24/ by CyQuant cell proliferation assay. Activation of lymphocytes
72 h) and metabolic activity by Alamar Blue after 24/72 h. PDT with hASCs has resulted in an increased attachment of immune
showed the lowest values in the control (MilliQ) and ddw, the cells to hASCs. Flow cytometry analysis revealed that activated
largest - in deuterated media, which indicates a decrease in the CD4+ T cells were capable to bind hASCs more effectively in
proliferation of the culture. The acute and chronic toxicity of comparison to non-activated. We showed that hASCs-dependent
deuterated water was shown (of 2 times in comparison with the suppression of activated T-cell proliferation is not associated with
control), as well as the chronic toxicity of ddw. Metabolic activ- the reduction of IL-2R (CD25) expression or apoptosis. The
ity decreased in the deuterated culture medium of 2 times in com- increased level of IDO mRNA was observed in hASCs cocul-
parison with the control. At the same time, in ddw there was an tured with activated PBMC in cell-cell contact system in compar-
increase in metabolic activity of 1.5–2 times in comparison with ison to separate cultures. Taken together our data support the
the control. The results show that deuterium plays an important substantial role of cell contact-dependent mechanism in hASC-
role in intracellular processes that are responsible for the metabo- mediated immune suppression.
lism and viability of ADSC in vitro, which can be used in further
research in the field of biology and medicine. The publication
was prepared with the support of the “RUDN University Pro-
gram 5-100”.
148 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Stem cells
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 149
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Stem cells POSTERS
150 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Stem cells
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 151
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Stem cells POSTERS
152 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Stem cells
Differentiated iPSC derivatives were resistant to allogenic CD8+ muscles. On the other hand, the analysis of the transcriptome of
T cell-mediated killing in vitro. Similar cytotoxic tests will be FSHD muscle biopsies revealed a high level of expression of pro-
conducted in co-cultivation with NK cells. This work was sup- teins associated with a nonspecific response to inflammation. Per-
ported by the Russian Science Foundation grant #17-75-10206. haps such a response is an attraction factor for mesenchymal
stromal cells (MSCs), which can differentiate into some types of
cells, leading to fibrosis. The limited proliferative capacity and
P.05-016-Mon inaccessibility of primary myoblasts (PM) hampers their use
Generation of folliculogenic human dermal in in vitro studies. Immortalization of cells allows obtaining an
papilla cells from induced pluripotent stem endless standardized resource for pathology study. IM cultures
cells (Institute of Myology, Paris) from healthy and FSHD donors;
PM cultures and MB transfected with DUX4, as control, were
E. S. Chermnykh1,2, E. P. Kalabusheva1,2, V. I. Sharobaro2,
used. The MSCs migration to myoblasts was analyzed in the
E. A. Vorotelyak1,2,3
1 Transwell system. To study the migration mechanism we used
Koltzov Institute of Developmental Biology, Russian Academy of
neutralizing antibodies to SDF1a, inhibitor CXCR4 AMD3100,
Sciences, Vavilov Street 26, 119334, Moscow, Russia, 2Pirogov
RT PCR, immunocytochemical method. MB from FSHD donors
Russian National Research Medical University, 1, Ostrovitianov
have morphological defects in in vitro differentiation. It was
str., 117997, Moscow, Russia, 3Lomonosov Moscow State
found that FSHD IM can stimulate MSC migration due to the
University, 1-12 Leninskie Gory, 119991, Moscow, Russia
secretion of SDF1, and the soluble factors of MSCs increase the
Hair follicle morphogenesis is a complex process that occurs dur- expression of SDF1 and VEGF in IM. In addition, increased
ing the development of skin and as a part of the hair cycle. An expression of a number of pro-inflammatory proteins was
important role in regulation of this process is played by hair folli- detected in FSHD MB. The results demonstrate that IM are
cle dermal papilla cells (DPCs). DPCs represent mesenchymal cell applicable as a model for researching the cellular mechanisms
subpopulation with stem properties which provide an opportu- fibrosis in FSHD and developing new targets for therapy. The
nity to apply them in the treatment of hair loss (alopecia). How- work is supported by the state program of fundamental scientific
ever the isolation and propagation of human DPCs for tissue research IDB RAS 0108-2018-0008.
engineering purposes remains a challenge because they quickly
lose intrinsic biological characteristics, especially hair inductive
capacity, with passaging, not allowing one to increase cell popu- P.05-018-Wed
lation sufficient for treatment. One of the promising and reason- Optimizing the preparation of MS1
able ways to develop cell products for hair loss treatment is to extracellular matrix for the amplification of
obtain trichogenic DPCs by differentiating them from induced
mesenchymal stem cells
pluripotent stem cells (iPSCs). iPSCs can differentiate into cells
D. S. Yang, I. H. Liu
of all three germ layers in vitro and in vivo. Here we develop a
Department of Animal Science and Technology, National Taiwan
strategy to differentiate human iPSCs into DPCs that can recon-
University, Taipei, Taiwan
stitute the mesenchymal component of the hair follicle. The
iPSC-derived DPCs show a similar marker expression signature Bone marrow-derived mesenchymal stem cells (BM-MSCs) have
as human DPCs directly isolated from human hair follicles. great potential for cell therapies. However, the scarcity and loss
Human iPSC-derived DPCs are capable of generating hair folli- of stemness during amplification has been a challenge for their
cles including the hair shaft, when transplanted into the skin of application. Our previous study indicated that de-cellularized
Nude mice. These results suggest an approach for generating a extracellular matrix (ECM) derived from microvessel endothelial
large amount of human DPCs for tissue engineering and new cell line Mile Sven 1 (MS1) can effectively preserve the BM-
treatments for hair loss. The work was funded by the Russian MSCs stemness including proliferation and differentiation capaci-
Science Foundation (Project No. 16-14-00204). ties. To optimize the preparation and application of this novel
material, we tested various methods to collect, homogenize and
coat MS1-ECM on the surface of culture dish, and then evalu-
P.05-017-Tue ated the re-gelification of MS1-ECM by scanning electronic
Applying of immortalized myoblasts for the microscopy. To test whether the optimized preparation can also
study of the cellular mechanisms of the preserve the stemness compared to the conventional culture con-
dition and/or the hypoxic culture condition, we evaluated the
muscle tissue replacement by connective
properties of MSCs including morphology, self-renewal, tri-linage
tissue in the in vitro model of differentiation capacities and immunomodulatory potential after
facioscapulohumeral muscular dystrophy two passages. We found that our optimized preparation of MS1-
(FSHD) ECM can provide BM-MSCs a better niche to preserve the stem-
O. Latyeva1,2, E. Kiseleva2, Y. Vassetzky2,3 ness. Moreover, the major content of MS1-ECM and the
1
Lomonosov MSU, Moscow, Russia, 2Koltzov Institute of responses of BM-MSCs cultured on various microenvironments
Developmental Biology of Russian Academy of Sciences, Moscow, will be assessed by proteomic and transcriptomic analysis to
Russia, 3UMR 8126, CNRS, Institut de Canc erologie Gustave- begin our journey to dissect the potential factors that are critical
Roussy, Villejuif, France for maintaining the stemness of BM-MSCs. In conclusion, we
have developed a novel and effective method to prepare and
The goal of this work was to describe immortalized myoblasts
apply the MS1-ECM for expanding the primary BM-MSCs.
(IM) and to investigate the cellular mechanisms of fibrosis in
FSHD. FSHD is a common autosomal dominant disorder that
presents progressive weakness of the skeletal muscles. The main
form - FSHD1 is associated with a reduction in the number of
repeat units of the D4Z4 array and two polymorphisms (4qA161
and 4qA). As a result, the expression of a number of proteins is
changed, in particular DUX4, which turns out to be toxic to the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 153
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Stem cells POSTERS
154 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Stem cells
anticancer drugs which signaling pathways are known. Two traumatic of peripheral nerve injury in diabetic neuropathy often
MSC cultures obtained from the desquamated endometrium from combined with diabetic peripheral vascular disease. This study was
different donors were used. Both lines secrete VEGF. Treatment supported by Russian Science Foundation (RSF Grant no. 16-45-03007).
of MSC with DX stopped proliferation, caused stress-induced
premature senescence, but did not reduce the secretion of VEGF
in both MSC lines. Selective ALK inhibitor of TGFb/Activin P.05-024-Wed
pathway (SB431542) inhibited secretion of VEGF and did not Differential effects of experimental hypoxia
reduce proliferation. Specific inhibitor of c-Jun N-terminal kinase conditions on cancer cell behaviours in HCC
JNK (SP600125) suppressed secretion and arrested proliferation. E. Kandemis1,2, N. Atabey2,3, E. Erdal2,3
p38 MAP kinase inhibitor (SB203580) also inhibited secretion, 1
Department of Molecular Biology and Genetics, Faculty of
but stimulated proliferation. Like DX, allosteric phosphoinosi- Engineering and Natural Sciences, Bahcesehir University (current
tide-dependent protein kinase-1 agonist (PS48) caused cellular address), Istanbul, Turkey, 2Department of Medical Biology,
senescence and maintained the secretion of VEGF in both MSC. Faculty of Medicine, Dokuz Eylul University, 35340-Inciralti,
PS48 is an activator of PDK1 and induces downstream path- Izmir, Turkey, 3Izmir Biomedicine and Genome Institute (IBG-
ways, primarily PI3K-Akt. Thus, PI3/Akt signaling in MSC can Izmir), Dokuz Eylul University, 35340-Inciralti, Izmir, Turkey
be the main regulator of survival and secretion of MSC after DX
treatment. However, due to the ability of MSC to migrate Hypoxic microenvironments are important for invasion, metasta-
towards tumor blood vessels, VEGF secreted by them can stimu- sis and drug resistance in solid tumors. In literature, two different
late angiogenesis and growth of certain tumors. This work was models have been using to create hypoxic conditions; CoCl2 which
supported by Russian Science Foundation project 14-50-00068. is a chemical inducer of hypoxia and hypoxia chamber or hypoxia
bags. In our experiments we performed these two methods on the
HuH-7 parental, cancer stem cell subpopulation (EpCAM+/
P.05-023-Tue CD133+) as well as non-stem cell subpopulation (EpCAM-/
Stimulation of peripheral nerve regeneration CD133). After magnetic separation, we constituted hypoxic condi-
tions with different hypoxic methods in these cells. We observed
after trauma by hepatocyte growth factor
that there were no differences in motility of HuH-7 presence of
gene therapy in mice CoCl2, whereas when we incubated HuH-7 cells in hypoxic bag
M. Boldyreva1,2, I. Bondar3, I. Stafeev1,2, P. Makarevich1,4, motility of HuH-7 was significantly increased. Since hypoxia is
I. Beloglazova1,2, E. Zubkova1,2, E. Shevchenko1, very important for regulation of cancer stem cells, we then exam-
Y. Molokotina1,5, K. Dergilev2, E. Ratner2, Y. Parfyonova1,2 ined motility of EpCAM+/CD133+ and EpCAM-/CD133- sub-
1
Faculty of Medicine, Lomonosov Moscow State University, populations under two different hypoxic conditions. In the
Moscow, Russia, 2National Medical Research Center for presence of CoCl2 motility of both EpCAM+/CD133+ and
Cardiology, Russian Ministry of Health, Moscow, Russia, EpCAM-/CD133- were decreased, contrary motility of these cells
3
Institute of Higher Nervous Activity & Neurophysiology, Russian were increased when hypoxic conditions were created with
Academy of Sciences, Moscow, Russia, 4Institute of Regenerative hypoxia bag. We next examined spheroid formation of these cells
Medicine, Lomonosov Moscow State University, Moscow, Russia, under two different hypoxic conditions and we determined that
5
Faculty of Medical and Biological Physics, Moscow Institute of spheroid numbers of HuH-7 was not changed by hypoxia treat-
Physics and Technology, Moscow, Russia ment. However, hypoxia created by CoCl2 were decreased spher-
Traumatic peripheral nerve injury is common and results to loss oid numbers in EpCAM+/CD133+ hepatic stem cell population,
of function and/or neuropathic pain. One of the most promising whereas there was no significant differences were observed in cells
non-surgical methods of peripheral nerve damage treatment is incubate in hypoxic chamber. In EpCAM-/CD133- subpopulation,
gene therapy with prolong local expression of neurotrophic and spheroid numbers were decreased with CoCl2, however spheroid
angiogenic factors. Hepatocyte growth factor (HGF) is one of numbers were increased in cells in hypoxia chamber. As a result,
the most promising pleiotropic factor for peripheral nerve gene methods of hypoxia have differential effects on the behaviours of
therapy due to its angiogenic, neuroprotective, antiapoptotic, HCC cells in context dependent. The biological consequences of
antifibrotic and anti-inflammatory effects. In our study, we exam- these differential effects needs further investigations.
ined the efficacy of HGF plasmid gene therapy (pC4W-hHGF) [Correction added on 25 April 2019, after first online publication:
to restore the effects of traumatic nerve damage in mice using a The abstract title of P.05-024-Wed has been corrected to “Differ-
model of unilateral traumatic crush injury of common peroneal ential effects of experimental hypoxia conditions on cancer cell
nerve (n. peroneus communis). Effects were assessed using electro- behaviours in HCC” in this version.]
physiological, histological and behavioral tests. Treatment of
pC4W-hHGF resulted in the restoration of the nerve structure
and functional recovery in comparison to similar parameters in P.05-025-Mon
control animals treated with empty vector. Compound action Fusion and reprogramming of human
potentials (CAP) in the experimental group treated with pC4W- cardiomyocytes with human mesenchymal
hHGF showed an increased amplitude and a decrease latency in stem cells
comparison with the control group. Immunofluorescent staining €
A. Karadag1, T. Ozkan 2
, A. Sunguroglu2
analysis Immunofluorescence staining analysis against the axon 1
Usak University, Faculty of Medicine, Department of Medical
marker protein NF-H showed axon marker protein NF-H Biology, Usak, Turkey, 2Ankara University, Faculty of Medicine,
showed a threefold increase in the number of axons in the nerve Department of Medical Biology, Ankara, Turkey
located distal to the crush region in HGF-treated mice compared
to the control (29.16 3.19 in HGF100 group vs. 9.17 0.55 in According to the classical belief, heart cells cannot be regenerated
control group, pcs/1000 lm2; P < 0.001). Furthermore, signifi- and repair the damaged tissue following irreversible damage.
cant functional recovery n. peroneus communis caused by Recently, cellular therapies have emerged as the most innovative
pC4W-hHGF gene therapy was observed using a trace analysis treatment strategies. In cardiovascular diseases, a clear under-
and sciatic function index (SFI). Results of present study provide standing of cardiac reprogramming and fusion mechanisms is
evidence for use of plasmid-based HGF gene therapy to treat necessary to achieve the goals of cellular therapy. The main
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 155
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Stem cells POSTERS
156 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Photosynthesis and plant biochemistry
performed. The expression pattern of uPA during the post-infarc- Photosynthesis and plant biochemistry
tion period showed significant increase during the early (3 days)
and remote periods (7 and 14 days after MI) at both transcrip-
tional and protein levels. An increase level of urokinase correlated
P.06-001-Mon
with activation of the c-kit+ CPC pool. Already on day 3, massive Influence of Chlorella vulgaris on the
infiltration of CPC occured in the peri-infarction area (289 +/- photosynthetic apparatus of rice plants under
154 cells / mm2) and the damage zone (252 +/- 118 cells / mm2). cadmium stress
Similar dynamics persists on day 7 after acute damage (264 +/- E. Yotsova1, A. Dobrikova1, M. Stefanov1, M. Kouzmanova2,
143 cells / mm2) and gradually decreased by the 14th day after E. Apostolova1
injury. In addition, an elevated level of urokinase caused an 1
Institute of Biophysics and Biomedical Engineering, Bulgarian
increase of CPC cell proliferation in the area of damage. In vitro Academy of Sciences, Sofia, 1113, Bulgaria, Sofia, Bulgaria,
studies have shown that CPC obtained from uPA-/- mice pos- 2
Department of Biophysics and Radiobiology, Biological Faculty,
sessed reduced proliferative capacity in comparison with CPC Sofia University “St. kliment Ohridski”, Sofia, Bulgaria, Sofia,
from wild-type mice. The present study demonstrates that uroki- Bulgaria
nase expression in the heart in response to injury may induce acti-
vation of CPC and increase their proliferation properties, which Cadmium (Cd) is one of the most toxic heavy metals due to its
may be important for starting a successful regeneration. This high solubility in water, its easy absorption through the roots and
work was supported by RSF grant 17-15-01368. its accumulation in plant tissues. The aim of this study was to
investigate the effect of the green algae Chlorella vulgaris on the
growth parameters and the functions of the photosynthetic appa-
P.05-029-Tue ratus in rice plants grown under Cd stress. PAM chlorophyll
Gene expression is modulated in human fluorescence, oxygen evolution (measured with Clark-type and
Joliot-type electrodes), P700 photo-oxidation measurements and pig-
umbilical cord Wharton’s jelly-derived
ment content were used to assess the changes in the functions of the
mesenchymal stem cells by TSH-TSHR photosynthetic apparatus under Cd stress (150 lM CdCl2) of rice
interactions seedlings grown hydroponically in the presence or the absence of
E. U. BAGRIACIK1,2, M. YAMAN2, N. ORUKLU2, Chlorella vulgaris (OD760 = 1.2). Several important markers associ-
M. BAYRAM3 ated with oxidative stress (lipid peroxidation, H2O2 and proline
1
Gazi University, Life Sciences Research Center, Ankara, Turkey, production) were also evaluated. Cadmium stress led to inhibition
2
Gazi University, Department of Immunology, Faculty of of the plant growth, the functions of the photosynthetic apparatus
Medicine, Ankara, Turkey, 3Gazi University, Department of and increased levels of the oxidative stress markers. Our data
Obstetrics and Gynaecology, Faculty of Medicine, Ankara, Turkey revealed that the presence of green algae Chlorella vulgaris, during
Cd exposure of the rice seedlings, improved notably the growth, the
We reported that TSH-TSHR interactions modulated gene expres-
photochemical activities of both photosystems, the electron flow
sion in human bone marrow-derived mesenchymal stem cells. In
from QA to plastoquinone, the kinetic parameters of the oxygen-
this study, we demonstrated that human umbilical cord Wharton’s
evolving reactions, pigment content and decreased lipid peroxida-
Jelly-derived mesenchymal stem cells expressed a functional thy-
tion, H2O2 and proline production. This study suggests that the
roid stimulating hormone (TSH) receptor (TSHR). To understand
green algae Chlorella vulgaris applied in the rooting medium has a
whether TSH-TSHR interactions has any effects in gene expression
protective effect against Cd toxicity in rice plants. The possible
of human umbilical cord Wharton’s Jelly-derived mesenchymal
molecular mechanisms involved in the defensive effect of the green
stem cells, we performed gene expression studies at mRNA levels
algae on the function of photosynthetic apparatus are shown. The
by using a gene array assay and RT-PCR gene profiling arrays.
authors thank to Ivan Iliev (IPPG-BAS) for providing the Chlorella
Human umbilical cord Wharton’s Jelly-derived mesenchymal stem
vulgaris.
cells were isolated from human umbilical cords. Alternatively, we
purchased human umbilical cord Wharton’s Jelly-derived mes-
enchymal stem from American Type Culture Collection (ATCC) P.06-002-Tue
as well. TSH treatment modified several gene expression of mes-
enchymal stem cells in all samples. We also found that in terms of
Distinct patterns of activation of papain-like
gene expression, response of human umbilical cord Wharton’s cysteine protease Triticain-a in vivo
Jelly-derived mesenchymal stem cells to the TSH treatment was dif- A. V. Balakireva1, V. G. Zgoda2, N. K. Tikhomirova3,
ferent from those of human bone marrow-derived mesenchymal L. V. Savvateeva1, E. Y. Zernii1,3, A. A. J. Zamyatnin1,3
1
stem cells. Based on these findings, we concluded that TSH-TSHR Institute of Molecular Medicine, Sechenov First Moscow State
interactions might contribute proliferation, migration, and differ- Medical University, Moscow, Russia, 2Institute of Biomedical
entiation of human mesenchymal stem cells. Chemistry (IBMC), Moscow, Russia, 3A.N. Belozersky Institute
of physico-chemical biology MSU, Moscow, Russia
Papain-like cysteine proteases (PLCPs) in plants and animals par-
ticipate in numerous physiological processes. They are synthe-
sized as preproenzymes consisting of N-terminal signal peptide,
inhibitory prodomain and proteolytic domain. The proenzyme
activation occurs through autocatalytic mechanism implying
intra- or intermolecular hydrolysis of prodomain or by other pro-
teases acting upstream in the proteolytic cascade. PLCPs com-
monly localize in lysosomes or vacuoles being activated and
stable at low pH levels. Despite recent progress in studying of
PLCPs, there is a lack of knowledge concerning in vivo properties
of these proteases, for most of which the enzymatic cascades,
subcellular localization, and specific substrates remain unknown.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 157
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Photosynthesis and plant biochemistry POSTERS
158 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Photosynthesis and plant biochemistry
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 159
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Photosynthesis and plant biochemistry POSTERS
P.06-009-Wed providing the plant with a robust root system. Also, the protein
and total free amino acid content together with the C/N ratio were
Light control of lipid metabolism in the brown
not significantly reduced in comparison with the other two experi-
alga Undaria pinnatifida: consequences for mental groups grown with inorganic forms of N. We determined
chloroplast ultrastructure and photosynthetic an increased concentration of free branched-chain (Ile, Leu) and
competence basic (Arg, His, Lys) amino acids in the plants supplemented with
N. Zhukova1, I. Yakovleva2 casein. Their proteomic analysis of the root exudates revealed three
1
Far Eastern Federal University, Vladivostok, Russia, 2National classes of proteases as being secreted to the medium, and that some
Scientific Center of Marine Biology, Far East Branch, Russian apoplastic proteases in the roots were downregulated, except for
Academy of Sciences, Vladivostok, Russia aleurain-like protease that was significantly upregulated. However,
most of proteins involved in N metabolism remained unchanged.
Light produces a variety of effects on algal lipid metabolism and
We detected that the plants supplemented with casein showed
therefore lipid composition. Glycolipids determine the physico-
inducible proteolytic activity at lower pH than neutral. Their high
chemical properties of the thylakoid membrane that are essential
activity of endo-1,3-b-glucanase in the roots was related to the cell
in supporting the photosynthetic capacity, and, hence, they
wall adjustment resulting in the loosening of the transport path-
should be included into the concept of light acclimation of pho-
ways for metabolites, including peptides and proteins. Taken
tosynthesis. Long-term exposure to varying light intensity (100,
together, our findings suggest that tobacco plants accumulate
290 and 400 lmol photons/m2/s) influenced the lipid and fatty
mainly branched-chain and basic amino acids from casein, secrete
acid composition of U. pinnatifida, as well as on growth rate and
proteases into the medium and break the peptides in the apoplast.
photosynthetic activity. The changes in total lipid content were
This project was supported by Charles University (UNCE 204025/
accompanied by variations in lipid classes. Each lipid class exhib-
2012).
ited a specific fatty acid composition and changed considerably
at a different light intensity. Under adaptation to low light,
besides the increased synthesis of glycolipids, monogalactosyldia- P.06-011-Tue
cylglycerol MGDG, sulfoquinovosyldiacylglycerol SQDG, and
Defining a metabolic core module of stress
phosphatidylglycerol PG we observed also active n-3 desaturation
with the formation of 18:3n-3, 18:4n-3 and 20:5n-3 acids. These response in Arabidopsis thaliana
changes were accompanied by the active synthesis of Chl a+c and urtauer1, A. Pschenitschnigg2, H. Scharkosi2,
L. F€
fucoxanthin pigments as well as a significant increase in the PSII W. Weckwerth2,3, T. Naegele1
1
maximum quantum efficiency and the electron transport rate. In LMU M€ unchen/Dept. Biology I, Munich, Germany, 2University
contrast, the high–light samples displayed a significant decline in of Vienna, Althanstrasse 14, A-1090 Vienna, Austria, 3Vienna
n-3 polyunsaturated fatty acid content, increase in the amounts Metabolomics Center ViMe, Althanstrasse 14, A-1090 Vienna,
of storage lipids, triacylglycerols, rich in 16:0 and 18:0 and a Austria
decline in photosynthetic activity accompanied with increase in Abiotic stress exposure of plants induces metabolic reprogram-
malondialdehyde content. The consequent accumulation of n-3 ming which is a tightly regulated and complex process affecting
fatty acids in glycolipids could facilitate the thylakoid membrane signalling cascades, transcriptional, translational and metabolic
fluidity, and, therefore, the velocity of electron flow involved in regulation. The complexity of resulting interconnected stress-
photosynthesis during light acclimation. Thus, a prolonged expo- responsive networks impedes the functional understanding of
sure to low and high light considerably affects lipid synthesis, molecular plant stress response compromising the design of
suggesting a correlation between the activity of photosystems and breeding strategies and biotechnological processes. Consequently,
the process of synthesis and desaturation of fatty acids. The work defining a molecular network enabling the prediction of a plant’s
was supported by the Russian Science Foundation (14-50-00034). stress mode promises to significantly promote the understanding
of stress-induced biochemical reprogramming and its technologi-
cal application. In the presented study, Arabidopsis wild type
P.06-010-Mon plants and three mutant lines with enzymatic deficiencies in the
What happens with tobacco metabolism if central carbohydrate metabolism were grown under ambient con-
protein casein is the only source of nitrogen? ditions as well as under low temperature/high light stress condi-
y2, H. Synkova3, H. Gielen4,
K. Belonoznıkova1, M. Cern tions. Stress-induced dynamics of metabolome and proteome
R. Valcke4, V. Hyskova1, O. Hodek5, T. Krızek5, were quantified in a mass spectrometry-based high-throughput
R. Schnablov a3, H. Ryslava1 experiment. Together with chlorophyll fluorescence parameters,
1
Department of Biochemistry, Faculty of Science, Charles which indicated a significant stress impact on photosystem reac-
University, Hlavova 2030, Praha, Czech Republic, 2Laboratory of tions, this multidimensional molecular data set was used to train
Plant Molecular Biology, Institute of Biophysics AS CR, v.v.i. and a machine learning algorithm to predict stress behaviour of all
CEITEC — Central European Institute of Technology, Mendel genotypes. Based on multivariate statistics and machine learning
University in Brno, Zemedelsk a 1, Brno, Czech Republic, 3Institute approaches on wild type data, a core module consisting of 20
of Experimental Botany, Academy of Sciences of the CR, proteins was identified enabling the prediction of the stress mode
Rozvojova 313, Praha, Czech Republic, 4Molecular and Physical of all mutant lines. Further, among these protein candidates a
Plant Physiology, Faculty of Sciences, Hasselt University, protein-protein interaction network was identified connecting
Agoralaan building D, Diepenbeek, Belgium, 5Department of transcriptional regulation with regulation of primary and sec-
Analytical chemistry, Faculty of Science, Charles University, ondary plant metabolism. In summary, the identified stress-
Hlavova 2030, Praha, Czech Republic responsive core module defines a tightly regulated molecular net-
work being preliminary for reprogramming of metabolism during
The well-known paradigm that plants must rely on soil fauna for
stress and for the predictability of complex biochemical regula-
the breakdown of organic matter has been challenged by many sci-
tion during environmental fluctuation.
entists now. We took a closer look on what happened with tobacco
metabolism if grown in vitro in sterile conditions with casein as a
sole nitrogen (N) source. Casein promoted the root growth,
160 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Photosynthesis and plant biochemistry
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 161
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Photosynthesis and plant biochemistry POSTERS
not involved in photosynthesis. Their biological function remains xanthophyll energy transfer. The study was funded by RFBR
unclear, but has been proposed to act as a carrier for Chl or Chl (grant no. 18-34-00700).
precursors/derivates in the Chl biosynthesis or metabolism path-
ways. WSCPs are remarkably stable towards high temperature,
harsh pH and protease treatments. Moreover, recombinant P.06-017-Tue
WSCP apoprotein can extract Chl from thylakoid membranes, Structural studies reveal protein dynamics
liposomes, detergent micelles and organic solvents. Upon Chl crucial for protein carotenoid uptake and
binding, the WSCP apoprotein forms a tetrameric complex with delivery
one Chl per apoprotein and no other pigments bound. In this
D. Harris1, A. Wilson2, F. Muzzopappa3, N. Sluchanko4,
study, we investigate the scope of pigments bound and the mech-
T. Friedrich5, E. Maksimov4, D. Kirilovsky3, N. Adir6
anism of the binding reaction in vitro employing recombinant 1
Technion - Israel Institute of Technology (IIT), Haifa, Israel,
WSCP. We developed an approach to monitor the pigment bind- 2
Institute for Integrative Biology of the Cell (I2BC), Gif sur
ing to WSCP by using its visible circular dichroism (Vis-CD).
Yvette, France, 3Institute for Integrative Biology of the Cell
Vis-CD measurements allowed rapid screening of various tetra-
(I2BC), CNRS, CEA–Saclay, Universit e Paris–Saclay, iBiTecS/
pyrroles as potential binding partners of WSCP. The protein was
SBIGEM, Laboratoire Stress Oxydant et Cancer,, Gif-sur-Yvette,
found to bind a broad range of tetrapyrroles including por-
France, 4Department of Biophysics, School of Biology, M.V.
phyrins, chlorines and bacteriochlorins. Furthermore, the kinetics
Lomonosov Moscow State University, Moscow, Russia, 5Technical
of the pigment-protein complex formation was measured by
University of Berlin, Berlin, Germany, 6Technion - Israel Institute
time-resolved Vis-CD. Interestingly, measurements with lipo-
of Technology, Haifa, Israel
somes revealed changes in the binding kinetics depending on the
lipid composition, suggesting that WSCP apoprotein interacts A family of soluble carotenoid proteins having the ability to
strongly with membranes containing negatively charged lipids. transfer its carotenoid to other proteins was recently described.
These results may help to understand the mechanism by which a These proteins are homologs of the C-terminal domain (CTDH)
water-soluble protein is able to extract Chls from membranes. of the Orange Carotenoid Protein (OCP) that is involved in
cyanobacterial photoprotection. CTDHs were suggested to have
a mediating role in a carotenoid transfer mechanism from the
P.06-016-Mon thylakoid membrane to the Helical carotenoid proteins (HCPs),
pH-induced conformational switching in LHCII- which are paralogs of the N-terminal domain of the OCP. In this
SR chimeric protein as a model for regulation work, the three-dimensional structure of a carotenoid-free CTDH
variant from Anabaena (Nostoc) PCC 7120 is presented. Obtain-
of light harvesting in photosystem II of green
ing this structure required the urea-induced disruption of a large,
algae crystallizable oligomeric form of the CTDH. This CTDH belongs
A. Belov, D. Khokhlov to clade 2, which contains a cysteine amino acid at position 103.
Lomonosov Moscow State University, Chemistry Department, Two dimer-forming interfaces were observed – one between the
Moscow, Russia two monomer’s disulfide-bond regions (head-to-head) and the
Stress-related pigment-protein complex LHCSR3 expressed in second between each monomer’s b-sheets domain (back-to-back).
high-light conditions is responsible for regulation of the non- SAXS measurements confirmed that this class of proteins has
photochemical quenching in green algae. It uses pH-sensitive C- two different kinds of dimeric interactions in solution that are
terminus as a switch button upon acidification of the lumen. In consistent with the oligomeric forms seen in the crystal structure.
the recent experiment, the recombinant protein LHCII-SR built In addition, the X-ray structure revealed that in apo-CTDH, the
from the LHCII main antenna by replacing the C-terminus with C-terminal tail (CTT) undergoes a dramatic change in its posi-
the LHCSR3 sequence exhibited pH-dependence of the fluores- tion. Mutational analysis revealed that the CTT facilitates both
cence decay (FD) rate. We studied pH-induced conformational carotenoid uptake and carotenoid delivery, thus playing an
changes in this protein and their impact on the structure of the important role in carotenoid transfer mechanism. Finally, the
exciton states and their properties. The 3D structure of the com- CTT movement seems to occur also in OCP upon photoactiva-
plex was reconstructed using the primary sequence for the tion, suggesting that it could also have a similar role in carote-
LHCII-SR and the tertiary structure for the LHCII. Constant- noid transfer as in CTDH.
pH molecular dynamics was used to reveal stable conformations
in the pH interval from 4.4 to 7.5 responsible for variations of
the FD rate. Exciton energies of the complex for stable confor- P.06-018-Wed
mations were modeled using exciton Hamiltonian method. Diag- Plastidial lysine acetyltransferases of
onal elements of the Hamiltonian were calculated as excitation Arabidopsis and their relevance for
energies of individual pigments; exciton couplings were evaluated photosynthesis
from their transition densities. Those properties were calculated A. Br€unje1, M. Koskela2, I. Lassowskat1, T. Dinh3, M. Wirtz4,
using QM/MM approach with TDDFT for QM part, and polar- W. Bienvenut5, C. Giglione5, D. Schwarzer6, P. Mulo2,
izable force field AMBER02 for MM part. Absorption, I. Finkemeier1
linear, and circular dichroism spectra were modeled based on 1
Westf€
alische Wilhelms Universit€
at M€ unster, M€unster, Germany,
eigenvectors of the Hamiltonian. Homogeneous spectral broaden- 2
University of Turku, Turku, Finland, 3Industrial University of Ho
ing was accounted for by using the Redfield theory, the inhomo- Chi Minh City, Ho Chi Minh City, Vietnam, 4University of
geneous one – by the random shift method. The spectra were Heidelberg, Heidelberg, Germany, 5University of Paris-Saclay,
compared with the experimental ones in order to check the valid- Gif-sur-Yvette, France, 6University of T€ubingen, T€
ubingen,
ity of the computations. Chlorophyll-xanthophyll exciton cou- Germany
plings responsible for FD were compared for conformations
obtained earlier demonstrating significant increase. These results Post-translational modifications, such as lysine acetylation, allow
can be combined in future with theories for quantum dissipative cells to rapidly respond to varying environmental conditions.
dynamics and lead to time-dependent description of chlorophyll- Lysine acetylation has the potential of altering localization, inter-
actions or enzymatic activities of proteins. Specific
162 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Photosynthesis and plant biochemistry
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 163
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Photosynthesis and plant biochemistry POSTERS
(811,811bw;L35, L35bw). Each pair comprised of “waxy” and at the same time even chains were lowered. Differences between
“waxless” line (“bw” suffix) bearing recessive mutation leading to the lines were noticed. The research was funded by the National
disorder in formation of wax coating. All lines were exposed to a Science Center, Poland, under project no. 2015/17 / B / NZ9 /
3-week drought stress, then the flag leaves were cut off, frozen 01694.
and lyophilized. The analysis of SSC and TPC content were con-
ducted. The experiment was carried out in 2 years. During both
years of experiment a decrease of both parameters was induced P.06-023-Tue
by water shortage. Among plants grown in water deficit during Actin depolymerization could result in
the first and the second year, the maximum TPC level was increased plant resistance to pathogens
observed for waxless line L35bw. The results obtained during H. Krutinova1, L. Trda1, T. Kalachova1, L. Lamparova2,
both years revealed significant differences among tested pairs. P. I. Dobrev1, R. Pospıchalova1, K. Malınska1, L. Burketova1,
Each of the differences (12–78%) indicated higher amount of O. Valentova2, M. Janda2
TPC in waxless lines over both treatments and each year of the 1
Institute of Experimental Botany AS CR, v. v. i., Prague, Czech
trial. The SSC content differed between the years. After the first Republic, 2University of Chemistry and Technology Prague,
year of experiment under both treatments waxless lines showed Prague, Czech Republic
higher amount of SSC than their waxy equivalents, line 811bw
had almost 3 times more soluble sugars than line 811. The second The integrity of actin cytoskeleton plays a key role in plant
year showed some changes in comparison to second year, under immunity, both by providing a physical barrier and by its
drought stress line 811bw accumulated higher SSC amount than involvement in the transport of callose, antimicrobial compounds
the waxy 811, whereas L35 exhibited opposite results by lower and cell wall components to the site of infection. Consequently,
accumulation of SSC than the L35. The research was funded by it is generally assumed that actin disruption reduces plant resis-
the National Science Center, Poland, under project no. 2015/17 / tance to pathogen attack. However, in a previous study, we
B / NZ9 / 01694. reported that actin depolymerisation triggers the salicylic acid
(SA) signalling pathway (Matouskova et al. 2014). Increased SA
level in plants is associated with enhanced plant resistance to
P.06-022-Mon pathogen attack. Here we present the attempt to solve this appar-
The mutation disrupting the wax layer ent inconsistency by showing that the relationship between actin
depolymerization and plant resistance is more complex than pre-
formation impact on photosynthetic pigments
viously thought. We investigated the nature of this relationship
content of rye near-isogenic lines under soil using two completely different plant pathosystems: i) a model
drought plant (Arabidopsis thaliana) and a bacterial pathogen (Pseu-
K. Kapłoniak1, A. Noga1, M. Dziurka1, B. Mysk ow2, domonas syringae), and ii) an important crop (Brassica napus)
I. Czyczyło-Mysza1 and its fungal pathogen (Leptosphaeria maculans). We demon-
1
The F. G
orski Institute of Plant Physiology PAS, Krakow, strated that actin depolymerization induces a dramatic increase
Poland, 2West Pomeranian University of Technology Szczecin, in SA levels and that in this case SA is biosynthesized by the iso-
Szczecin, Poland chorismate synthase pathway. In both pathosystems, disruption
Drought caused by soil water deficit is one of the most important of actin filaments led to significantly increased resistance toward
the infection.
constraint for crop improvement all over the world. Rye has a
number of interesting features, such as outstanding cold hardi- References
sek, V., Kocourkova,
Matouskova, J., Janda, M., Fiser, R., Sa
ness, strong disease resistance and valued excellent drought toler-
D., Burketova, L. & Valentova, O. (2014). Changes in actin
ance. The aim of the study was to analyze the differentiation of
dynamics are involved in salicylic acid signaling pathway. Plant
near-isogenic lines (NILs) of rye in terms of selected biochemical
parameters as a result of the mutation disrupting the wax layer Science, 223, 36–44.
formation impact. Two pairs of NILs were tested (811,811bw;
L35,L35bw), each one consisted of the typical line (“waxy”) and P.06-024-Wed
the one with recessive mutation causing disorder in forming
proper wax coating (“waxless”, denoted as “bw” suffix). Tested Arsenic accumulation and speciation in Pteris
lines were exposed to a 3-week drought stress, then the wax layer cretica and Pteris straminea
was collected, later on weighted (mg per g of leaf Drought M. Popov1, V. Zemanova2, P. Kotrba1, M. Pavlık2,
Weight) and analyzed by GC-FID. The flag leaves were cut off, D. Pavlıkova3
1
frozen and lyophilized for photosynthetic pigments content Department of Biochemistry and Microbiology, University of
(PPC) analysis. All lines subjected to drought stress reacted by Chemistry and Technology, Prague, Czech Republic, 2Isotope
lowering the PPC level: the concentration of chlorophyll a (Chl Laboratory, Institute of Experimental Botany, Academy of
a), chlorophyll b (Chl b), total chlorophyll a+b (TChl) and caro- Sciences of the Czech Republic, Prague, Czech Republic,
3
tenoids (Car). The differences between waxless and waxy lines Department of Agro-Environmental Chemistry and Plant
under water deficit was observed within all parameters and lines, Nutrition, Faculty of Agrobiology, Food and Natural Resources,
biggest variety occurred between the concentrations of Chl a. Czech University of Life Sciences, Prague, Czech Republic
Waxless line 811bw demonstrated significantly higher values of Arsenic (As) contaminated soils are becoming major global envi-
Chl a, Chl b and TChl than the waxy 811, whereas Chl a and ronmental and human health risks due to ubiquity of As and its
Car content was higher for the waxy line of pair L35,L35bw. The carcinogenicity. The identification of natural hyperaccumulators
wax content varied among lines, from 13 to 15 mg/g DW and of As opens the door for phytoremediation of the arsenic con-
from 12 to 22 mg/g DW under control and reduced soil moisture taminant and sparked the research interest in the biology of As
conditions, respectively. Almost all lines increased wax content in (hyper)accumulating ferns. Here we report on As accumulation
under drought conditions. The wax composition stated as hydro- and speciation analysis in two cultivars of Pteris cretica and in
carbon chain length showed higher accumulation of odd chain Pteris straminea. The concentrations of As accumulated in ferns
hydrocarbons by lines bearing mutation under both conditions, grown in greenhouse in soils amended with 20, 100 and 250 mg
164 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biotechnology
As per kg dry weight (dwt) followed the order P. cretica cv. Whereas the catalase (CAT) activity increased on 7th day of expo-
Albo-lineata >> P. straminea ≥ P. cretica cv. Parkerii; As in roots sure to cadmium at normal temperature and in combination with
and fronds of cv. Albo-lineata was more than 10 times greater heat and dropped under combination of cadmium with low tem-
than in P. straminea. The highest As concentrations were perature. Moreover guaiacol - dependent peroxidase (G-POD)
observed in cv. Albo-lineata grown for 90 days in the presence of activity did not changed under combination cadmium with heat
250 mg As per kg. The extracts obtained from disrupted cells of and increased at cadmium and its combination with cold. Under
roots and fronds of investigated ferns were fractionated by using all stress conditions on 7th day of experiment the decrease in hy-
the size exclusion chromatography, followed by ICP-OES and drogen peroxide content was observed that can be a result of
RP-HPLC analyses of eluted As species. These analyses identified effective antioxidant enzymes activation. It was shown that com-
all As eluting in late fractions corresponding to unbound As. bine effect of cadmium and unfavorable temperatures did not have
Our analyses excluded the possibility of As binding with peptida- additive negative effect on some parameters. Due to involvement
ceous ligands, such as phytochelatins, and indicate that investi- of the general mechanisms of tolerance, in particular the activation
gated ferns deposited As in cells as inorganic or methylated of antioxidant enzymes, the plant realizes a protective cascade
species, irrespective of their accumulation status. This study was reactions directed at the survival of plants under these conditions.
supported by Czech Science Foundation, Grant No. 17-10591S.
Biotechnology
P.06-025-Mon
Ubiquitination activities of SPL1 and SPL2, P.07-001-Mon
two E3 ligases of chloroplast outer membrane Surface functionalization dependent
A. Szczurek1, W. Białek2, A. Szczepaniak2 subcellular localization of superparamagnetic
1
Uniwersytet Wroclawski, Wroclaw, Poland, 2Faculty of nanoparticle in plasma membrane and
Biotechnology, University of Wroclaw, Wroclaw, Poland
endosome
Ubiquitination plays a vital role in defining a faith of a protein, D. B. Thimiri Govinda Raj1, N. Ali Khan2
1
thus affecting growth and development of a plant. Ubiquitination NCMM-EMBL, Oslo, Norway, 2KU Leuven, Leuven-Heverlee,
depends on the activity of three enzymes: E1 activator, E2 conju- Belgium
gating enzyme and E3 ubiquitin ligase. The presence of different
In our research, we elaborate the application of thermal decompo-
E3 ligases have been confirmed in many eukaryotic organelles,
sition based Fe3O4 superparamagnetic nanoparticle (SPMNP) for
but only recently SP1, a RING-type ubiquitin E3 ligase, has been
subcellular fractionation in cell biology context. Here, we used
discovered in the chloroplast outer membrane of a model plant
thermal decomposition based seed mediated growth for the synthe-
Arabidopsis thaliana (Ling & Jarvis, 2012). The protein regulates
sis of 8 nm Fe3O4 core SPMNP. We performed surface functional-
the TOC (translocon on the outer chloroplast membrane)
ization of SPMNP with phospholipids (Pl) and dimercaptosuccinic
machinery in response to abiotic stresses by UPS (ubiquitin-pro-
acid (DMSA). Surprisingly, we observed surface functionalization
teasome system) (Ling & Jarvis, 2015). In addition, two homo-
dependent SPMNP localization in subcellular compartments such
logs of SP1, SPL1 and SPL2, have also been identified. To
as plasma membrane, endosomes and lysosomes. By using
confirm their putative enzymatic activities we heterologously
SPMNP based subcellular localization with pulse-chase methodol-
expressed both proteins and conducted in vitro ubiquitination
ogy, we could use SPMNP for high pure-high. Yield organelle
assays. We also present our approach to identify their substrates
(plasma membrane, endosomes and lysosome) fractionation. Previ-
and thus their physiological role in the plant cell.
ously, several groups have used SPMNP based subcellular fraction-
ation to isolate endosomes and lysosomes with high purity-yield
P.06-026-Tue for subcellular omics. For example using Dextran coated SPMNPs,
several research groups were able to decipher endosomal traffick-
Effect of cadmium and its combination with
ing in lysosomal storage disorders. Due to generic nature of
low and hight temperature on activity of SPMNP, our methodology has additional advantage in isolating
antioxidant enzymes in wheat subcellular compartments such as plasma membrane, endosome
N. Repkina, A. Ignatenko, V. Talanova and lysosome from any given adherent cells. With additional opti-
Institute of Biology Karelian Research Centre of Russian Academy mization step, our methodology could be applied for suspension
of Sciences, Petrozavodsk, Russia cells. In addition, our methodology does not include any acidic
treatment or antibody based pulldown and subcellular compart-
Wide range stresses directly or indirectly lead to development of
ments are isolated under native physiological conditions. Hence,
oxidative stress in plants. Plants possess a number of protective
this methodology would facilitate enzymatic studies, isolating
mechanisms, including activation of enzymatic and non-enzy-
intact membrane protein complexes and structural stud-
matic compounds of antioxidant system that serve to neutralize
ies. Thus, SPMNP that are distinctly localized in subcellular com-
reactive oxygen species (ROS) and prevent oxidative stress. The
partments can be used as technology for subcellular fractionation
comparative study of antioxidant enzymes activity in wheat
that can complement existing tools for cell biology research. As a
plants in response to cadmium (Cd) influence in combination
future perspective, our methodology can be used to isolate subcel-
with normal (22°C), low (4°C) and high (37°C) temperatures was
lular compartments in primary cells and can be extended to in vivo
performed. 7-days-old wheat seedlings (cv. Moskovskaya 39) sub-
analysis for biochemical and structural biology studies.
jected to cadmium (100 lM) treatment or its combination with
low (4°C) of high (37°C) temperature for 1 week. According to
the results of electrolyte leakage, MDA content, Cd accumulation
in wheat leaves, it was found that in combination of cadmium
with low or high temperature, the temperatures have dominant
effect, and reaction of plants was similar with the response to sep-
arate effect of cold or heat. The activity of superoxide dismutase
(SOD) increased in wheat leaves under all stress conditions.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 165
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biotechnology POSTERS
166 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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P.07-006-Wed P.07-008-Tue
Efficient DNA-free nanoparticle mediated Identification of major bee venom allergen Api
genome editing of potato using CRISPR-Cas9 m 1 IgE epitopes and characterization of their
RNP complex corresponding mimotopes
A. Zahirovic1, A. Koren2, P. Kopac2, J. Luzar1, B. Strukelj1,
A. Khromov1,2, V. Makarov1,2, A. Makhotenko1,2,
P. Korosec2, M. Lunder1
S. Makarova1,2, E. A. Snigir2, T. P. Suprunova2, 1
Faculty of Pharmacy, University of Ljubljana, Ljubljana,
N. O. Kalinina1,2, M. E. Taliansky2,3
1 Slovenia, 2University Clinic of Respiratory and Allergic Diseases
Lomonosov MSU, Moscow, Russia, 2DokaGene Ltd, Rogachevo,
Golnik, Golnik, Slovenia
Russia, 3The James Hutton Institute, Dundee, United Kingdom
Immunotherapy with bee venom extracts is associated with high
Due to its simplicity, practicality and efficiency CRISPR–Cas9 is
rate of side effects including severe systemic reactions. Short pep-
rapidly developing as the emerging technology for genome edit-
tides that mimic IgE epitopes (mimotopes) offer an opportunity
ing on various eukaryotic systems including higher plants and
for development of safer immunotherapeutics. Due to their small
animals. Here we describe new approach for delivery of pre-
size, they do not possess the ability to cross-link IgEs on effector
assembled Cas9–sgRNA ribonucleoproteins into dissected potato
cells. Moreover, in combination with a suitable immunogenic car-
(Solanum tuberosum, cv Chicago) shoot apical meristems by chi-
rier, they are able to stimulate T cell immune response. Peptides
tosan nanoparticles using vacuum infiltration and regeneration of
were selected from phage-displayed random peptide libraries and
plants with edited alleles. As a model gene for genome editing,
used to computationally map IgE epitopes. Peptide mimotopes of
we have selected a gene encoding coilin, a major protein of Cajal
identified epitopes were synthesized. Additionally, peptide mimo-
bodies(CBs). In additional to its traditional role in assembly of
topes fused to bacteriophage coat protein pIII were isolated from
CBs, coilin also participates in biotic (virus attack) and abiotic
Escherichia coli periplasm and tested for IgE binding with sera of
(high salinity) plant stress responses. Using this method of
bee venom-allergic patients in the immunodot assay. Basophil
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 167
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168 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 169
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170 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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P.07-018-Wed Detection limit of the probe is about 5 ng/mL and detection sen-
sitivity could be significantly increased using spacer-enhanced
Quantitation of alpha-ketoglutaric acid in
peptides, which designed with the concepts based on the length
athletic supplements by using D-phenylglycine and charge of peptides. The chymase reaction would be obvi-
aminotransferase ously inhibited by adding a specific chymase inhibitor, chymo-
T. Rojanarata1, W. Laiwattanapaisal2, T. Ngawhirunpat1, statin. The specificity of AuNPs-peptide probe was also identified
P. Opanasopit1 by trypsin and chymotrypsin reactions. The AuNPs-peptide
1
Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, probe has been applied in detecting chymase activity as an indi-
Thailand, 2Faculty of Allied Health Sciences, Chulalongkorn cator of nephropathy in a mouse model induced by aristolochic
University, Bangkok, Thailand acid I (AAI) challenge. The probe was successfully used for the
Alpha-ketoglutaric acid (AKG) is a biologically important com- detection of renal chymase activity, and the results show that
pound acting as an intermediate in Krebs cycle, a substrate for pathogenically increased chymase activity in the kidney tissue of
the synthesis of glutamic acid and glutamine, and a transporter nephropathic mice treated with AAI. This is the first study using
of intracellular nitrogen to the urea cycle. Currently, supplements an AuNPs-peptide probe for the detection of chymase activity.
containing AKG are commercially available e.g. capsules and
used for building muscle and maintaining the balance of nitrogen
P.07-020-Tue
or ammonia levels in the human body. To determine AKG con-
tents in these products, a novel method was developed based on Identification of novel genes coding for cold-
the conversion of AKG to a product with high absorbance active glycosidases with biotechnological
namely 4-hydroxybenzoylformic acid using D-4-hydroxyphenyl- potential
glycine as a co-substrate and D-phenylglycine aminotransferase P. Vodickova, E. Benesova, P. Lipovova
(D-PhgAT) as a catalyst. By this route, the initial rates of the UCT, Prague, Czech Republic
reaction which were directly proportional to AKG concentrations
Due to many unique properties of cold-active enzymes such as
were spectrophotometrically measured at 25°C by the increase of
high catalytic efficiency at low temperatures associated with a
absorbance at 334 nm during the first 30 s and used for the con-
high thermosensitivity, they have a significant potential to partici-
struction of standard curve. The method showed good linearity
pate in various industrial processes. In this work, we were search-
(AKG of 20–160 lM, r2 = 0.9994) with the limits of detection
ing for genes coding for new cold-active amylases and cellulases
and quantitation of 4.09 and 13.62 lM, respectively. It was accu-
from cold-adapted microbial strains. In the previous experiments,
rate, precise and repeatable and free from the interference by the
activities of the selected glycosidases at low temperatures were
excipients in capsules. Furthermore, it gave the results in agree-
tested in different samples containing microorganisms originating
ment with HPLC method. Hence, the proposed assay is an effi-
from various cold areas. Due to the obtained temperature pro-
cient and facile alternative method for the quality control of
files, two yeast (Mrakiella aquatica and Cryptococcus victoriae)
these products.
and one bacterial (Arthrobacter sp. C1-1) strains, showing the
highest amylase or cellulase activities at low temperatures, were
P.07-019-Mon chosen for the identification of responsible genes in their gen-
omes. First, gene-specific (GS) primers were designed based on
Detection of chymase activity using the
the similarities found in available sequences of amylases or cellu-
AuNPs-peptide probe, a specific peptide probe lases from the organisms most phylogenetically related to the
conjugated onto gold nanoparticles chosen isolates. The microbial strains were then cultivated at 15
Y. Sun1, F. Yeh2, I. Tseng2, C. Chang3, C. Chen4, C. Lin2 °C and used for the isolation of bacterial genomic DNA (gDNA)
1
Aquatic Technology Laboratories, Agricultural Technology or yeast RNA. For the amplification of bacterial target genes, we
Research Institute, Hsinchu, Taiwan, 2Department of Biological performed PCR reactions using the GS primers and the gDNA
Science and Technology, National Chiao Tung University, as a template. In the case of the yeasts, a template DNA was
Hsinchu, Taiwan, 3Division of Nephrology, Department of Internal obtained by reverse transcription of the isolated RNA. The com-
Medicine, Changhua Christian Hospital, Changhua, Taiwan, bination of 3’RACE and 5’RACE methods was used to sequen-
4
Division of Nephrology, Department of Internal Medicine, tially amplify the whole sequence of target genes using the set of
Hsinchu Mackay Memorial Hospital, Hsinchu, Taiwan GS primers and the prepared DNA. At the moment, we are still
Gold nanoparticles (AuNPs) with the characteristics of fluores- in the process of mapping the gene sequences. In future, the
cence resonance energy transfer have the ability of quenching flu- cold-active glycosidases coded by the identified genes will be pro-
orescence with distant dependence. Based on this, we have duced in the form of recombinant proteins and deeply character-
established a sensitive and efficient biosensing method by modify- ized.
ing peptide-probe onto the AuNPs to detect enzyme activity. The
method is designed for chymase activity detection and applied in
P.07-021-Wed
renal disease diagnosis. In this study, 15-nm AuNPs were used to
establish the AuNPs-based fluorescence peptide probe (AuNPs- Effective T2R and G alpha co-expression
peptide probe). The peptide sequence is FITC-Acp- system using IRES bicistronic vector
DRVYIHPFHLDDDDDC which contains a fluorophore at the I. Shimomura, M. Jotaki, T. Yamamoto
C-terminal end, enzyme (chymase) substrate (DRVYIHPFHL), a Japan Tobacco Inc., Yokohama, Japan
spacer (DDDDD) and a cysteine (C) used for conjugating to Human taste receptor type 2 (T2R) consists of 25 members
AuNPs. When the enzyme catalyzed the substrate sequence, fluo- belonging to the GPCR superfamily, which mediates bitter taste
rophore drifted away from AuNPs and the fluorescence emitting perception and protection against xenobiotic substances. The
signal can be excited at 485 nm and detected at 515 nm. The binding of ligands to T2R causes the increase in intracellular cal-
results show, detection time of the AuNPs-peptide probe used for cium level via activation of G protein a subunit (Ga) in taste
activity detection of chymase is only needed 15 min and a linear cells. Thus, T2R and Ga co-expressing cells have been used to
correlation from 10 to 120 ng/mL of chymase is acquired. evaluate the function of T2R in vitro. To date, several
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 171
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biotechnology POSTERS
172 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 173
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biotechnology POSTERS
conjugates consist of polymer particle that contains on its surface their basis. Two main methods are employed for the preparation
specific bio-ligands capable of affinity binding to a detectable of nucleoside analogues. One of them is based on the modifica-
component (antigen, antibody). We took the IgG antibody as a tion of natural compounds. In another one, heterocycle or
bioligand. The immune activity of this biomolecule depends on monosaccharide are modified followed by the formation of N-
how the polymer attaches to antibody and whether the active site glycosidic bond. Enzymatic methods for the formation of a gly-
of the protein is blocked. Therefore, it was proposed to immobi- cosidic bond complement chemical procedures and, in several
lize gold nanoparticles on the antibody surface in various concen- cases, have obvious advantages. We found that 7-methyl-20 -deox-
trations to select the optimal conditions for conjugate creation. It yguanosine (7MedGuo) is an efficient and novel donor of the 2-
is expected that, due to their small size, they will not block the deoxyribose moiety in the enzymatic transglycosylation for the
antibodies active sites. Using the chemical reduction of tannin, synthesis of purine and pyrimidine 20 -deoxyribonucleosides in
we obtained gold nanoparticles of size 40 nm. To prepare excellent yields. 7MedGuo + Ura ↔ 7MeGua + dUrd. We have
biononconjugates gold nanoparticles were mixed with antibodies studied the stability of 7MedGuo at various pH values, different
to thyroglobulin in various concentrations. These conjugates were temperatures, as well as a reagents’ ratios (7-Me-dGuo: Base:
studied using the Transmitting Electron Microscope. The pho- phosphate) in order to find optimal enzymatic transglycosylation
tographs show that nanoparticles on the surface of the protein conditions. Using the developed methodology several practically
are so small that they do not block its active site and do not important purine and pyrimidine 20 -deoxyribonucleosides were
interfere with its normal functioning. The publication was sup- prepared in high yields. This work was supported by Russian
ported of the “RUDN University Program 5-100” and the Min- Science Foundation (project No. 16-14-00178)
istry of Education and Science of the Russian Federation (the
Agreement No. 02.A03.21.0008)
P.07-030-Wed
Functional and structural characterization of a
P.07-028-Mon novel low molecular weight asparaginase
Bioethanol production by consortium of L. Schultz1, C. Angelica Ottoni1, P. Leo2, A. Pessoa-Jr3,
Trichoderma reesei and yeast from cellulose G. Monteiro4, M. A. de Oliveira1
1
B. S. Kim, J. Zheng, A. Deshmukh, P. Saha UNESP - S~ ao Paulo State University -Biosciences Institute -
Chungbuk National University, Cheongju, South Korea Coastal Campus, S~ ao Vicente, Brazil, 2Institute for Technological
Research, S~ao Paulo, Brazil, 3University of S~ao Paulo, S~
ao Paulo,
Industrial cellulosic ethanol production is still a challenge due to
Brazil, 4Universidade de S~
ao Paulo, S~ao Paulo, Brazil
the high cost of cellulase for hydrolysis after using lignocellulosic
materials as feedstock. In this study, bioethanol was produced Bacterial asparaginases (ASNAse) from Escherichia coli and
from cellulose by consortium of Trichoderma reesei and yeasts Erwinia chrysanthemi are high molecular weight tetrameric
(Candida molischiana or Saccharomyces cerevisiae). Different con- enzymes (140 kDa) considered as gold biopharmaceuticals largely
centrations of cellulose (Avicel) was hydrolyzed by a fully enzy- used in hematological malignancies as acute lymphoblastic leuke-
matic saccharification process using T. reesei. The reducing sugar mia (ALL). In ALL the neoplastic cells are unable to synthesize
produced during the process was further used as carbon source adequate amounts of asparagine (Asn) and are dependent of
for bioethanol production using C. molischiana or S. cerevisiae. extracellular sources. The ASNAse administration depletes Asn
C. molischiana could utilize both glucose and cellobiose, while S. from serum, resulting in the death of the neoplastic cells by Asn
cerevisiae could utilize only glucose. Consortium of T. reesei with starvation. Meanwhile, several side effects are associated with
two yeasts revealed that C. molischiana led to efficient bioconver- their administration, including immunological reactions, neuro-
sion of reducing sugars to ethanol than that of S. cerevisiae. The toxicity and coagulation abnormalities. The high molecular
maximum reducing sugar, glucose, and ethanol yields were 42%, weight of these enzymes (140 kDa) and a secondary glutaminase
26%, and 20%, respectively, from 20 g/L Avicel. At higher Avi- activity are frequently related to the side effects and the search
cel concentration of 50 g/L, the maximum concentrations of for novel enzymes with better characteristics is important.
reducing sugar, glucose, and ethanol were 10.9 g/L, 8.57 g/L, Recently, using molecular biology techniques we characterize a
and 5.95 g/L, respectively, with consortium of T. reesei and C. novel asparaginase named ASNAseM. Size exclusion chromatog-
molischiana. raphy experiments revealed a monomeric enzyme presenting low
molecular weight (~ 45 kDa) and circular dichroism spectroscopy
analysis revealed an alpha/beta structure containing ~ 34% of
P.07-029-Tue beta sheets and 16% of alpha helix. Thermal analysis also
Use of 7-methyl-2’-deoxyguanosine for the showed that ASNAseM is thermal resistant, maintaining the sec-
preparation of 20 -deoxynucleosides ondary structure in temperatures higher than 50°C. Through bio-
C. S. Alexeev, M. S. Drenichev, S. N. Mikhailov chemical assays we determined the kinetic parameters as the
Engelhardt Institute of Molecular Biology, Russian Academy of catalytic efficiency over asparagine as ~ 1.08 9 104 M1 s1 and
Sciences, Vavilova Str., 32, 119991, Moscow, Russia that the enzyme presents allosteric behaviour with S0.5 to Asn
determined as ~ 0.35 mM. Additionally, biochemical approaches
Enzymes are widely used in industry for carrying out various revealed that the enzyme is free from glutaminase activity.
transformations and producing useful substances and materials. Finally, preliminary cytotoxicity assays using HUVEC (normal
Enzymatic transglycosylation - a transfer of the carbohydrate cells) and MOLT4 (leukemic cells) revealed that the ASNAseM
moiety from one heterocyclic base to another - is being actively induced apoptosis in leukemic cells. Together the data presented
developed and applied for the synthesis of biologically active here indicates that ASNAseM is a promising enzyme to be used
nucleosides. This reaction is catalyzed by nucleoside phosphory- as a biodrug in ALL. Financial Support: FAPESP.
lases (NPs), which are responsible for reversible phosphorolysis
of nucleosides (Nuc) to yield the corresponding heterocyclic bases
and monosaccharide 1-phosphates. Nuc + pi ↔ Sug-p + Base
Nucleoside area is one of the most fruitful part of medicinal
chemistry. About 100 nucleoside-based drugs were developed on
174 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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176 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 177
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FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 179
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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strain produced up to 11.4 g/L of 3HP under fed-batch condition 6. Thus, comprehensive analysis of metabolites and proteins
from cellobiose with a yield of 13% (g-3HP/g-sugar). excreted by Rhodococcus sp. S10 under iron-limited conditions
will reveal the mechanisms of adaptation to geochemically
extreme conditions and their potential role in the serpentinite
P.07-047-Tue biomineralization. This work was performed within the Program
Metabolic design of Escherichia coli for of Competitive Growth of Kazan Federal University and sup-
muconic acid production ported by RFBR (grant no. 16-34-60200) and the scholarship of
R. Fujiwara1, S. Noda2, T. Tanaka1, A. Kondo1,2 the President of the Russian Federation for young scientists and
1
Kobe University, Kobe, Japan, 2RIKEN, Yokohama, Japan graduate students.
180 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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P.07-050-Tue P.07-052-Mon
Ligands in the active site of carbohydrate Purification and properties of insulin receptor –
oxidase from Microdochium nivale related receptor ectodomain expressed in
P. Kolenko1,2, J. Duskova2, M. Maly1,2, T. Koval’2, T. Skalova2, mammalian cell culture
L. H. Østergaard3, J. Dohnalek2 A. Mozhaev1,2, I. Deyev1, A. Orsa1, A. Gavrilenkova1,
1
FNSPE CTU, Prague, Czech Republic, 2Institute of D. Kaluzhny3, A. Petrenko1
Biotechnology CAS, v.v.i., Vestec, Czech Republic, 3Novozymes 1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry
A/S, Bagsvaerd, Denmark Russian Academy of Sciences, Moscow, Russia, 2A.V. Shubnikov
The carbohydrate oxidase from Microdochium nivale is a flavoen- Institute of Crystallography of Federal Scientific Research Centre
zyme with bicovalently linked FAD molecule. The enzyme is “Crystallography and Photonics” of Russian Academy of Sciences,
preferentially active towards disaccharides such as cellobiose and Moscow, Russia, 3Engelhardt Institute of Molecular Biology
lactose. The enzyme activity is attractive for application in indus- Russian Academy of Sciences, Moscow, Russia
trial processes. We investigated binding of various saccharide The Insulin receptor-related receptor (IRR) was originally discov-
substrates in the active site of the enzyme using single crystal ered due to its high homology to the other family members (in-
diffraction analysis. We observed multiple modes of enzyme:li- sulin receptor and insulin-like growth factor 1 receptor),
gand interactions. Moreover, we have observed two additional however, no endogenous ligands of a peptide or protein nature
ligand binding sites on the protein surface that have not been have been found for IRR. We determined that IRR can be acti-
observed previously. Surprisingly, high resolution crystal struc- vated by mildly alkaline extracellular media and has typical fea-
tures of old crystals (more than 6 months) shows specific FAD tures of the ligand-receptor interaction, including its specificity
degradation that has not been observed before. Nevertheless, the and dose dependence. Since pH-sensitive properties of IRR are
old enzyme is capable of substrate binding and is very likely to determined by its ectodomain; therefore, we chose as option
be still active. Analysis of all available crystal structures of carbo- study the soluble an extracellular portion IRR. Based on the
hydrate oxidase:ligand complexes together with crystal structures CHO-K1 cell line, an effective system of constitutive heterolo-
of functionally related proteins revealed the key residues in the gous expression of IRR ectodomain was developed, which pro-
active site that modify the enzyme specificity. This publication vides a yield of 0.6 mg of recombinant protein per liter of
was supported by MEYS CR (LM2015043 CIISB), by the ERDF conditioned media. We determined optimal conditions for culti-
fund (CZ.02.1.01/0.0/0.0/16_013/0001776), and by GA CTU vation of the cell line were selected with the replacement of the
(SGS16/246/OHK4/3T/14). nutrient medium for serum-free. A purification protocol has been
developed that allows obtaining IRR ectodomain with a purity
of more than 99%, according to the results of analytical gel fil-
P.07-051-Wed tration and gel electrophoresis. The identity of the protein was
Bio-active polycaprolactone nanofibers as a confirmed by the Western blot analysis and MALDI mass spec-
potential fluoxetine filter trometry. It was also shown that approximately 25% of the mass
€
H. Taslak, O. Ozcan, B. G€urel-G€
okmen, T. Tunali-Akbay of recombinant IRR ectodomain is comprised carbohydrates.
Marmara University, Istanbul, Turkey The analysis of IRR ectodomain by circular dichroism, repro-
ducible conformational changes have been shown upon changes
Fluoxetine is a widely used serotonin reuptake inhibitor for the
of the solution pH. The spectra suggest that the structure of the
treatment of postpartum depression. Adverse effects such as
protein corresponds predominantly to stacking with b-sheet, and
colic, fussiness, and drowsiness have been reported in some
at a wavelength below 260 nm the absolute ellipticity of the ecto-
breastfed infants of fluoxetine using mothers. In this study, bio-
domain IRR at alkaline pH is lower than at neutral. The
active polycaprolactone nanofiber was developed for the removal
obtained spectra correlate with the conformational changes
of fluoxetine from the milk. The efficiency of the fluoxetine filter
observed in insulin receptor ectodomain, IRR close homolog,
was tested by using spiked goat milk samples. Goat milk is used
upon its activation by insulin. This work was financially sup-
instead of breastmilk, as its content is nearly similar to the
ported by the Russian Science Foundation (grant N_ 14-50-
breastmilk. Fluoxetine was added to the goat milk in 25, 50, 75
00131).
and 100 ng/mL concentrations. Filter is incubated with the
spiked milk samples for fluoxetine removal. Lactose, creamatocrit
and protein levels were also measured to determine the milk com- P.07-053-Tue
position before and after the incubation. Within-run repeatability
Multimodal magnetic-luminescent
was tested with the same branded goat milk for ten days. Fluox-
etine levels were determined with ELISA method following incu- nanocomplexes based on upconversion
bation. The fluoxetine filter effectively removed fluoxetine from nanoparticles for theranostics
milk and did not significantly change the milk composition. As a P. Demina1,2, N. Scholina3, D. Khochenkov4, I. Asharchuk1,
conclusion, different types of bio-active nanofibers can be devel- V. Rocheva1, B. Chichkov5, A. Generalova2, E. Khaydukov1,3
1
oped for the sensing of chemicals found in the breastmilk. Scientific Research Centre “Crystallography and Photonics” RAS,
Moscow, Russia, 2Shemyakin-Ovchinnikov Institute of Bioorganic
Chemistry RAS, Moscow, Russia, 3I.M. Sechenov First Moscow
State Medical University, Moscow, Russia, 4Federal State
Scientific Institution N.N. Blokhin Russian Cancer Research
Center, Moscow, Russia, 5Laser Zentrum Hannover, Hannover,
Germany
The development of synthesis technology made it possible to
obtain new non-toxic luminescent nanoparticles with anti-Stokes
photoluminescence, also known as upconversion nanoparticles
(UCNPs). Such particles are able to convert near-infrared
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 181
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biotechnology POSTERS
radiation (NIR) (975 nm), falling into the “transparency win- spheroids – spherical and ellipsoidal morphology with a necrotic
dow” of biological tissues, into the photons of the visible and center induced by hypoxia. Acknowledgements to PN-III-P2-2.1-
ultraviolet spectral range, avoiding the autofluorescence of the PED-2016-0904 project.
tissues and significantly improving the sensitivity of detection.
The unique optical properties of anti-Stokes nanoparticles allow
us to consider them as a universal platform for multimodal P.07-055-Mon
nanocomplexes, whose biocompatibility and biological inertness Heterologous expression of bacterial phytase
remain under activation by light. Such complexes provide a from Pantoea sp. 3.5.1 in Pichia pastoris
unique functional system for a deep photodynamic and pho- D. Troshagina, A. Suleimanova, M. Sharipova
tothermal therapy, as well as for multimodal diagnosis of tumors. Kazan (Volga Region) federal university, Kazan, Russia
We prepared magnetic-luminescent UCNP, suitable for in vivo
applications, by embedding magnetite in polymer shell based on Phytase catalyzes the stepwise removal of phosphate from phytic
an amphiphilic copolymer poly(maleic anhydride-alt-1-octade- acid. Microbial phytases are the most commonly used feed addi-
cene) for UCNP surface hydrophilization. Multimodal nanocom- tive for monogastric animals. However no single phytase can per-
plexes were also obtained by incorporating magnetic and UC form effectively under various conditions of digestive systems
nanoparticles simultaneously in preformed polystyrene particles. and dietary compositions. That’s why there is a constant search
Third approach was based on magnetite encapsulation into poly- of new phytase-producers and efficient systems for phytase
mer shell on the UCNP surface formed by NIR-induced pho- expression. The methylotrophic yeast Pichia pastoris has been
topolymerization using UCNP luminescence as internal source used for several years as effective expression system of heterolo-
for initiator activation. The enhanced permeability and retention gous protein production. The aim of this study was to clone and
effect (EPR), which was increased due to application of the exter- express phytase from Pantoea sp. 3.5.1 in P. pastoris. The
nal magnetic field, enabled accumulation of magnetic-luminescent sequence of histidine acid phytase gene of Pantoea sp. 3.5.1
nanocomplexes in tumor. Nanocomplexes were injected intra- (agpP) was used. For the correct expression in yeast the codon-
venously while neodymium magnet was attached to the tumor. optimization of the nucleotide sequence was performed. Cloning
Efficacy of nanoparticle accumulation in the tumor increased of bacterial gene into integrative yeast vectors pPINK-HC and
more than two times. We developed a new strategy for pho- pPINK-LC was carried out by three-way ligation of a vector, a
tothermal therapy using magnetic-luminescent nanocomplexes. phytase gene and a signal sequence of Kluyveromyces maxianus
inulinase gene. Genetic constructs containing agpP gene under
the control of AOX1 promoter were obtained and transformed
P.07-054-Wed into P. pastoris cells by electroporation. The expression of bacte-
Generation of 3D in vitro breast cancer models rial phytase in yeast culture media was detected by immunoblot-
ting and analysis of phytase activity. After screening for the
by different techniques and their biological
highest phytase activity, one colony was selected for shake flask
characterization expression. After 48 h of methanol induction, culture showed an
M. A. Badea, M. Balas, A. Dinischiotu extracellular phytase activity of 0.82 U/mL. Recombinant phy-
University of Bucharest, Faculty of Biology, Department of tase was isolated and purified by affine chromatography. The
Biochemistry and Molecular Biology, 91-95 Splaiul Independentei, study of effect of temperature on the stability of the enzyme
Bucharest, Romania showed increase in thermal stability of recombinant phytase in
The aim of this study was to generate multicellular 3D breast comparison with the native enzyme, which might be associated
cancer spheroids using different culture techniques and to charac- with glycosylation of the protein by yeasts. This work was per-
terize their biological properties in order to be used as models formed in accordance with the Russian Government Program of
for cancer research purposes. Triple negative breast cancer cell Competitive Growth of the Kazan Federal University and sup-
line MDA-MB-231 was used to generate spheroids by hanging ported by Russian Science Foundation (project no. 16-16-04062).
drop and liquid overlay (single and multiple spheroids) tech-
niques using different cell densities (500 – 8000 cells/spheroid).
The morphology and cellular viability were evaluated up to P.07-056-Tue
7 days through LIVE/DEAD assay. Spheroids’ diameters were Antimycoplasmal activity of the enzyme
also measured over time. Hypoxia (Nrf2 and Hsp70) and prolif- L-lysine-a-oxidase
eration capacity (PCNA) were assessed by Western blot analysis. N. A. Shevkun, I. P. Smirnova, E. V. Neborak, S. P. Syatkin,
Reduced glutathione (GSH) content and the level of lactate dehy- G. I. Myandina, T. A. Lobaeva, E. V. Kharlitskaya,
drogenase (LDH) were analyzed by colorimetric methods. The T. V. Maksimova
breast cancer spheroids’ morphology was characterized by a high Peoples’ Friendship University of Russia (RUDN University),
compaction of cells. Therefore, the diameter of spheroids gener- Moscow, Russia
ated by hanging drop and liquid-overlay-single spheroid tech-
The concentrate of cultural liquid of Trichoderma harzianum
niques was dependent by cell density (~ 500 lm for 5000 cells)
Rifai being producers of the enzyme L-lysine-a-oxidase was
and decreased over time, while those generated through liquid-
gained after cultivation in the equipment of Experimental techno-
overlay-multiple spheroids technique had various diameters (150
logical installation of the G.K. Skryabin Institute of Biochemistry
- 550 lm). The biological evaluation showed that breast cancer
and Physiology of Microorganisms (Pushchino, Russia). The con-
spheroids develop a hypoxic center over time with low cellular
centrate has an activity 0.54–0.56 U/mL. The influence of this
viability and high release of LDH in a time-dependent manner,
concentrate on the growth of mycoplasmas has been studied for
the highest level of LDH being obtained for the liquid-overlay-
the first time: two species of the family Mycoplasmataceae: Myco-
multiple spheroids technique. Furthermore, a decrease of PCNA
plasma hominis (Mh) and Mycoplasma fermentans (Mf) and one
expression and a significant increase of Hsp70 and Nrf2 ones in
species of the family Aholeplasmataceae: Aholeplasma laidlawii
correlation with the increase of GSH intracellular level were
(Al). For the growth of mycoplasmas was used broth (Difco
observed in breast cancer spheroids generated by both methods.
PPLO Broth, Becton, Dickinson, USA) with adding of 20%
In conclusion, through both techniques similar breast cancer 3D
horse serum, 2% fresh yeast extract, 1% arginine or 1% glucose
models were generated, expressing characteristics of multicellular
182 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 183
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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184 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 185
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biotechnology POSTERS
caused by both the action of sorbents and the effect of pH shift P.07-068-Tue
in the alkaline side. The possibility of photobacteria adhesion
Obtaining and characterization of a single-
and introduction were also studied. According to the luminescent
analysis it was detected that sorbents are effectively populated by chain variable fragment alkaline phosphatase
photobacteria. A direct indication of the photobacteria’s adhe- fusion proteins and specific polyclonal
sion on sorbents was obtained from electron microscopic studies. antibodies for detection of IFN-beta and
Photobacteria on sorbents formed colonies. Therefore it was interleukin-10
established, that the toxicity of sorbents and the adhesion of pho- M. Usenko1, O. Gorbatiuk1, O. Okunev2, V. Kordium1
tobacteria on it depend on a polymerization process, a structure 1
Institute of Molecular Biology and Genetics, NAS of Ukraine,
and concentration of a crosslinking agent and a stability of a rhe- 150, Akademika Zabolotnoho Str., 03680, Kyiv, Ukraine, 2State
ological parameters of the sorbent in salt solutions. Institute of Genetic and Regenerative Medicine, NAMS of
Ukraine, 67, Vyshgorodska Str., 04114, Kyiv, Ukraine
P.07-067-Mon Interferon-b 1b (IFN-b) is used as first line treatment for multiple
Rational design of formate dehydrogenase sclerosis (MS). The incidence of MS has been increasing world-
wide. One of the proved effects of IFN-b longitudinal MS treat-
from the thermotolerant yeast Ogataea
ment is an increase in the number of interleukin-10 (IL-10)
parapolymorpha secreting cells. Development of new effective test systems for
S. Zarubina1,2, A. Pometun2,3, S. Kleymenov3,4, S. Savin2,3, detection of IL-10 and IFN-b is of current importance. The aim
V. Tishkov1,2,3 of our research is the development of method for IL-10 and
1
Lomonosov Moscow State University, Moscow, Russia, IFN-b detection using single-chain variable fragment (scFv) con-
2
Innovations and High Technologies MSU Ltd, Moscow, Russia, jugates with alkaline phosphatase (AP) and specific polyclonal
3
Federal Research Centre “Fundamentals of Biotechnology” of antibodies. ScFv were chosen due to their obvious advantages,
RAS, Moscow, Russia, 4Koltzov Institute of Developmental such as small size, possibility of easy obtaining in high concentra-
Biology, Russian Academy of Sciences, Moscow, Russia tions in bacterial systems of expression. Besides, they can be
Formate dehydrogenase (EC 1.2.1.2, FDH) is widely used for genetically fused to the marker protein, that allows to use them
coenzyme regeneration of NADH or NADPH in different biocat- for one-step immunodetection of target molecules. ScFv specific
alytic processes. The gene of a novel NAD+-dependent formate to IFN-b and IL-10 were obtained from combinatorial libraries
dehydrogenase from the thermotolerant methylotrophic yeast of cDNA V-genes of murine IgG using phage display technology.
Ogataea parapolymorpha DL-1 was cloned in our laboratory. AP coding DNA sequence was subcloned into plasmid vectors
Nucleotide sequence of the opafdh gene was modified at N-termi- pCANTAB-scFv(IL-10) and pCANTAB-scFv(IFN-b). E. coli
nus with additional Gly codon. It was shown that recombinant BL21(DE3) cells were transformed by obtained plasmid vectors.
wild-type OpaFDH had the lowest values of Michaelis constants ScFv(IFN-b)-AP and scFv(IL-10)-AP synthesis was induced by
(KM) both for NAD+ and formate among all described FDHs adding IPTG. Proteins of interest were accumulated in the
from yeast and bacteria. The enzyme also demonstrated high periplasmic space of E. coli. Functional activity of scFv(IFN-b)-
thermal stability, which was lower only in comparison with the AP and scFv(IL-10)-AP was confirmed with dot blot analysis
FDHs from bacteria Pseudomonas sp. 101 and Staphylococ- and ELISA. The possibility of long-term storage of scFv(IFN-b)-
cus aureus. According to the multiple amino acid alignment AP and scFv(IL-10)-AP as a periplasmic extracts at -20°C
Tyr286 residue placed in front of conserved motive PQP was without loss of their functional activity was shown. Polyclonal
selected and substituted by rational design for improving thermal antibodies with high specificity and affinity to IFN-b and IL-10
stability of wt-OpaFDH. Four plasmids encoded mutant forms were produced via immunization of laboratory mice. Application
of OpaFDH with change of Tyr286 with Glu, Asp, Ser and Ala of polyclonal antibodies for antigen capture and scFv-AP for
were obtained, mutans were expressed in E. coli cells, purified antigen detection is promising for the development of new sand-
and characterized. Substitution with Glu and Asp resulted in 7- wich ELISA system for the determination of human IL-10 and
and 15-fold increases of thermal stability, respectively. Thermal IFN-b levels.
inactivation kinetics and differential scanning calorimetry were
used to study thermal stability. Mutation Cys230 (analog of
Cys255 in PseFDH) to Ala increased chemical stability by 100- P.07-069-Wed
fold. Double mutant OpaFDH C230A/Y286D showed both Site-directed mutagenesis of the recombinant
increase of thermal and chemical stability. This work was sup- alpha-amino acid ester hydrolase from
ported by Russian Foundation for Basic Research, grant 17-04- Xanthomonas rubrilineans
01469. A. Stepashkina1,2, E. Fedorchuck2,3, V. Fedorchuck2,3,
A. Sklyarenko4, S. Yarotsky4, M. Khrenova2,5, A. Pometun1,2,
S. Savin2,3, V. Tishkov1,2,3
1
A.N. Bach Institute of Biochemistry, Research Center of
Biotechnology RAS, Russian Academy of Sciences, Moscow,
Russia, 2Innovations and High Technologies MSU Ltd, Moscow,
Russia, 3Department of Chemical Enzymology, Faculty of
Chemistry, M.V. Lomonosov Moscow State University, Moscow,
Russia, 4State Research Institute for Genetics and Selection of
Industrial Microorganisms GosNIIgenetika, Moscow, Russia,
5
Department of Physical Chemistry, Faculty of Chemistry, M.V.
Lomonosov Moscow State University, Moscow, Russia
Alpha-amino acid ester hydrolase (EC 3.1.1.43; AEH) catalyzes
the synthesis and the hydrolysis of the amide bond in semi-syn-
thetic b-lactam antibiotics and displays the preference for the
186 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biotechnology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 187
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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188 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 189
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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190 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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extracts and the samples were freeze dried. The extracts were re- P.07-083-Tue
dissolved in appropriate solvent to investigate their fatty acid,
Yeast isogenic system as a tool for study
carotenoid and phenolic acid profile by GC and HPLC analysis
under gradient conditions. The antioxidant capacity and total protein interaction with DNA structures
phenolic content of extracts were evaluated using TEAC and the V. Brazda1, B. Tomanova1, J. Cechova2, E. B. Jagelska2,
Folin-Ciocalteu method respectively. Finally, the antimicrobial J. Coufal2
1
activities of the extracts were investigated by means micro-broth Brno University of Technology, Brno, Czech Republic, 2The
dilution method against E. coli and S. aureus species. As a result, Czech Academy of Sciences, Institute of Biophysics, Brno, Czech
the major carotenoids and phenolic acids were determined as b- Republic
carotene, b-cryptoxanthin, catechol and rutin for Gracilaria sp. Protein-DNA interaction is the fundamental part of life. There
Also the strain is a reassuring source for EPA and DHA fatty are many techniques for detection and evaluation of the protein
acids. Promising results were obtained in the case of antimicro- DNA binding from in vitro to in vivo assays. Here we presented
bial activity studies as up to 98% inhibition of growth. Within application of yeast isogenic system for in vivo transactivation
the results, it can be declared that the macro-algal compounds studies in chromosomal context. This variable system is applica-
proved the applicability of these strains in industrial applications ble for analyses of influence of DNA structures to protein-DNA
with further standardization techniques. binding in vivo. We used a panel of S. cerevisiae haploid isogenic
strains, except for the different p53 target site with different
DNA structures located upstream of the luciferase reporter gene.
P.07-082-Mon The targeting of p53 target sequence of interest by the replace-
Why is G6PD enzyme activity measured as ment of the ICORE cassette, using transfected single strand
zero? oligonucleotides, was performed following the Delitto Perfetto
unastı, U. K€
B. G€ okbas, K. Kartlasmıs, A. Tuli technique. Correct targeting events were isolated exploiting the
Department of Biochemistry, Faculty of Medicine, Çukurova counter-selectable and the reporter selection markers of the
University, ADANA, Turkey ICORE cassette and confirmed by colony PCR across the modi-
fied locus and Sanger DNA sequencing. yLFM isogenic deriva-
Glucose-6-phosphate dehydrogenase (G6PD) enzyme is the first
tive yeast strains constructed for this study were transformed
and key enzyme of the hexose monophosphate shunt. In this
with different plasmids for the expression of wild-type human
enzymatic reaction, G6PD reduces nicotinamide adenine dinu-
p53 or p73 proteins. Our results show that transactivation in vivo
cleotide phosphate (NADP) to its reduced form (NADPH) while
correlated more with the relative propensity of a response ele-
oxidizing glucose-6-phosphate (G6P) and producing a proton.
ment to form cruciforms than to its expected in vitro DNA bind-
Conventional test used to quantitatively measure G6PD activity
ing affinity. Structural features of target sites are therefore an
are spectrophotometric methods based on measuring the amount
important determinant of its transactivation function.
of NADPH generated. This test is a standardized protocol in
centralized laboratories. Currently available methods can mea-
sure G6PD enzyme activity as zero. But, since the enzyme is P.07-084-Wed
housekeeping enzyme, the activity of G6PD enzyme cannot be
Ultrahigh-throughput screening techniques for
zero. We aimed to clarify by using an enzyme biosensor that the
cause of G6PD enzyme activity was measured as zero in our investigation of bacterial interactions in
study. We needed optimization and characterization studies in microbiota community
order to improve the optimization of the working conditions of S. Terekhov, I. Smirnov, A. Nazarov, A. Gabibov
G6PD enzyme activity. Enzyme concentration, pH, temperature, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the
presence of activator and inhibitors, cofactor presence were stud- Russian Academy of Sciences, Moscow, Russia
ied in optimization studies. In the characterization studies, the Intimate interactions between different bacteria in microbiota
use of the analogues of the substrates of the G6PD enzyme was community represent one of the most important “hot spots” of
studied. Triple electrode system used in the study. The compo- common microbiology. Nevertheless, classical microbiological
nents of our enzyme biosensor consist of gold electrode, reference techniques suffer from a shortage of productivity of analysis. Con-
electrode and counter electrode. Proton production from a chem- sequently, bacterial interactions between rare and slow-growing
ical reaction catalyzed by G6PD was measured to reflect G6PD species are often ignored or misinterpreted. The development of
activity in a blood sample. We determined that the activity of next generation ultrahigh-throughput screening (uHTS) techniques
G6PD enzyme is not absolutely zero at the end of the study. The is of particular interesting as it allows direct detection of biological
improved enzyme biosensor serves as a specific results to G6PD activity of even minor components of specific microbiota commu-
and G6P interaction. This new procedure can be suggested as an nities. Moreover, uHTS techniques allow investigating the dynam-
alternative to G6PD enzyme activity measure methods. Enzy- ics of microbiota community that is very important in complex
matic Biosensors show high performance characteristics with mixtures of different species with highly distributed growth param-
broad detection ranges, short measuring times, and good speci- eters and sensitivity to the external influence of environmental con-
ficities. Thus, we provide new approaches to specific enzyme ditions. Utilization of uHTS techniques based on cultivation of
biosensors that are fast, accurate and easy to use for the determi- bacterial species in droplets of microfluidic double emulsion
nation of G6PD enzyme activity. (MDE) enabled us to perform highly sensitive single-cell selection
of predesigned activity and the exploration of pairwise interactions
between different bacteria unperturbed by other microbiota spe-
cies. Thus, individual droplets of MDE serves as isolated micro-
containers for cultivation and analysis. uHTS of MDE with
encapsulated bacteria, enable us to isolate culturable bacterial
strains and predict effectors inhibiting growth of model pathogenic
bacteria. This work was supported by grant RFMEFI60716X0145
from the Ministry of Education and Science of Russia.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 191
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biotechnology POSTERS
192 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biotechnology
to reduce the cost of feeding rations and increase the digestibility molecular weight was estimated 50 KDa. In conclusion, because
of nutrients from cheaper components. The use of an enzyme of noticeable pectinase production of P. indica by using sugar
protease becomes topical in these conditions. The main condition beet pulp as a suitable and cheap substrate, economically is valu-
for the successful carrying out of the technological process of able and moreover cause to decrease environmental pollution.
obtaining the enzyme is the selection of a highly effective strain.
We tested a number of recombinant strains for obtaining subtil-
isin-like proteinase (AprBp) B. pumilus: MRB044, B. subtilis P.07-090-Wed
MRB045, B. subtilis MRB046 (containing optimized LIKE sys- The implementation of the CRISPR-Cas system
tem), B. subtilis MRB073 (containing the constitutive promoter for Kluyveromyces lactis genome editing
PDegQ, B. subtilis pCS9 (containing multicopy plasmid pCS9, con- A. Gedvilaite, D. Ziogiene, M. Norkiene, M. Valaviciute
structed on the basis of the vector pCB22) and B. pumilus labora- Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
tory streptomycin resistant strain. The dynamics of growth and
activity of the strains were studied. It was shown that the maxi- Kluyveromyces lactis are amongst the most frequently used and
mum activity of the AprBp was at 22–28 h of growth in all well-studied non-conventional yeast. It serves as an expression
strains. We also studied the specific activity of all strains. The platform for recombinant proteins including secreted proteins
most effective strain was B. subtilis pCS9. We also developed and produces industrially relevant enzymes. Gene targeting effi-
optimal conditions for growth and cultivation. Purification of the ciencies in K. lactis are low and differ greatly depending on the
subtilisin-like proteinase was carried out on a column of car- integration locus and the design of the homology cassette. In this
boxymethylcellulose (“Sigma”). SDS-PAGE in 12.5% was per- study the implementation of the CRISPR-Cas system as a tool
formed, the protein fraction after chromatography showed the for highly efficient genome editing was used for improvement of
presence of a protein with a molecular weight of 28 kDa, which gene targeting efficiency and introduction of mutations in K. lac-
corresponds to the molecular mass of the AprBp. Physicochemi- tis genome without additional selection markers. The endonucle-
cal, biochemical and enzymatic properties of individual enzymes ase Cas9 (human-codon optimized and fused to a SV40 nuclear
will be established to assess the possibility of their use the subtil- localization signal at its N and C-terminus) was expressed either
isin-like proteinase as feed additives. This work was performed in under the control of the constitutive PGK1 promoter, or induci-
accordance with the Russian Government Program of Competi- ble LAC1 promoter in the plasmid which also included the
tive Growth of Kazan Federal University. The experiments on gRNA cassette(s) and pKD1 stabilizing element for expression in
selection of the optimal strain and purification of serine pro- K. lactis. The gRNA was expressed as a chimeric guide RNA
teinase were supported by the Russian Science Foundation (Pro- from either of S. cerevisiae or of K. lactis the SNR52 promoter
ject No. 16-16-04062). and both gRNA expression cassettes terminated by the S. cere-
visiae SUP4 terminator. The high targeting efficiency was reached
by introducing mutation(s) in one essential and four non-essential
P.07-089-Tue genes even though no selection marker was used for integration
Optimization of pectinase production from at the site of modification. In case of co-transformations of the
CRISPR-Cas9 system and donor cassettes, the donor DNA frag-
sugar beet pulp as carbon source by
ments efficiently integrated by homologous recombination. When
Piriformospora indica the donor DNA fragment was not used the Cas9 endonuclease
P. Fathi Rezaei, S. Kiani, S. Shahabi double strands brakes were repaired by NHEJ and introduced
University of Maragheh, Maragheh, Iran indels. The efficient CRISPR-Cas9 system generated for K. lactis
Pectinases, as a large group of enzymes, catalysis break down of genome editing was also successfully applied for S. cerevisiae.
pectin to smaller molecules including galacturonic acid, and they
are widely produced by many microorganisms. Nowadays, abun-
dant waste from agriculture and fruit processing industries P.07-091-Mon
including sugar beet pulp (SBP), apple and citrus pomace as rich Electrochemical detection of aspartate
source of pectin are employing to induce pectinase production by transaminase using a multienzyme-modified
many microorganisms. The aim this study was optimization of biosensor
pectinase production of Piriformospora indica fungus by using K. Kartlasmıs1, U. K€ okbas2, B. G€
unastı2, A. Tuli2, L. Kayrın2
sugar beet pulp (SBP) as a carbon source. P. indica was cultured 1
Çukurova University Medical School Biochemistry Department,
in Kaefer medium supplemented with SBP, then biomass and Adana, Turkey, 2Çukurova University, Adana, Turkey
spore production, enzyme activity and total protein content were
measured for 10 days. One unite of pectinase activity was defined AST and ALT are considered to be two of the most important
as the amount of enzyme that catalyzed the formation of 1 lmol tests to detect liver injury, although ALT is more specific for the
galacturonic acid per min under the assay condition. According liver than is AST and is more commonly increased than is AST.
to the results, maximum biomass production and pectinase activ- Sometimes AST is compared directly to ALT and an AST/ALT
ity were detected 6.125 g/L and 3.2 U/mL in the presence of SBP ratio is calculated. This ratio may be used to distinguish between
at 6th day, respectively. Furthermore effect of different concen- different causes of liver damage and to distinguish liver injury
trations of glucose and ammonium sulphate and their interaction from damage to heart or muscle. We aimed to design a biosensor
on enzyme activity was investigated. Based on the results by for the quantitative determination of AST enzyme in a short time
increasing the amount of them alone the activity was increased and an affordable cost. Polymerization of the biosensor active
but in the case of their interaction with sugar beet pulp the activ- layer developed for the AST assay was immobilized with UV,
ity was decreased. Next, by using Response Surface Methodology BSA, gelatin, malate dehydrogenase, AST and glutaraldehyde. L-
(RSM), pectinase production was 19.4 U/mL which increased by aspartate changing in the range of 1.5–15 mM and a-oxogluta-
5.84 times compared to non-optimized conditions. Then, the rate 25–100 mM were investigated in the reaction medium. The
enzyme were purified by 80% ammonium sulphate and dialysis results were compared with spectrophotometric results. Optimiza-
and the enzyme activity and specific activity were 12.64 U/mL tion of the bioactive layer used for the designed biosensor were
and 55.46 U/mg, respectively. The pectinase purification was con- performed. The response current in the range of 0.2–1.2 V was
firmed by SDS-PAGE and silver nitrate staining, and its realized in the cyclic voltammogram where the scanning speed
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 193
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Host–pathogen interactions POSTERS
194 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Host–pathogen interactions
and overcome host defences mechanisms, which is as important Label-free quantitative LC-MS was used to evaluate abundance
progress to the understanding of the pathways underlying bacterial profiles of proteins along the gel lane which allowed to determine
virulence. changes in complex stoichiometry based on profiles likelihood. We
found difference in composition of protein complexes in control
mycoplasma laboratory strain and mycoplasma cultivated after
P.08-004-Mon being extracted from host cells. Besides, we observed the forma-
Isolation and characterization of tion of novel non-canonical complexes in post-infectional myco-
bacteriophages infecting uropathogenic plasma. This study was funded by Russian Science Foundation
(14-24-00159).
Escherichia coli strains
B. Szalaiova1, M. Kajsık2, V. Kadlicekova1, A. Lichvarikova1,
na1,2, H. Drahovska1
J. Tur P.08-006-Wed
1
Department of Molecular Biology, Faculty of Natural Sciences,
Comenius University in Bratislava, Bratislava, Slovakia,
Prediction and validation of the structural
2
Comenius University Science Park, Bratislava, Slovakia features of Ov58GPCR as an immunodominant
antigen of Onchocerca volvulus: implications
Urinary tract infections (UTIs) caused by Escherichia coli are
among the most common bacterial infections in humans. Despite in onchocerciasis control efforts
effective antibiotic therapy, these infections tend to recur. The R. A. Shey1, F. N. Njume1, S. M. Ghogomu2,
prevalence of UTIs caused by antibiotic-resistant UPEC is steadily L. O. T. GAINKAM1, J. Souopgui1,3
1
increasing, highlighting the need to find an alternative for the con- Institut de Biologie et de M
edecine Mol
eculaires, Gosselies,
trol of pathogenic bacteria. In this study we characterized Escheri- Belgium, 2University of Buea, Buea, Cameroon, 3Molecular and
chia coli isolates obtained from patients with urinary tract Cell Biology Lab., University of Buea, Buea, Cameroon
infection and supplemented with eight collection strains. Strains Onchocerciasis, also called river blindness is a devastating yet
were differentiated by nonconventional phylogrouping (quadru- neglected tropical disease that creates a remarkable stigma, gener-
plex PCR), K1 antigen detection and two locus MLST - CH ates and perpetuates poverty, and is a major impediment to
(fumC/fimH) typing. Phylogenetic grouping revealed that group socioeconomic development/progress of affected communities. It
B2 was the most common type (n = 15). PCR-based detection of is the second leading infectious cause of blindness worldwide,
neuC (K1-specific gene) identified two clinical strains as K1 posi- affecting about 15.5 million people including 12.2 million with
tive. A remarkable level of haplotype diversity identified by CH onchocercal skin disease (OSD), 1.025 million with vision loss
typing among 28 clinical isolates was observed (n = 21). fumC/- and an additional 172 million people in need preventive
fimH allele combination 40/30 was found to be most common chemotherapy. Currently, Ivermectin (IVM) is the only drug used
(n = 6). Few representative strains were chosen as a hosts to iso- for mass treatment against the disease. Ivermectin, however has
late bacteriophages from sewage infecting E. coli. Four phages limited effects on adult parasites, so treatments must be expanded
(vB_EcoP_KMB13, vB_EcoP_KMB14, vB_EcoP_KMB15 and over at least 12–15 years, corresponding to the reproductive lifes-
vB_EcoP_KMB16) were then isolated which were able to lyse pan of the adult worm when exposed to drug pressure, hoping
53% of a subset (36) of the E. coli strains. The phage genome no further infection occurs within this time. The WHO’s ambi-
sequences were obtained, annotated and this resulted in classifica- tious goal of eliminating the disease by 2025 will not be achieved
tion of vB_EcoP_KMB13 and vB_EcoP_KMB14 as the Podoviri- without the development of new tools. Measures to certify dis-
dae family and vB_EcoP_KMB15 and vB_EcoP_KMB16 were ease elimination and surveillance are of dire need for health sys-
classified as the Siphoviridae family. New isolated phages still need tems in countries where onchocerciasis is a public health
to be characterized in terms of interactions with different bacterial problem. Here, we report that (i) O. volvulus antigen Ov58 is a G-
hosts, but they represent a promising candidates for phage therapy protein coupled receptor (GPCR) conserved in nematodes, (ii) syn-
targeted specific for infections caused by uropathogenic E. coli. thetic peptides predicted in the extracellular domain (ECD) of
Ov58GPCR elicit positive responses to sera from onchocerciasis
patients, (iii) synthetic peptide cocktails discriminate sera from
P.08-005-Tue infected, treated patients and normal African controls, (iv) poly-
Protein complexes as a mechanism of clonal antibodies against the recombinant ECD revealed a single
adaptation in bacteria with a reduced genome band on blots of O. volvulus total extracts, corresponding to the
O. Bukato, O. Pobeguts, D. Rakitina, I. Butenko, M. Levites, expected size of the endogenous native antigen, (v) Ov58GPCR is
D. Matyushkina, V. Ladygina, D. Evsyutina, G. Fisunov, transcriptionally activated in both the larvae and adult parasite, (vi)
V. Govorun humoral immune responses depict declines with IVM treatment
Federal Research & Clinical Center of Physical-Chemical Medicine compliance. Hence, we conclude that the ECD of Ov58GPCR har-
of Federal Medical Biological Agency, Moscow, Russia bors antigenic features of interest in the context of onchocerciasis
control efforts.
M. gallisepticum is a simplistic organism with significantly reduced
genome. Adaptation mechanisms based on differential gene expres-
sion are generally reduced in mycoplasmas due to the loss of P.08-007-Mon
majority of respective proteins (i.e. transcription factors). Alternate
Interplay between L-A and M viruses in yeast
adaptation pathways may involve reorganization of protein com-
L. Aitmanait_e1, A. Konovalovas1, E. Servien_e2,3, S. Serva1,3
plexes without significant changes in abundance of participating 1
Vilnius University, Vilnius, Lithuania, 2Nature Research Centre,
proteins. To explore this possibility we tested model mycoplasmal
Vilnius, Lithuania, 3Vilnius Gediminas Technical University,
intracellular infection of eukaryotic cells (chick erythroblast cell
Vilnius, Lithuania
line HD3). To study protein complexes we used protein correlation
profiling (PCP) as main approach with verification by tandem Totiviridae family dsRNA viruses are commonly spread among
affinity purification (TAP) for complexes of interest. PCP included fungi and protozoa kingdoms. Special kind of these viruses - L-A -
fractionation of complexes by centrifugation in sucrose gradient are found among Saccharomycetales and other yeasts. Some yeast
with subsequent native electrophoresis of each gradient fraction. strains together with L-A helper virus carries smaller satellite
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 195
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Host–pathogen interactions POSTERS
196 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Host–pathogen interactions
domain, represent the only exception. In WAMP homologues, at of P. syringae is HopU1. HopU1 is an ADP-ribosyltransferase
position 34 of the mature peptide Ala and Val were detected. interacting with RNA-binding proteins, thereby suppressing plant
Using heterologous expression in E. coli cells, the recombinant immunity. It acts as a “saboteur” that ruins plant immunity at
WAMPs were produced. In vitro antimicrobial assays showed the posttranscriptional level. In this regard, we hypothesized that
that WAMP homologues differ in antifungal activity. The results the suppression of the functions of this effector protein should
obtained expand our knowledge on the biodiversity of genes lead to an increase in plant resistance to the phytopathogens that
encoding WAMPs in cereals. use the effector proteins of the third transport system. In order
to suppress HopU1 we have proposed the utilization of the RNA
aptamers specifically binding this protein. The approach of the
P.08-011-Tue utilization of RNA aptamers to suppress the function of proteins
Peculiarities of BCG vaccine performance in is known for a long time, but has never been tested on plants.
B10.M (H2f) mice First we have expressed a heterologous recombinant effector pro-
M. Korotetskaya1,2, P. Baikuzina3, M. Kapina2, A. Apt2 tein HopU1 of P. syringae bacteria. Using the SELEX method
1
I.M. Sechenov First Moscow State Medical University, Institute we have selected RNA aptamers for HopU1 protein. Next we
of Molecular Medicine, Moscow, Russia, 2Central Institute for have characterized the selected RNA aptamers, described their
Tuberculosis, Moscow, Russia, 3Lomonosov Moscow State secondary structure and evaluated the dissociation constants
University, Moscow, Russia (Kd) of the RNA-aptamer-HopU1 complexes by the MST
method. The values of Kd for aptamers H1 and H2 were Kd
Pathogenesis of tuberculosis (TB) infection is a multistage, com- (H1) = 6.71 +/- 0.69 nM and Kd (H2) = 6.41 +/- 0.48 nM,
plex sequence of events involving a plethora of cell populations respectively, indicating a high degree of binding. In our future
and molecules. Earlier, it was demonstrated that generally vacci- work we plan to obtain transgenic plants constitutively express-
nation with bacillus Calmette-Guerin (BCG) prior to Mycobac- ing selected RNA aptamers and use them for phytotests. We
terium tuberculosis infection significantly prolongs survival time expect that such plants will have increased resistance to patho-
of genetically resistant and susceptible inbred mice. The B10.M genesis by P. syringae This work was supported by a grant from
(H-2f) mouse strain appeared to be an interesting exception: the Russian Foundation for Basic Research No. 16-04-01002
these mice exhibited intermediate resistance to primary infection
but their survival time even slightly decreased if intravenous chal-
lenge was applied after BCG vaccination. In the present study, P.08-013-Mon
we addressed: (i) possible immune mechanisms behind this para- The diversity of putative antimicrobial
doxical phenotype, and (ii) assessed whether the “fail-of-vaccina-
peptides revealed in wheat by high-
tion” phenotype is expressed after challenging mice via
respiratory tract. Our experiments confirmed previously reported throughput next-generation transcriptome
defect in B10M mice: after intravenous challenge with 2 x sequencing
106 CFU of M. tuberculosis H37Rv post-infection life span and A. Kovtun1, A. Shelenkov2, T. Odintsova2
1
cachexia levels were identical in non-vaccinated mice and mice Moscow Institute of Physics and technology, Dolgoprudny,
vaccinated with 5 x 107 BCG CFU. Moreover, vaccination did Moscow Region, Russia, 2Vavilov Institute of General Genetics
not change mycobacterial loads at weeks 4 and 8 post challenge, RAS, Moscow, Russia
the ratio of various cell types, and cytokine profiles in the lung
Antimicrobial peptides (AMPs) are important components of the
tissue. We also applied a more “natural” TB model based upon plant immune system. Diverse in structure, biological activity and
aerosol infection (result 600 CFU) to B10.M and B10/Sn control the mode of action they offer a rich repertoire of biologically
mice; the latter have the same genetic background but differ by
active molecules for practical applications: in agriculture to pro-
the H2 haplotype (H-2b) and readily respond to BCG vaccina-
duce disease-resistant crops and in medicine to develop next-gen-
tion in the intravenous challenge model. We still accumulate eration antimicrobials. This study is focused on AMP mining in
immunologic data concerning vaccination effect in these to transcriptomes of the wheat species Triticum kiharae Dorof. et
mouse strain, but it is already clear that lung cells of infected
Migusch, a synthetic allopolyploid, which is highly resistant to
B10.M mice produce significantly lower amounts of IFN-c in pathogens. For the first time RNA-seq analysis of healthy,
response to mycobacterial antigens compared to B10/Sn cells, infected with Fusarium oxysporum, treated with elicitor, and trea-
and that this phenotype does not depend upon BCG vaccination. ted with elicitor followed by infection with Fusarium oxysporum
This result suggest that immune response in infected lungs of
wheat seedlings, as well as de novo transcriptome assembly and
B10M mice might be less Th1-biased. annotation, are presented. To identify pAMPs a hidden Markov
model based on conserved cysteine motifs characteristic to differ-
P.08-012-Wed ent AMP families was created followed by searching, quality con-
trolling and sorting out the results. Using the developed pipeline
RNA aptamers for inhibition of the effector 563 unique putative AMPs belonging to different families – defen-
protein HopU1 of phytopathogenic bacteria sins, thionins, hevein-like peptides, LTPs, snakins, and cysteine-
P. syringae rich peptides with novel cysteine motifs, were predicted. Further-
A. Vyacheslavova, L. Maloshenok, S. Bruskin, I. Abdeeva more, other cysteine-rich peptides with defensive role, such as
Vavilov Institute of General Genetics Russian Academy of Science, Bowman-Birk inhibitors and Proteinase inhibitors II, as well as
Moscow, Russia RALF peptides involved in defense signalling were identified,
together with other CRPs, such as MEG Ae1 and Pollen Ole e1,
It is known that phytopathogenic bacteria are capable of sup-
whose role in immunity is still unclear. For the first time three
pressing both basic and induced plant immunity due to the secre-
types of hevein-like peptides that differ in the number of cysteine
tion of the virulence factors or effector proteins inside the plant
residues and the length of the C-terminal prodomain were discov-
cell. It is established that the main players in the pathogenesis of
ered in wheat. The antimicrobial activity of a number of predicted
gram-negative bacteria are the effector proteins secreted by bacte-
AMPs was proven by in vitro assays of recombinant peptides.
ria via the third type transport system (TTS) into the cells of the
Thus, the developed pipeline of AMP search disclosed an amazing
host plant. One of the key effector proteins in the pathogenesis
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 197
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Host–pathogen interactions POSTERS
complexity of AMP array in wheat. This work was supported by Methyl L-arginine acetate (NMLAA) and pentamidine) to check
the Russian Science Foundation (grant no. 16-16-00032). their effect on ADR. L. donovani promastigotes and THP-1
infected amastigotes were analyzed in this study. We identified
that pentamidine and canavanine downregulated ADR response;
P.08-014-Tue While NMLAA had no effect on ADR both in promastigotes
Outer surface protein C (OspC) from Borrelia is and intracellular amastigotes. Additionally, pentamidine and
capable of fibrinogen binding canavanine also mimicked the presence of arginine thus downreg-
P. Bierwagen, K. Szpotkowski, M. Jask olski, A. Urbanowicz ulating ADR response in arginine starved promastigotes and
Institute of Bioorganic Chemistry, PAS, Poznan, Poland intracellular amastigotes. Our data shows that show that arginine
sensing and transport are uncoupled. The arginine sensor
Pathogenic bacteria differ widely in their strategies for evading responds to both arginine deprivation and sufficiency by either
immune surveillance. In the case of Borrelia burgdorferi sensu activating or deactivating ADR by modulating the expression of
lato, which is the causative agent of Lyme disease, such a strat- AAP3.
egy relies on producing a multitude of surface proteins showing
high serological divergence among pathogenic strains and capable
of escaping the immune response. In addition, some of the sur- P.08-016-Mon
face proteins recruit host factors to perturb the host homeostatic The protective capacity of anti O:4 antigen
system or cover the spirochete with an “invisibility cloak”. Outer
antibodies against Salmonella infection is
surface protein C (OspC) is one of the most abundant surface
lipoproteins produced by the spirochete during the early stages of influenced by the presence or absence of O:5
infection and its DNA coding is much more variable than of antigen
other genes from these bacteria. A few specific functions have M. Elsheimer-Matulova, Y. Masukagami, Y. Ogawa, Y. Shimoji,
already been proposed for OspC, such as binding of host comple- M. Eguchi
ment factors, interaction with tick immunoprotective salivary NARO, Tsukuba, Japan
protein Salp15, plasminogen recruitment, or protection from O-antigen antibody such as anti O:4 antigen IgG induces protec-
phagocytosis by macrophages. Our report adds a new target to tive immunity against Salmonella enterica serovar Typhimurium
the OspC interaction network. We observed that recombinant
(S. Typhimurium) infection. Although S. Typhimurium belongs
OspC proteins from different human-infectious Borrelia species to the group O:4, it can be classified into two serological vari-
are capable of fibrinogen binding with high affinity. We also pro- ants, factor O:5 positive strain (O5+) and factor O:5 negative
pose a fibrinogen binding site of OspC, present some structural
strain (O5-). In this study, to determine whether anti O:4 antigen
features of the complex in solution, and evaluate the influence of IgG induces protective immunity against both O5+ and O5- S.
the OspC-fibrinogen complex formation on fibrinogen polymer- Typhimurium infection, we compared S. Typhimurium x3306-
ization. Finally, we discuss the implications of our findings in the oafA (O5+) with x3306-pBR322 (O5-) using mouse model. High
context of Borrelia survival at the early stage of the mammal
dosage of anti O:4 antigen IgG induced protective immunity
host infection. This work was supported by grant no. UMO- against both O5+ and O5- S. Typhimurium infection. However,
2015/17/B/NZ1/00873 from the National Science Center low dosage of anti O:4 antigen IgG was able to induce protective
(Poland). immunity against O5- S. Typhimurium infection only. The affin-
ity of O:4 antigen with O:4 antigen IgG was found to be stronger
in the O5- strains compared to O5+ strains. Next, macrophages
P.08-015-Wed
infected with O5- strains treated with anti O:4 antigen IgG
Arginine sensing and transport is uncoupled in showed enhanced pathogen uptake compared to counterparts
Leishmania donovani parasites infected with O5+ strains treated with anti O:4 antigen IgG.
H. Pawar1, M. Puri2, R. Madhubala2, D. Zilberstein1 Moreover, these macrophages produced elevated levels of nitric
1
Faculty of Biology, Technion—Israel Institute of Technology, oxide, indicating that O5- strains treated with anti O:4 antigen
Haifa, Israel, 2School of Life Sciences, Jawaharlal Nehru IgG enhanced more pronounced macrophage activation than
University, New Delhi, India O5+ strains treated with anti O:4 antigen IgG. We suggest that,
Visceral leishmaniasis is a deadly disease caused by Leishmania although anti O:4 antigen IgG has the potential to protect
donovani. During infection, parasites up-regulate macrophage against S. Typhimurium infection, the effects of anti O:4 antigen
nitric oxide synthase and arginase activity, both of which use IgG in protection against Salmonella infection might differ
arginine as a substrate. These elevated activities depleted macro- depending on the presence of O:5 antigen.
phage arginine pools, a situation that invading Leishmania can-
not tolerate as this is an essential amino acid. L. donovani
P.08-017-Tue
imports exogenous arginine via a mono-specific amino acid trans-
porter (AAP3) and utilizes it primarily to provide precursors for Modulating host metabolic sensors for
trypanothione biosynthesis. The depletion of arginine from pro- targeting TB
mastigote and amastigote growth media induced a rapid up-regu- C. Cheng, A. Singhal
lation in AAP3 expression and activity, as well as a few other Singapore Immunology Network, Singapore, Singapore
genes by activation of a signaling cascade of arginine deprivation
An effective host immune response is important to control
response (ADR) pathway. Arginine is a positively charged amino Mycobacterium tuberculosis (Mtb) growth, and to contain its
acid with a guanidine cap at the distal end. Based on previous latent persistence. Using a chemical genetics approach we have
arginine transport analysis data from our group we postulated demonstrated that activating AMP-activated protein kinase
that some of best known arginine transport inhibitors have con-
(AMPK) and Sirtuin 1 (SIRT1) axis, regulator of whole-body
served guanidino groups in their side chains and some of these energy metabolism, by FDA approved drugs / supplements could
transport inhibitors were also structural analogues of arginine. control inflammation and Mtb infection. This indicates the deep
To understand whether arginine sensing and transport are cou-
engagement of Mtb pathogenicity with host cell immunometa-
pled; we used these arginine transport inhibitors (canavanine, N- bolic machinery. We hypothesize that the functional connections
198 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Host–pathogen interactions
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 199
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Host–pathogen interactions POSTERS
200 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Host–pathogen interactions
study was funded by Russian Foundation for Basic Research phosphorylation of RNAP II. Ku overexpression reduced Tat-
(grant No. 16-34-60217). mediated activation of elongation and Ku depletion increased the
effect of Tat protein. Moreover, the effect of Ku depletion on
transcription from LTR promoter was abolished by Tat expres-
P.08-024-Wed sion. These data demonstrate the role of TAR RNA in the posi-
Comparative analysis of secondary tive regulation of transcriptional elongation from LTR promoter
metabolites with antimicrobial activities from by Ku. This work was supported by the RSF grant 17-14-1107.
mosses and liverworts
L. Valeeva, I. Agabekyan, L. Nigmatullina, Y. Shakirov P.08-026-Tue
Kazan Federal University, Kazan, Russia
Human adenovirus type 26 uses av integrin for
Plants have evolved efficient systems to counteract microbial phy- infection of human epithelial cells
topathogens, including production of various antimicrobial sec- D. Nestic1, T. Uil2, J. Ma3, R. Soumitra2, J. Velinga2, A. Baker4,
ondary metabolites. Bryophytes (mosses, liverworts and J. Custers2, D. Majhen1,2
hornworts) are the ancient group of land plants with many 1
Laboratory for Cell Biology and Signalling, Division of Molecular
unique antimicrobial compounds, both intracellular and secreted. Biology, Ruder Bo skovic Institute, Zagreb, Croatia, 2Viral Vaccine
Antimicrobial potential of many bryophyte-derived compounds is Discovery and Early Development, Janssen Vaccines and
well-known but there is no comprehensive data about their nat- Prevention BV, Leiden, Netherlands, 3Glasgow Cardiovascular
ure and composition. To begin analysis of bryophytes’ secondary Research Centre College of Medical, Veterinary and Life Sciences,
metabolites with antibacterial and antifungal activity we exam- University of Glasgow, Glasgow, United Kingdom, 4Institute of
ined natural compounds from mosses Physcomitrella patens, Cer- Cardiovascular and Medical Sciences, College of Medicine,
atodon purpureus and Sphagnum fallax and a liverwort Veterinary and Life Sciences, University of Glasgow, Glasgow,
Marchantia polymorpha. Specifically, we aim to determine chemi- United Kingdom
cal composition of the most biologically active against plant
pathogens bryophyte compounds from water-, methanol- and Human adenovirus vectors based on type 5 (HAdV5) are the most
hexane-soluble extracts. In addition, we analyze bryophytes’ commonly investigated adenoviral vectors for gene therapy and
extracellular peptidomes and secreted enzymes. Overall, our study vaccination. Due to activation of the host innate immune
has a unique potential to help identify metabolites suitable for responses as well as preexisting immunity to AdV5 development
further development of new plant-derived biotechnological strate- of new strategies to evade undesired anti vector host immune
gies to control plant pathogens and improve crop quality and responses is needed. One approach is the search for other types of
yield. This study is supported by the Russian Foundation for adenoviruses that occur at low prevalence in human populations,
Basic Research grant 18-016-00146a. KFU is supported by the such as human adenovirus type 26 (HAdV26). HAdV26 belongs
Russian Government Program of Competitive Growth. to subgroup D and its natural tropism is still unknown. While
HAdV26 immunogenicity in vivo is rather well described, basic
biology of this virus is less studied. Hence, the main objective of
P.08-025-Mon this study was to investigate transduction efficacy and cell entry of
TAR RNA-dependent mechanism of HAdV26 in epithelial cells. As a cell model we used A549 and SK-
OV-3 cells which have different expression of known adenovirus
Ku-mediated transcription regulation of HIV-1
receptors: CAR, CD46 and av integrin. We showed that HAdV26
genes transduces SK-OV-3 cells 4 times better than A549 indicating that
O. Shadrina, E. Knyazhanskaya, M. Gottikh SK-OV-3 cells might express molecule/s that HAdV26 uses as a
Lomonosov Moscow State University (MSU), Moscow, Russia receptor. Thus, we downregulated CAR, CD46 and av integrin in
The Ku protein plays a key role in the DNA double-strand break A549 and SK-OV-3 cells and subsequently measured transduction
repair by non-homologous end-joining mechanism. In addition, it efficacy and binding of HAdV26. Downregulation of av integrin
participates in transcription, telomere maintenance, V(D)J-recom- significantly decreased transduction efficacy and binding of
bination and some other processes. Ku is a participant of human HAdV26 in both A549 and SK-OV-3 cells. We obtained the same
immunodeficiency virus-1 (HIV-1) replication at the stages of effect by measuring transduction efficacy of HAdV26 after anti-
integration and transcription although the exact mechanism of body-mediated blocking of av integrin Namely, blocking av inte-
Ku-dependent transcriptional regulation is unclear. We identified grin in A549 cells efficiently decreased transduction efficacy of
RNA structural motifs important for the interaction with Ku. HAdV26. These results were further confirmed in A549 cells with
The highest Ku affinity was detected toward a hairpin RNA increased expression of av integrin. We stably transfected A549
structure containing bulk bulge close to the loop. TAR RNA cells with av integrin expression plasmid and showed that overex-
forms a hairpin at the 5’-end of HIV mRNA and has a Ku pre- pression of av integrin increases binding and transduction efficacy
ferred structure. TAR RNA lacking the bulge had significantly of HAdV26 in A549 cells. Based on our results we conclude that
lower affinity towards Ku. We constructed a set of reporter vec- HAdV26 uses av integrin for infection of epithelial cells.
tors containing firefly luciferase gene either under the control of
wild-type HIV LTR or its deletion mutants: lacking the region
coding for the whole TAR RNA or the region corresponding to P.08-027-Wed
the bulge of TAR RNA. CRISPR/Cas9 depletion of Ku led to Virulence factors of Achromobacter ruhlandii
significant decrease in luciferase expression from HIV LTR and important for the host-pathogen interaction
only slightly influenced the mutant constructs. Overexpression of M. Kunda, O. Voronina, N. Sharapova, N. Ryzhova,
Ku protein in Ku-knockdown cells partially restored luciferase E. Aksenova, A. Gintsburg
expression from WT LTR. TNF-a mediated induction of HIV N.F. Gamaleya National Research Center for Epidemiology and
transcription initiation had no significant impact on Ku-depen- Microbiology, Moscow, Russia
dent regulation. Tat is an HIV trans-activating protein participat-
In the era of antibiotic resistance there is very important task to
ing in activation of transcription elongation that binds to TAR
RNA and recruits P-TEFb complex to the promoter for the develop new antibacterial drugs. Bacterial virulence factors (VF)
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 201
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Host–pathogen interactions POSTERS
are the candidates for potential targets. So investigation of VF of product of the BP1253, in agreement with the higher activity of
dangerous strains by genomic approach is the first step of medi- the enzyme towards GMP. Moreover, BP1253 turns out to be
cine elaboration. Achromobacter ruhlandii SCCH3:Ach33-1365 also prevalently expressed in clinical isolate strains compared to
ST36 (Arul ST36), the Russian epidemic strain, was the subject Tohama. Overall these data suggest that the 6-O-methylguanine
of our analysis, because of Arul ST36 is transmissible, and could have a real impact on virulence during infection and that
decreases the lung respiratory function of the cystic fibrosis BP1253 is a cytokinin-activating enzyme showing for the first
patients. Type III secretion system (T3SS) and motility factors time the presence of a phosphoribohydrolase activity in Borde-
attracted our attention. Genome of Arul ST36 (Accession Num- tella pertussis.
ber CP01743) was analyzed by the set of bioinformatic tools.
Only type IVb pili were revealed in Arul ST36 in contrast to
famous Burkholderia pathogen. At least 12 tight adherence (tad) P.08-029-Tue
genes are required for IVb pili formation. The tad genes of Arul The structure of rhizospheric and rhizoplanic
ST36 genome were organized in operon consisted of 18 genes. fungal microbiome of potato in different crop
Flp prepilin of Arul ST36 had typical structure for Flp subclass. rotation
It was 58 aa long and consisted of 3 parts (leader peptide,
M. Lutfullin1, G. Hadieva1, E. Shagimardanova1, P. Vankov1,
hydrophobic and C-terminal regions). The leader peptide was 14
S. Vologin2, Y. A. Akosah1, M. Sharipova1, A. Mardanova1
aa long. Flp motif ITALEY, crucial for the assembly of pili VIb, 1
Kazan Federal University Institute of fundamental medicine and
was characterized by adjacent E(+5) and Y(+6) residues, and G
biology, Kazan, Russia, 2Tatar Research Institute of Agriculture,
residue in (-1) position, that was essential for a cleavage of the
Kazan, Russia
signal peptide. Conservative F reside was revealed in C-terminal
region. T3SS was detected in Achromobacter genome for the first The present study analyzed the root-associated fungal micro-
time. T3SS of Arul ST36 contained 31 ORFs encoded by the biomes of potato (Solanum tuberosum), cultivated under different
23920 bp region, and separate virB1 gene, and belonged to the agricultural rotation on 3 fields; previously, peas, wheat and
EscN/YscN/HrcN family defined an extracellular pathogen. 570 potato had been grown on them. DNA of microbes in the Rhi-
candidate effectors of T3SS were revealed in Arul ST36 genome. zosphere (RS) and rhizoplane (RP) were isolated using “Fas-
9 effectors were established important for the regulation of innate tDNA SPIN Kit for Soil@”. The fungal ITS fragments 5.8S
immune response. Analyzed VF of Arul ST36 revealed new tar- rRNA genes were amplified and sequenced by the Illumina
gets for the antibacterial drug elaboration and demonstrated the MiSeq platform. The sequence data were further analyzed using
permission of using new drug Ftortiazinon for the suppression of the bioinformatics pipeline QIIME, version 1.9.1. The mean of
secretion of Arul ST36 effectors. Shannon index was 5.35 0.37 for the RS of fungal metage-
nomic data, while that of RP was 4.43 0.3. Fungal communi-
ties were classified into 6 phyla, 31 classes, 91 orders, 191
P.08-028-Mon families, and 329 genera. Ascomycota, Basidiomycota and
BP1253, a phosphoribohydrolase enzyme in Zygomycota were the 3 most dominant phyla in the potato’s RS
Bordetella pertussis and RP, accounting for 98–99%. Their frequency did not signifi-
F. Moramarco1,2, P. Pezzicoli1, L. Salvini3, M. Scarselli1, cantly change between the samples: averagely, the respective con-
R. Leuzzi1, W. Pansegrau1, E. Balducci4 stituents of RS and RP for Ascomycota was 81.4% and 81.7%;
1
GSK, Siena, Italy, 2Department of Pharmacy and Biotechnology, Basidiomycota - 9.7% and 9.8%, while Zygomycota - 8.5% and
University of Bologna, Bologna, Italy, 3Toscana Life Sciences 6.7%. The genus Fusarium was dominant in majority of the sam-
Foundation, Siena, Italy, 4School of Biosciences and Veterinary ples but their highest occurrence was seen in the RS and RP of
Medicine, University of Camerino, Camerino, Italy potatoes, whose precursor for the same field was also potato. 30
isolates of Fusarium were obtained from tubers, affected with
Notwithstanding the existence of a vaccine, the Whooping cough dry rot as well as latently infected tubers. Isolates with high vir-
infection caused by the Gram-negative bacterium Bordetella per- ulent properties were selected for molecular genetic identification.
tussis (Bp), is re-emerging. This recurrence could be the reflection 9 isolates were identified as Fusarium oxysporum; 3–Fusarium
among others of a waning immunity, epidemiological changes in solani; 3- Fusarium sambucinum; 2–Fusarium sp. and 1–Fusarium
the circulating strains and lack of herd immunity. New features redolens. It has been shown that the level of latent infection of
related to Bordetella pathogenesis and microbiology could be rel- tubers increases with the absence of crop rotation and in the cul-
evant to defeat the infection. BP1253 from Bp is a predicted tivation of potatoes. The work is performed in accordance with
exported protein erroneously classified as lysine decarboxylase. the Russian Government Program of Competitive Growth of
Nevertheless BP1253 contains high sequence homology with Kazan Federal University.
some newly structurally characterized “Lonely Guy” proteins.
These phytohormone cytokinins-activating enzymes share with
BP1253 a nucleotide-binding Rossmann fold, a highly conserved P.08-030-Wed
Motif PGGxGTxxE and the catalytic core complying with the The interaction between HIV-1 integrase and
peculiar distance among the catalytic Arg and Glu. SEC and
Ku70 facilitates postintegrational DNA repair
MALS analysis revealed a main dimeric form as functional unit.
A. Anisenko1, E. Knyazhanskaya2, T. Zatsepin3, M. Gottikh4
Studies by Biacore demonstrated that BP1253 has the ability to 1
Lomonosov Moscow State University, Faculty of Bioengineering
selectively bind monophosphate nucleotides such as AMP and
and Bioinformatics, Moscow, Russia, 2Lomonosov Moscow State
GMP. Time-course and dose-response experiments carried out
University, Chemistry Department, Moscow, Russia, 3Skolkovo
with an enzymatic assay in the presence of AMP and GMP as
Institute of Science and Technology, Moscow, Russia, 4Lomonosov
substrate, clearly showed that BP1253 is able to cleave the N-gly-
Moscow State University, Belozersky Institute of Physico-
cosidic linkage between the base and the ribose, leading to the
Chemical Biology, Moscow, Russia
formation of free bases. As a matter of fact this phosphoribohy-
drolase activity is the crucial reaction in producing active cytoki- The integration of the HIV DNA into the host genome is com-
nins. Through LC-MS/MS we identified on growth medium of pleted by cellular factors that repair the short gaps flanking the
Tohama strain the 6-O-methylguanine as the physiological proviral DNA. We have shown earlier, that viral integrase can
202 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Host–pathogen interactions
directly interact with Ku70 repair factor, and this interaction is P.08-032-Tue
weakened by E212A/L213A substitutions in integrase. The trans-
Interaction forces between the extracellular
duction of HEK 293T cells by a single-round HIV-1 vector is
found to be decreased in cells stably depleted of the DNA-PK matrix and variants of borrelial decorin
repair components (Ku70, Ku80 or DNA-PKcs). We have estab- binding protein A probed by atomic force
lished a qPCR-based approach for quantitative measurement of microscopy
postintegrational gap repair and shown that depletion of any M. Strnad1, Y. Oh2, M. Vancova1, P. Hinterdorfer2,
DNA-PK component reduces gap-repair efficiency in our system. L. Grubhoffer1, R. O. Rego1
1
The same effects have been detected in presence of specific DNA- Institute of Parasitology, Biology Centre of the Academy of
PKcs inhibitor Nu7441. Viral vector carrying E212A/L213A inte- Sciences of the Czech Republic, Ceske Budejovice, Czech Republic,
2
grase substitutions demonstrates decreased gap-repair and a Johannes Kepler University in Linz, Linz, Austria
reduced sensitivity to the DNA-PK components depletion while
The spirochetal bacterium Borrelia burgdorferi, the etiologic
its integrational capacity remains at the wild-type level. We spec-
agent of Lyme disease (LD), infects mammalian hosts via the bite
ulate that integrase recruits DNA-PK complex to gap cites and
of Ixodes spp. ticks. Unlike in North America, where the infec-
facilitates gap-repair through a direct interaction with Ku70 sub-
tion is caused only by B. burgdorferi sensu stricto, in Europe
unit. This work was supported by RFBR grant 17-04-01178 and
there are a number of B. burgdorferi genospecies that are associ-
RSF grant 17-14-1107 (development of the gap repair approach).
ated with human LD. Different genospecies display different pat-
terns of host specialization and tissue tropism and are associated
P.08-031-Mon with distinct spectra of clinical manifestations. It is well estab-
lished that B. burgdorferi modulates its gene expression as it
Adhesive properties and cytotoxicity of
cycles between the tick vector and the mammalian host they
Klebsiella spp. for urothelial T24 cells infect. Upon host invasion, B. burgdorferi has avoid vigorous
A. Gilyazeva1, I. Abaseva2, A. Mardanova1 host immune response in order to colonize and persist in the
1
Institute of Fundamental Medicine and Biology, Kazan Federal host. B. burgdorferi binds various components of the host extra-
University, Kazan, Russia, 2Bacteriological laboratory, Republican cellular matrix (ECM). Arguably the most well studied borrelial
Clinical Hospital # 2, Kazan, Russia adhesin is decorin-binding protein A (DbpA). DbpA was identi-
Catheter-associated urinary tract infections (CAUTIs) account fied as the adhesin responsible for binding to the proteoglycan
for 80% of cases of nosocomial urinary tract infections (UTIs). decorin. Interestingly, DbpA amino acid sequences are highly
The ability for long-term persistence of UTIs can be attributed polymorphic between the genospecies, with sequence similarities
by bacterial ability to invade urothelial cells and proliferate inside as low as 40–60%. This sequence heterogeneity results in struc-
of them. Mannose-sensitive type I fimbriae are shown to be tural variations that contribute to large differences in the adhe-
important for adhesion and invasion of uropathogenic Escheri- sion activities of the protein, which ultimately influences the
chia coli. Using NCBI (https://www.ncbi.nlm.nih.gov/) and tissue tropism and disease manifestation of the genospecies.
ASAP (https://www.genome.wisc.edu/tools/asap.htm) services Using atomic force microscopy, we measured specific binding
genomic locus containing genes responsible for expression of type forces of single bio-molecular adhesive interactions between vari-
I fimbriae was identified at K. oxytoca and K. pneumoniae. ants of borrelial DbpA and several ECM components, and local-
Strains had the same structural genes of the locus organized in ized the interactions on the surface of Borrelia.
fim-operon but their regulatory foundations varied. K. pneumo-
niae had fimB recombinase gene placed ahead of fim-operon and
K. pneumoniae had another regulatory gene conservative in the P.08-033-Wed
species. Yeast cells were used for detection of fimbriae on the Cross talk between nuclear PML, SUMO and
surface of bacteria. Agglutination of the yeast cells by K. oxytoca caspase 8 in response to interferon a and
was more distinct than that of K. pneumoniae and in both cases TGF-b
it was not inhibited by mannose. The effect of mannose could be F. El Asmi, L. Dianoux, M. Chelbi-Alix
masked by mannose-resistant agglutination supported by type III INSERM UMR1124, Universit e Paris Descartes, Paris, France
fimbriae characteristic for Klebsiella. Despite results of yeast cell
agglutination assay, K. pneumoniae was more adhesive towards ProMyelocytic Leukaemia protein (PML), an interferon-stimu-
urothelial T24 cells than K. oxytoca. It should be noted that both lated gene product, is essential for the formation of nuclear
strains did not have a cytopathic effect for T24 cells after 2 h of matrix-associated organelles named PML nuclear bodies (NBs).
coincubation and during further 23 h of incubation with amika- Covalent modification of PML by the Small Ubiquitin-like Modi-
cin. The fact that bacteria do not damage T24 cells may indicate fier (SUMO) is necessary for PML NB function. PML NBs are
that K. pneumoniae and K. oxytoca use urothelial cells for dynamic structures harboring numerous transiently and perma-
defence, intracellular proliferation and as a source of secondary nently localized proteins and have been associated with the regu-
infection. Differences in the fimbrial regulatory genes may lation of several cellular functions including apoptosis. Due to
explain dissimilar adhesive properties of K. pneumoniae and K. alternative splicing, many PML isoforms are synthesized from a
oxytoca. The work is performed in accordance with the Russian single gene that are classified into seven groups, designated PML
Government Program of Competitive Growth of Kazan Fed- I-VII, they share the N-terminal region, whereas they differ in
eral University. their C-terminal region. The variability in the C-terminal region
confers specific functions to each isoform. Most of PML isoforms
(PMLI to PMLVI) contain the nuclear localization signal (NLS)
and are localized in the nucleus, whereas PMLVII (cPML) lacks
the NLS and is found in the cytoplasm. It has been reported pre-
viously that PML-null primary cells are resistant to TGF-b-
dependent induction of apoptosis and that cPML is an essential
activator of TGF-b signaling. The defects in TGF-b responses
are rescued by the restoration of a cPML. Here we show that
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 203
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Host–pathogen interactions POSTERS
interferon (IFN) a potentiates TGF-b-mediated apoptosis. Inter- myeloid phagocytic target cells. Firstly, one class of CyaA con-
estingly, TGF-b induces the nuclear PML conjugation to SUMO formers forms small cation-selective pores that can cause colloid-
and the shift of PML from the nucleoplasm to the nuclear osmotic lysis of target cells. In parallel, other CyaA conformers
matrix. This shift leads to the recruitment of caspase 8 within translocate the adenylate cyclase (AC) enzyme domain of the
PML NBs where PML and caspase 8 were found colocalizing. toxin across the membrane of target cells, thus effectively sub-
This process is followed by caspase-dependent PML degradation verting the bactericidal functions of cells by unregulated catalysis
leading to NB disruption. Our findings add a new complexity to of cytosolic ATP conversion into cAMP, a key signaling mole-
the cooperation between TGF-b and PML NBs in mediating cule. The dual function CyaA toxin thus belongs to the RTX
apoptosis. (Repeats-in-ToXin) family of pore-forming toxins. However, it
forms notably smaller pores than other pore-forming toxins from
the RTX cytolysin family. Here we investigated the specific seg-
P.08-034-Mon ments of CyaA involved in the reduction of the pore conductance
Conserved RNA secondary structure of the NS and diameter. Using planar lipid bilayers made of DPhPG we
segment as a possible additional influenza investigated single pores formed by CyaA and several mutant
viral pathogenicity factor toxin variants and their pore selectivities. By measuring pore con-
ductance in the presence of non-electrolytes of known diameter,
I. Baranovskaya1,2, M. Sergeeva2, A. Brodskaia1,2, A. Taraskin1,2,
we have studied the overall shape of CyaA pores. Our results
A. Vasin1,2
1 suggest that CyaA pores adopt two distinct states with distinct
Peter the Great St. Petersburg Polytechnic University, Saint-
pore selectivities.
Petersburg, Russia, 2Research Institute of Influenza, Saint-
Petersburg, Russia
Nowadays, RNA secondary structure elements are well known to P.08-036-Wed
be vitally important for the life cycle of RNA-containing viruses, Study of synergistic antibacterial action of
including influenza virus. Here, we investigated a predicted hair- host defense peptides with varied antibiotic
pin structures in the (+)-RNA of the influenza NS-segment (nu-
compounds and their combined action on
cleotide positions 82–148 and 497–564). Both regions are located
near the 50 and 30 splice sites: the first region within the NS1 mammalian cells in vitro
open reading frame, while the second – within the NS1 and NEP O. Shamova1,2, M. Zharkova1, O. Golubeva3, A. Kolobov4,
open reading frames. The secondary structures in these regions M. Smirnova4, G. Afinogenov2, A. Afinogenova2,
differ in various influenza strains. The highly pathogenic H5N1 E. Romanovskaya2, D. Orlov1
1
strain has more stable secondary structure in the second region FSBSI Institute of Experimental Medicine, Saint Petersburg,
compared to other strains. Since evolution of the NS gene is Russia, 2Saint Petersburg State University, Saint Petersburg,
strongly associated with host adaptation, it was intriguing to Russia, 3Institute of Silicate chemistry RAS, Saint Petersburg,
investigate these structures as an additional factor of the Russia, 4State Research Institute of Highly Pure Biopreparations,
pathogenicity of the influenza virus. Based on RNA secondary Saint Petersburg, Russia
structure predictions of the NS-segments of more than 25,000 Innate immune system plays a pivotal role in realization of host–
sequences from different influenza A viruses, we chose single pathogen interactions. Among the key effector molecules of the
nucleotide positions which may be important in hairpin structure innate immunity there are host defense peptides (referred also as
formation and stability. We obtained several combinations of antimicrobial peptides - AMPs) containing mainly in neutrophils
mutations in the corresponding regions of the NS-segment by and epithelial cells. Most of AMPs posses a potent activity
site-directed mutagenesis. Then, we experimentally demonstrated towards pathogenic microorganisms. The synergy of antibacterial
the existence of the previously predicted RNA secondary struc- action of AMPs with different antibiotic compounds including
tures by electrophoresis in the native and denaturing conditions. other AMPs or proteins of neutrophil granules as well as with
To directly test the functionality of conserved stem-loop struc- clinically used antibiotics (AB) has been reported before. It is
tures, we undertook reverse genetic experiments and obtained known that most of natural AMPs exert some toxic effects for
viral strains based on A/Puerto Rico/8/1934(H1N1) virus with human cells, but the toxicity of the combinations of antimicrobial
different secondary structural characteristics on the (+)-RNA NS- compounds for the host cells is much less investigated. The aim
segment. According to preliminary data, the infectious titer of of this study is an exploration of a combined action of several
the strain containing the stable secondary structures was ten-fold structurally different AMPs (beta-hairpin protegrin 1 (PG1), lin-
higher in comparison to others. The pathogenicity and other ear proline-rich caprine bactenecin, alpha-helical human catheli-
characteristics of novel assembled strains are under further inves- cidin LL-37, alpha- and beta-defensins) with varied conventional
tigation. AB towards bacteria and mammalian cells in vitro. Using broth
microdilution assay and checkerboard titration approach for
fractional inhibitory concentrations determination we have shown
P.08-035-Tue that more often the cases of synergistic antibacterial action were
Mapping the inner profile of pores formed by stated for combinations of AMPs with AB that act via inhibition
adenylate cyclase toxin of Bordetella pertussis of protein or nucleic acids synthesis in bacterial cells. Effects of
A. Sukova1, R. Fiser1,2, P. Sebo1, J. Masin1 AMPs – AB combinations on bacterial membranes integrity and
1
Institute of Microbiology of the Czech Academy of Sciences, bacterial metabolism have been explored. In most cases the toxic-
Prague, Czech Republic, 2Faculty of Science, Charles University, ity of combinations of AMPs with AB for normal mammalian
Prague, Czech Republic cells was not increased in comparison with effects of individual
substances. But we revealed a synergy for PG1 and doxorubicin
The whooping cough agent, Bordetella pertussis, secretes the
toward several types of mammalian cultured tumor cells, and
adenylate cyclase toxin-hemolysin (CyaA, ACT, AC-Hly), a
studied a mechanism of their combined action. The obtained data
major virulence factor that plays a crucial role in bacterial colo-
point for the future prospect of combined application of AMPs
nization of host respiratory tract. CyaA exhibits two distinct and
mutually independent biological activities on CD11b expressing
204 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Host–pathogen interactions
based drugs and AB to combat infections or cancer. This work quality without genetic modifications are of importance. The aim
was supported by RFBR grant 17-04-02177. of this research is to investigate the effects of a range of Zn con-
centrations on PSD resistance in the soybean cultivar Galina. As
an essential micronutrient, Zn is required for the function of
P.08-037-Mon transcription factors, enzymes, and structural proteins. The
Enzymatic desialylation of lung epithelium hypothesis is that Zn distribution in the roots and leaves, and
increases binding of lectin PAIIL, activation of defence signalling pathways may affect the severity
Pseudomonas aeruginosa virulence factor of P. longicolla infection. To investigate this, soybean plants were
grown hydroponically under different Zn availability (deficient
P. Hodek1, B. Kubıckova1, J. Rychnova1, K. Dostalova1,
0.03 lM up to sublethally toxic 10 mM) for 4 weeks prior to
K. Vyhnalov a1, J. Mr
azkova2, V. Mandys3, M. Wimmerova2,
start of infection. Distribution of Zn and other elements in the
M. Stiborov a1
1 leaves and roots was measured in vivo by imaging micro X-ray
Department of Biochemistry, Faculty of Science, Charles
fluorescence (lXRF) and related to the level of infection. In addi-
University, Prague, Czech Republic, 2Department of Biochemistry,
tion, combined effects of Zn and P. longicolla on photosynthetic
Faculty of Science, Masaryk University, Brno, Czech Republic,
3 performance were determined by imaging measurements of
Department of Pathology, Third Faculty of Medicine, Charles
chlorophyll fluorescence kinetics (OJIP and Kautsky transients,
University, Prague, Czech Republic
QA reoxidation). The results should contribute to better under-
Cystic fibrosis (CF) is one of the most common human autosomal standing of the mechanisms of metal-induced plant immunity
recessive diseases. This genetic disorder is associated with and the cross-talk between abiotic and biotic stress, as well as to
increased susceptibility of lungs to bacterial infections, which fre- the importance of biofortification of crops for securing maximal
quently develop to life-threatening conditions for CF patients. In yield and food production.
this regard Pseudomonas aeruginosa (PA) is the most dangerous
bacterial pathogen. Among PA virulence factors lectins seem to
play crucial role in the PA adherence on CF airway epithelium. P.08-039-Wed
PA lectin, PAIIL, showing high affinity for L-fucose, is assumed Type IIA topoisomerase in Entamoeba is
to be namely involved in the bacteria binding. Likely, the low-sia- essential for its encystation
lylation of glycoconjugates on CF epithelial cells provides exposed S. S. Varghese1, S. K. Ghosh2
saccharide residues suitable for the PAIIL interaction. To study 1
Indian Institute of Technology Kharagpur, Kharagpur, India,
the PAIIL involvement in the adherence of PA on airway epithe- 2
Department of Biotechnology, Indian Institute of Technology
lium the mouse model mimicking CF lung conditions was devel- Kharagpur, Kharagpur - 721302, India
oped. The status of low-sialylation was induced by the
intratracheal instillation of neuraminidase, the enzyme cleaving Topoisomerases are a group of ubiquitous enzymes, associated
terminal sialic acid. For our experiments PAIIL was recombi- with all key DNA processes and are often involved in stress
nantly expressed and coupled with high-performance fluorescent response across the biological world. Owing to its indispensable
label, DyLight 488. The saccharide binding of the DyLight 488 nature for cell survival, it has been extensively explored as a drug
labeled PAIIL was checked by using a red blood cell agglutination target for various diseases including cancer as well as infectious
assay. The PAIIL labeling did not alter the lectin saccharide bind- diseases. The genome of E. histolytica, the causative organism of
ing affinity. Next, tissue slices of lungs from neuraminidase-treated amoebiasis, has seven unexplored putative topoisomerases and
and untreated mice were mounted on microscope slides and incu- five of them have orthologs in the reptilian counterpart, E.
bated with DyLight 488 labeled PAIIL. Finally, tissue specimens invadens. The stage conversion of Entamoeba from a pathogenic
were examined on an inverted fluorescence microscope (Nikon trophozoite to an infective cyst is responsible for the host-to-host
Eclipse TE2000-U). In the neuraminidase-treated group the cuboi- transmission of the disease. Formation of a tetranuclear stage in
dal epithelium lining the surface of respiratory bronchioles showed the late hours of encystation is one of the key features. However,
an apparent fluorescence when compared with untreated mice. the exact process behind this phenomenon is still not understood.
These results support the assumption that PAIIL mediates the PA In this study, we have identified a Type IIA topoisomerase of
binding on CF airway epithelium. Thus, lectin PAIIL is a candi- Entamoeba that is highly upregulated both at transcription as
date target in preventing PA lung infections of CF patients. well as protein level during the later stages of encystation and
various other stress conditions, like heat shock and oxidative
stress. Silencing of that gene has shown a significant impact on
P.08-038-Tue the transcription of meiosis-specific genes as well as on the viabil-
The role of zinc in soybean resistance to ity, and encystation efficiency of Entamoeba.
Phomopsis longicolla
F. Morina, A. Mijovilovich, I. Koloniuk, H. K€ upper
Biology Centre, Academy of Sciences of the Czech Republic,
Institute of Plant Molecular Biology, Ceske Budejovice, Czech
Republic
Soybean is among the most important crops for global agricul-
ture, food and nutrient security. The major agent affecting soy-
bean yield and seed quality is the pathogenic fungus Phomopsis
longicolla, which causes PSD (Phomopsis seed decay) disease all
over the world. Favourable factors for PSD spread are frequent
rainfalls and high humidity during warm periods of soybean mat-
uration and at this time P. longicolla infects all plant organs.
Although several soybean cultivars have been described as toler-
ant to PSD, the search for resistant traits is still ongoing, which
is why alternative means of improving plant vigour and seed
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 205
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Host–pathogen interactions POSTERS
P.08-040-Mon disulfide bond, the study of the role of these cysteines in virus life
cycle is of a high interest. To determine the importance of M-
Mutations of selected amino acids of Mason-
PMV CA cysteines, we introduced their mutations in both bacte-
Pfizer monkey virus capsid protein and their rial and proviral vectors and evaluated the impact of these muta-
effects on its life cycle tions in vitro and also in M-PMV transfected cells. In in vitro
ckova1, B. Vokata1, R. Pıchalova1, V. Todorovova1,
L. Ctvrte assembly system we observed the formation of immature virus-
P. Klımova1, A. Dostalkova2, I. Krızova2, M. Rumlova2, like particles and mature cores and in tissue culture cells we
P. Ulbrich1, T. Ruml1 focused on intracytoplasmic immature particle formation, their
1
Department of Biochemistry and Microbiology, University of budding, maturation and virus infectivity. Our data revealed that
Chemistry and Technology Prague, Technick a 3, 166 28, Prague 6, the presence of both C193 and C213 of M-PMV CA is necessary
Prague, Czech Republic, 2Department of Biotechnology, University for immature and mature particle assembly and virus infectivity.
of Chemistry and Technology Prague, Czech Republic, Prague, However, if we mutated only one of conserved cysteines, we did
Czech Republic not observe any serious defects in M-PMV assembly and mature
Retroviruses belong to the group of enveloped RNA viruses, core formation. We concluded that the presence of both con-
causing lifelong, sometimes life-threatening infections. To inhibit served cysteines is important for keeping proper CA structure
their life cycle it is important to understand its steps into the and virus particle stability and that their mutations probably
detail. The potential target for new antivirals can be protein-pro- cause virus arrest at some process occurring shortly after the
tein interaction interfaces within retroviral structural proteins, virus entry.
thus inhibiting the assembly and/or influencing stability of imma-
ture and mature retroviral particle. The most important interac-
P.08-042-Wed
tions are mediated mostly by amino acids present in retroviral
capsid protein (CA), that consists of two domains, N-terminal Differential expression of Fusarium oxysporum
domain (NTD) and C-terminal domain (CTD), connected by genes upon infection of susceptible and
unstructured flexible linker. Based on 3D structure of Mason-Pfi- resistant flax genotypes
zer monkey virus (M-PMV) immature particle, we predicted R. Novakovskiy1, G. Krasnov1, T. Rozhmina1,2, P. Kezimana1,3,
amino acids in both NTD and CTD that seemed to be critical E. Borkhert1, N. Melnikova1, A. Dmitriev1
1
for interactions in immature and mature retroviral particle, Engelhardt Institute of Molecular Biology, Russian Academy of
namely N89, G119, E175, D179, Y180 and E187 (numbered from Sciences, Moscow, Russia, 2All-Russian Research Institute for
the first amino acid of CA). Then we mutated these amino acids Flax, Torzhok, Russia, 3Peoples’ Friendship University of Russia
and studied the effect of these mutations on in vitro formation of (RUDN University), Moscow, Russia
immature-like virus particles and also on selected virus life cycle
Fusarium oxysporum sp. lini is one of the most harmful pathogens
steps in infected cells. N89 and G119 mutants did not form any
of flax, which leads to fusarium wilt and causes more than 20%
immature particles in vitro or in vivo, suggesting the critical role
reduction in yield. Flax varieties differ considerably in their resis-
of these two CA NTD amino acids in CA-CA interactions.
tance to fusarium wilt. Alterations in the expression of a number
Mutations of amino acids present in CA CTD, i.e. E175, D179,
of flax genes between resistant and susceptible genotypes under
Y180 and E187, did not inhibit immature particle formation, but
the infection were shown. However, it is unknown whether the
these particle were often unstable and with different morphology
expression of F. oxysporum genes differs when this pathogen
in the comparison with wild type virus. Except E187 mutant they
affects flax genotypes with diverse resistance. Our work was
did not bud from infected cells, thus confirming the importance
devoted to study this issue. Flax seedlings of four flax varieties
of studied amino acids in virus life cycle. Description of interac-
with different resistance (resistant – Dakota and # 3896, suscepti-
tion interfaces in CA-CA in both immature and mature virus
ble – AP5 and TOST) were inoculated with isolate #39 of F.
particle, followed by targeted design of molecules blocking these
oxysporum from the collection of the All-Russian Research Insti-
amino acid motifs may lead to finding of new inhibitors of retro-
tute for Flax. Forty-eight hours after infection, the roots were
virus life cycle.
harvested and total RNA was isolated. RNA samples were
obtained in duplicate for each variety. The concentration and
P.08-041-Tue quality of RNA were assessed using Qubit 2.0 fluorometer and
Agilent 2100 Bioanalyzer. To prepare DNA libraries for high-
Study of the role of cysteines present in
throughput sequencing, the TruSeq Stranded Total RNA Sample
Mason-Pfizer monkey virus capsid protein Prep Kit (Illumina) was used. A total of 8 cDNA libraries were
B. Vokat a1, R. Pıchalova1, T. F€
uzik2, A. Dostalkova3, obtained, and their quality was evaluated using Agilent 2100 Bio-
I. Krızov
a3, P. Ulbrich1, M. Rumlova3, T. Ruml1 analyzer. The libraries were mixed and sequenced on NextSeq
1
Department of Biochemistry and Microbiology, University of 500 sequencer (Illumina) using 80 nucleotides pair-end reads.
Chemistry and Technology Prague, Technick a 3, 166 28, Prague 6, About 25 million pair-end reads were obtained for each type of
Prague, Czech Republic, 2CEITEC - Central European Institute of the experiment, and 30–40% of reads were mapped on F. oxyspo-
Technology, Masaryk University, Brno, Czech Republic, rum genome, while other reads belonged to flax. The number of
3
Department of Biotechnology, University of Chemistry and reads for each F. oxysporum transcript was assessed, and expres-
Technology Prague, Czech Republic, Prague, Czech Republic sion analysis was performed using the edgeR software. Fungus
Retrovirus immature particle assembly is driven mostly by the genes with differential expression upon infection of susceptible
oligomerization of their structural polyprotein Gag. Gag-Gag and resistant flax genotypes were revealed that brings the novel
interactions are mediated by inter and intra protein interactions knowledge on plant-pathogen interaction. This work was finan-
among their capsid (CA) domains. Mason-Pfizer monkey virus cially supported by the Russian President Grant MК-
(M-PMV) CA contains three cysteines at positions C82, C193 5828.2018.4.
and C213, counted from the first amino acid of CA. Since C193
and C213 are highly conserved among the majority of retro-
viruses and their distance enables them to form intramolecular
206 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
P.09-001-Mon
P.08-044-Tue
A predicted unstructured C-terminal loop
Epstein–Barr Virus LMP1 induces IL-8
domain of SIRT1 is required for cathepsin B
expression via regulation of the NF-kB
cleavage
pathway in human gingival epithelial cells
A. Kumar1, Y. Daitsh1, L. Ben Aderet1, O. Qiq1, J. Elayyan1,
K. Imai
G. Batshon1, E. Reich1, S. Engel2, M. Dvir-Ginzberg1
Department of Microbiology, Nihon University School of 1
The Hebrew University of Jerusalem, Jerusalem, Israel, 2Ben
Dentistry, Tokyo, Japan
Gurion University of the Negev, Beer-Sheva, Israel
Periodontitis is a major public health problem that effects over
Previous reports demonstrated that a SIRT1 C-terminal trunca-
half of the adult population worldwide. Although traditional
tion, generating a 75 kDa SIRT1 polypeptide (75SIRT1), is facili-
microbiological research on periodontitis has focused on putative
tated by cathepsin B at amino acid (a.a.) H533. Here we identified
bacteria, emerging evidence implicates an association between
N-terminally intact Sirt1 variants at the 75 kDa range, which
Epstein–Barr virus (EBV) and periodontitis. However, the molec-
appeared to be augmented with ageing in various mice tissues.
ular mechanism underlying the role of EBV in the pathogenesis
Variable N-terminally intact SIRT1 variants, were increased in
of periodontitis is unknown. This study investigates the effect of
lung, liver, brain, heart and kidney as a function of age, and corre-
EBV-encoded latent membrane protein 1 (LMP1) on IL-8 protein
lated with increased cathepsin B levels. To examine whether the
expression in human gingival epithelial cells. We performed RT-
H533 amino acid is essential for cathepsin B cleavage, we generated
PCR and luciferase assays to investigate the effects of LMP1
SIRT1 H533A (in the P1 site of SIRT1), which did not affect the
transfection on IL-8 expression and NF-kB activation in the
extent of cathepsin cleavage. Next, a SIRT1 mutant protein lacking
human gingival cell line Ca9-22. Western blot analysis and
a portion (a.a. 528–543) of the predicted C-terminus loop domain
enzyme-linked immunosorbent assay were used to detect protein
of SIRT1 (denoted “DSIRT1”), was generated, and presented rela-
expression. LMP1 induced dose-dependent production of IL-8
tive resistance to cathepsin B cleavage, compared to full-length
and activation of NF-kB. LMP1-induced IL-8 expression was
SIRT1 (flSIRT1) in-vitro and in cell cultures. Moreover, DSIRT1
inhibited by dominant negative mutant of IkBa. In addition,
displayed similar enzymatic activity as flSIRT1, based on analysis
mutated LMP1 lacking the NF-kB activation domain failed to
of H3K9/14 and RelA acetylation. Cells expressing DSIRT1 were
induce IL-8 expression. LMP1 activates NF-kB, leading to the
more likely to apoptose under pro-inflammatory stress, given that
induction of IL-8 expression in human gingival epithelial cells.
it possessed lower capacity to bind cytochrome C, compared to
Our findings suggest that LMP1 is involved in the pathogenesis
75SIRT1. In all, the data support that the predicted unstructured
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 207
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
15aa loop motif embedded in the C-terminal domain of SIRT1, is processes driving to fibrosis and chronic kidney disease (CKD).
susceptible to proteolytic cleavage, leading to the formation of Men are more prone to AKI and CKD than women and it is
variable N-terminally intact SIRT1 truncated isoforms in various accepted that androgens participate on that. The molecular
tissues, which may contribute to cell survival under inflammatory mechanisms involved in regeneration as well as in gender-related
insult. outcomes upon injury remain to be elucidated. We postulate that
the identification of differentially expressed genes in male and
female kidney pigs, both in basal and in IRI conditions might
P.09-002-Tue unravels genes and pathways useful to understand the different
Spherical particles of tobacco mosaic virus outcomes observed in men and women. We aim to provide new
enhance the immunogenic effect of the candidate repair regulators able to promote restoration of kidney
inactivated rabies vaccine function. Renal IRI was performed in female and male pigs and
mice to identify genes of translational relevance for humans that
E. Ryabchevskaya1, I. Matveeva2, E. Trifonova1, E. Donchenko1,
could be also studied in mouse models. Pre-ischemic, ischemic
N. Nikitin1, J. Atabekov1, O. Karpova1
1 and post-ischemic kidney tissues from male and female pigs were
Lomonosov Moscow State University, Moscow, Russia, 2All-
collected for microarray assays. Moreover, systems biology-based
Russian Research and Technological Institute of Biological
mathematical models for the analysis of microarray data were con-
Sciences Industry, Shchelkovo, Russia
ducted. The most promising targets that exhibit sexual dimorphism
Spherical particles of the tobacco mosaic virus (TMV SP) are along the injury/regeneration process have been selected and char-
obtained by thermal denaturation and remodeling of the TMV acterized. The mRNA levels, the protein expression and localiza-
virions. We have previously described a number of specific and tion in the kidney have been assessed. The early results strongly
promising properties of TMV SPs such as their high stability and suggest that the selected targets are potentially androgen-regulated.
biosafety. Moreover, TMV SPs were demonstrated to adsorb tar- In order to further study the molecular mechanisms of these tar-
get proteins of different nature on their surface. Complexes of gets, in vitro IRI models of pig and human PTEC cultured cells
TMV SPs and proteins were shown to dramatically enhance the are currently under development. An in vitro model of kidney
immune response in mice against the target antigenic protein, sup- organoids where IRI is induced is also being established.
posing the TMV SPs to act both as an antigen-presenting plat-
form and an adjuvant. These facts make TMV SP a promising
candidate for a new safe adjuvant of the modern vaccines. We P.09-004-Mon
continue the study of the adjuvant properties of TMV SPs testing Does vitamin D receptor play a role in the
of their potential to enhance the immunity against the rabies pathogenesis of Behcet’s disease?
virus. For comparative study of the immune-stimulating (IS) N. E. Dal1, G. Keskin2, P. Cerci2, U. Olmez2
potency of TMV SPs, the standard NIH test was performed with 1
Dokuz Eylul University Institute of Health Sciences Department
a reference inactivated rabies vaccine based on the vaccine strain of Molecular Medicine, Izmir, Turkey, 2Ankara University, School
“Shchelkovo-51” and the currently licensed adjuvant-free IRV of Medicine, Department of Clinical Immunology, Ankara, Turkey
(RabikanTM, Russia). RabikanTM is highly effective and success-
fully applied for rabies prevention. For comparison RabikanTM Behcet’s disease(BD) is an autoinflammatory disorder with
was adjusted with TMV SPs or Freund’s incomplete adjuvant unknown etiology. Vitamin D has a crucial role for immune sys-
(IFA). According to the NIH test protocol all vaccine variants tem and the functions of vitamin D depends on vitamin D recep-
were applied for intraperitoneal immunization of mice (immu- tor(VDR). The VDR effects many genes through vitamin D-
nized twice: on days 0 and 7). Then mice were subjected to the VDR ligand and the defects of this pathway may be associated
lethal challenge against the rabies virus of CVS strain, injected with pathogenesis of autoimmune diseases. In this research
intracerebrally. According to the obtained results 300 lg of TMV rs1544410(G>A), rs2228570(T>C), rs7975232(G>T) and rs731236
SPs enhance the IS potency of the RabikanTM by 0.2 IU and the (T>C) single nucleotide polymorphisms(SNP) in VDR gene were
IFA enhances it by 0.3 IU, while the IS potency of the intact studied in BD patients in Turkish population. 150 patients with
RabikanTM was 1.5 IU. This result demonstrates the ability of BD and 150 healthy controls (HCs) were included and genotyp-
TMV SPs to efficiently stimulate the immune response (compara- ing of each SNP was carried out by PCR/RFLP. Then as a pilot
ble with the IFA) and thus provides further evidences of the high study, soluble VDR levels were measured by ELISA in 32 BD
clinical potential of this novel immunostimulatory compound. patients(12 active, 20 remission) and 30 HCs. There are signifi-
Funding: Russian Science Foundation (grant No. 14-24-00007). cant differences between patients and HCs in rs1544410,
rs2228570 and rs731236 genotypes (respectively; P = 0.04,
P = 0.007, P = 0.01). The alleles of rs1544410 and rs2228570
P.09-003-Wed were evaluated with clinical features and patients with ocular
Renal ischemia-reperfusion injury in a pig lesion had higher percentage of rs1544410 A allele (P = 0.013)
model reveals gender-specific expressed genes and patients with oral aphthae, positive pathergy test and arthri-
tis had more rs2228570 C allele than patients without this clinical
as potential new biomarkers of renal injury/ features (respectively; P = 0.027, P = 0.037, P = 0.02). In the
regeneration processes driving to chronic analysis of the VDR expression; mean VDR concentrations were
kidney disease 18.69 8.77 ng/mL for patients in active period;
S. Nemours, L. Castro, D. Ribatallada, M. Aranda, M. Ferrer, 31.73 11.05 ng/mL for patients in remission period and
J. Morote, A. Meseguer 32.49 18.12 ng/mL for HCs. Patients in active period had sig-
Vall d’Hebron Research Institute, VHIR, Barcelona, Spain nificantly lower serum VDR level than patients in remission per-
iod and HCs (respectively; P = 0.002, P = 0.04). Patients
Kidney diseases are a global public health problem, that is reach-
carrying rs2228570 C allele were found to had lower VDR level
ing epidemic proportions. Renal ischemia/reperfusion injury
than patients with TT genotype (P = 0.033). In conclusion VDR
(IRI) is a major cause of acute kidney injury (AKI) leading to
may have possible role in the pathogenesis of BD and VDR
injury of proximal tubule epithelial cells (PTEC). After injury,
SNPs may be affecting VDR level. Furthermore rs1544410-
the kidney can either regenerate or be engaged in remodeling
208 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
rs2228570 SNPs may associated with increased risk of several protein, ferritin, and the most potent glycation agent, methylgly-
clinical manifestations. oxal. In these experiments, commercially available horse spleen
ferritin was in vitro glycated with methylglyoxal; fluorescence of
glycation products, iron uptake, reductive iron release and hydro-
P.09-005-Tue gen peroxide production were assayed. Glycation of ferritin
Vitamin D3 modulates impaired upstream NF- resulted in detectable structural changes, but the ferroxidase
jB-associated signalling pathways and NF-jB activity, iron release in classical reductive assay or the diameter
downstream target genes in rat bone marrow of pores in ferritin were not altered. However, the interaction of
methylglyoxal with ferritin protein shell did increase production
after chronic glucocorticoid treatment of hydrogen peroxide, especially in the presence of iron. In con-
O. Lisakovska, I. Shymanskyi, D. Labudzynskyi, V. Vasylevska, clusion, ferritin is a stable protein resistant to functional alter-
M. Veliky ation due to glycation, this modification can nevertheless increase
Palladin Institute of Biochemistry of the National Academy of oxidative stress in the cell. Possibly this can represent one of the
Sciences of Ukraine, Kyiv, Ukraine mechanisms through which long-term complications of diabetes
Osteoporosis is the common side effect of glucocorticoid (GC) develop.
therapy. NF-jB is considered as integrating link between GC
receptor (GR) and RANK (receptor activator of NF-jB)⁄
RANKL (RANK ligand) signalling pathways, involved in bone P.09-007-Mon
remodeling. Little is known about GC-evoked disturbances of The role of adiponectin in streptozocin-
the NF-jB-associated axes in bone marrow (BM) cells and ways induced diabetic rat placental development
to prevent them by modulating vitamin D3 (D3) receptor (VDR) Z. Avcil1, A. hanikoglu1, E. T. Korgun2, D. Kipmen-Korgun1
signalling. We studied GC-induced changes in upstream NF-jB- 1
Department of Biochemistry, Akdeniz University, School of
associated pathways – GR, RANK⁄RANKL and NF-jB down- Medicine, Antalya, Antalya, Turkey, 2Department of Histology
stream target genes – VEGF and TNFa in BM depending on D and Embryology, Akdeniz University, School of Medicine, Antalya,
bioavailability. Female Wistar rats received prednisolone (5 mg⁄ Antalya, Turkey
kg b.w.) with and without 1000 IU⁄kg b.w. of D3 (for 30 days).
A healthy pregnancy is closely associated with normal placental
Western blotting, RT-qPCR, ELISA, flow cytometry and confo-
cal microscopy were used. Prednisolone caused a decrease in GR development. Inadequate placental development may cause clini-
protein level and the number of GR+ BM cells. GC-induced cal complications such as intrauterine growth retardation,
preeclampsia and diabetes. In order to prevent the adverse preg-
increase in RANKL, RANK protein levels and the number of
RANK+ osteoclast precursors (OCPs) in BM, peripheral blood nancy outcomes, it is important to understand the hormonal
and spleen were seen. In response to RANK activation we found changes and altered glucose metabolism that are associated with
an increase in the level of phospho-NF-jB and decrease in IjB. diabetes mellitus during pregnancy. Adiponectin, an adipokine,
regulates glucose and lipid metabolism and its concentration is
VEGF and TNFa were shown to be declined after GC treatment
while caspase-3 elevated. These changes were accompanied by negatively correlated with diabetes. This study aims to evaluate
D-deficiency and down-regulated CYP27B1 and VDR expression. the role of adiponectin on placental development in diabetic con-
Notably, we observed VDR/RANK co-localization in OCPs. ditions. This study comprises diabetic and control groups. The
control group underwent injection of saline. Diabetes was pro-
Vitamin D3 administration elevated GR level and prevented dis-
turbances of the RANK⁄RANKL in BM, which may be associ- vided by injection of streptozotocin (STZ, 50 mg/ kg). Female
ated with improved D bioavailability and VDR signalling. D3 rats whose blood glucose value is higher than 200 mg/dL were
considered as diabetic. Adiponectin, its receptors (AdipoR1 and
lowered pNF-jB and inhibited its translocation to the nucleus
AdipoR2), and its downstream molecules (PPARa, PPARc) were
contributing to normalization of VEGF, TNFa, caspase-3 levels
and OCPs pool. Thus, GC-induced changes in upstream NF-jB- detected in the control and STZ-induced rat placentas on day 14,
associated signalling pathways and NF-jB downstream target 16, 18, and 20 of pregnancy by using Western Blot and RT-PCR
techniques. Serum concentrations of adiponectin were assessed
genes are associated with abnormal vitamin D auto⁄paracrine sys-
tem in BM and can be ameliorated by D3 treatment. by ELISA method. We showed that there is an inversely associa-
tion between adiponectin, PPARs and diabetes. In the present
study, we observed that adiponectin and its receptors levels
P.09-006-Wed reduced in diabetic conditions. Furthermore, maternal serum adi-
Ferritin modified with methylglyoxal produces ponectin levels reduced during the pregnancy in diabetic rats. We
also showed that the levels of PPARa, PPARc and RXRa
reactive oxygen species but otherwise resists expression were lower in diabetic rats compare to control groups.
functional alteration Based upon those observations above, we believe that adiponec-
A. Rybnik arov
a, J. Pl
atenık tin has a significant role in placental development. In conclusion,
Institute of Medical Biochemistry and Laboratory Medicine, First pregnancy and diabetes is associated with adiponectin levels.
Faculty of Medicine, Charles University, Prague, Czech Republic
Diabetes mellitus affects about 8. 5 per cent of the world’s popu-
lation and leads to long term complications such as blindness,
amputation, neuropathic pain and renal failure. With diabetes,
glycolysis overflows and its metabolites convert into alternative
products such as methylglyoxal. Simultaneously, a relatively free
and reactive iron in the cell called labile iron pool (LIP), is
increased. Some studies indicate that methylglyoxal and LIP play
an important role in the pathogenesis of diabetic complications,
but how these two factors are related is not yet known. This
study focuses on the interaction of the main cellular iron storage
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 209
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
210 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 211
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
P.09-014-Tue hypoxia. Taken together, these results indicate that iEPR mediate
hypoxia-induced apoptosis in PTC. Due to their high metabolic
Stress-protective action of Dalargin intranasal
activity, PTC have a large oxygen demand so that they become
form, shown in the dynamics of average readily hypoxic in several types of both acute and chronic kidney
weight molecules index distribution in blood disease. Our work suggests that the apoptotic death of hypoxic
serum of laboratory rats subjected to stress PTC might be prevented by targeting of the PGE2 uptake trans-
ulcerogenesis porter.
I. Cheretaev, V. Nikolskaya, E. Minina, E. Birukova, E. Chuyan,
M. Ravaeva
V.I. Vernadsky Crimean Federal University, Simferopol, Russia
P.09-016-Mon
Unusual inorganic pyrophosphatase from
There is convincing evidence pointing to expressed biological
Mycobacterium tuberculosis: from structural
effectiveness of intranasal Dalargin application (10–9–10–11 M) in
case of overcoming various complications of physiological stress and functional features to effective inhibitors
in an organism [Minina, Cheretaev, 2016]. Definition of average R. Romanov1, L. Vainonen1, M. Shuvalov1, D. Yanvarev2,
weight molecules (AMW) [Nikolskaya, 2012] widely used as an E. Rodina1
1
integral marker of the biochemical state of the body at various Lomonosov Moscow State University, Moscow, Russia,
2
pathologies and stress. The aim of this work was to test the pres- Engelhardt Institute of Molecular Biology, Moscow, Russia
ence of stress-protective action Dalargin intranasal form by Soluble inorganic pyrophosphatases (PPases) are enzymes essen-
studying the dynamics of average weight molecules (AWM) in tial for all organisms. They hydrolyze inorganic pyrophosphate,
blood serum of laboratory rats in stress-induced ulcerogenesis. thus providing the driving force for key biosynthetic reactions.
The experiments performed on 32 male Wistar rats weighing Most of well-characterized Family I PPases share highly con-
180–220 g, which divided into 4 groups. Rats in the control served structure of their active site; however, some PPases have
group (n = 8) and in group II (n = 8) was administrated within unique functional properties. One of them, PPase from Mycobac-
28 days intranasally 0.1 ml of 0.9-% NaCl solution. Animals of terium tuberculosis (Mt-PPase), is the possible target for the
groups I (n = 8) and III (n = 8) received intranasally 0.1 ml of rational design of anti-tuberculosis agents. Our previous studies
Dalargin (0.2 mkg/kg) within 28 days. On day 28 the animals have shown that Mt-PPase is evolutionary isolated from the
of groups II (n = 8) and III (n = 8) were subjected to stress majority of Family I PPases and has several unique features. In
ulcerogenesis for 60 min [Porsolt, 1993]. Index distribution the present work, these functional peculiarities were studied in
(ID280/254) of AWM in the serum all groups of rats using the detail. Enzyme kinetics was used to describe interactions of Mt-
method, based on the precipitation of protein components of PPase with known effectors of other Family I PPases. Sedimenta-
20% trichloroacetic acid [Gabrielian et al., 1987] was determined. tion analysis was used to explore different oligomeric forms of
It is shown that in the blood serum of laboratory rats after expo- Mt-PPase. Site-directed mutagenesis was used to understand the
sure Dalargin ID280/254 was significantly reduced by 54% (n = 8; role of specific structural features of Mt-PPase. Molecular dock-
p≤0.01) compared to control, and under the influence of stress ing and molecular dynamics was used to simulate particular steps
ulcerogenesis (n = 8; p≤0.01) – by 38.73%. With the combined of catalytic mechanism, binding of effectors, and to predict the
effects of stress ulcerogenesis and Dalargin the ID280/254 almost location of effector binding sites. Virtual screening and in vitro
back to the values of the control group and slightly higher, high-throughput screening was used to find novel effectors. We
amounting to 112% (n = 8; p≤0.01). Thus, we can talk about a found that in contrast to most of other Family I PPases, calcium
stress-protecting dalargin effect, which helps to maintain a natu- and fluoride ions do not inhibit Mt-PPase, while substrate ana-
ral relation of physiologically important AWM fractions in an logs (bisphosphonates) activate Mt-PPase under various condi-
organism under stress, showing the activity of stress-realizing tions. Oligomeric forms of Mt-PPase with unusual properties
(254 nm) and stress-limiting systems of the organism (280 nm). were obtained after treatment of hexameric form with water-
organic solutions. Three symmetry-related cavities for binding
positively charged compounds were predicted in Mt-PPase hex-
P.09-015-Wed amer. Enzyme-substrate complex was modeled and the possible
Role of PGE2 in hypoxia-induced death in interconnection between the structural and functional peculiari-
human proximal tubular cells ties of Mt-PPase was suggested. Two novel classes of effectors
C. Garcıa-Pastor1, S. Benito-Martınez1, A. Fernandez-Martınez2, were predicted in silico. Three new inhibitors and one new activa-
J. Lucio-Caza~ na1 tor of Mt-PPase were discovered in vitro.
1
University of Alcal a de Henares, Spain, 2Universidad
a, Alcal
Autonoma de Madrid, Madrid, Spain
Prostaglandin E2 (PGE2) is produced by the inducible enzyme
cyclooxygenase-2 (COX-2), and acts through EP receptors (EP1,
EP2, EP3 and EP4), which are located at the cell membrane.
Recently, we have found that intracellular EP receptors (iEPR)
also mediate several PGE2 effects, so that they are prevented by
the inhibitor of PGE2 uptake transporter bromosulfophthalein
(BS). Since tissue hypoxia is an important factor in renal patho-
physiology, here we studied the role of PGE2 in hypoxia (1%
O2)-induced apoptosis in human proximal tubular cells (PTC).
Hypoxia-induced increase in the expression of both caspase-3
and annexin V (which were respectively assessed by Western blot
analysis and flow cytometry) was prevented by COX-2 inhibition,
antagonism of EP receptors or BS treatment. Of note, COX-2
expression as well intracellular PGE2 levels were up-regulated by
212 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 213
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
214 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
P.09-023-Tue P.09-024-Wed
High-fat diet compromises male fertility that is Mitochondria-targeted antioxidant SkQ1
not surpassed by reversion to a controlled diet improves corneal healing after UV-induced
L. Crisostomo1,2,3, L. Rato4, I. Jarak1,5, B. M. Silva4, damage in rabbits
J. F. Raposo6,7, P. F. Oliveira1,2,3, M. G. Alves1 V. Tiulina1,2, E. Zernii1, V. Baksheeva1, O. Gancharova1,
1
Department of Microscopy, Laboratory of Cell Biology, and Unit E. Kabanova2, L. Sotnikova2, A. Zamyatnin3, P. Philippov1,
for Multidisciplinary Research in Biomedicine (UMIB), Institute I. Senin1
of Biomedical Sciences Abel Salazar (ICBAS), University of 1
A.N. Belozersky Institute of Physico-Chemical Biology,
Porto, Porto, Portugal, 2Department of Genetics, Faculty of Lomonosov Moscow State University, Moscow, Russia, 2Federal
Medicine, University of Porto, Porto, Portugal, 3Instituto de State Budgetary Educational Institution of Higher Education
Investigac~ao e Inovac~ao em Sa ude i3S, Universidade do Porto, “Moscow State Academy of Veterinary Medicine and
Porto, Portugal, 4Faculty of Health Sciences, University of Beira Biotechnology – MVA by K.I. Skryabin”, Moscow, Russia,
Interior, Covilh~a, Portugal, 5Department of Life Sciences, Faculty 3
Institute of Molecular Medicine, Sechenov First Moscow State
of Sciences and Technology and Centre for Functional Ecology Medical University, Moscow, Russia
(CFE), University of Coimbra, Coimbra, Portugal, 6NOVA
Medical School - Faculdade de Ci^ encias M edicas, Lisboa, Cornea absorbs most of daily ultraviolet (UV) light. An excess of
Portugal, 7APDP - Associac~ ao Protectora dos Diab eticos de UV damages results in not only keratopathy and cataract but
Portugal, Lisboa, Portugal also maculopathy. Previously, it was demonstrated UV exposure
are associated with mitochondrial dysfunction and oxidative
Lifestyle is a major cause for the onset of metabolic diseases stress in cultured corneal epithelial cells in vitro. In this work,
(MD), notably obesity and type II diabetes (T2DM). Men suffer- the feasibility of mitochondria-targeted antioxidant SkQ1 based
ing from those diseases also display poorer fertility outcomes, therapy for enhancement of corneal regeneration after UV irradi-
although the cause-effect relation between lifestyle and impaired ation was demonstrated in vivo. A model of corneal damage
fertility is still arguable. Besides, there is no evidence that lifestyle caused by UV radiation was developed. It was demonstrated that
changes can restore the fertility potential in individuals who suf- 312 nm light irradiation of the cornea triggers the development
fered MD. Herein we studied MD onset caused by diet (fat-rich of corneal lesions in rabbits. In particular, loss of epithelium,
diet), its effect on male reproductive potential and the effect of a stromal edema and apoptosis of keratocytes and endothelium
diet reversion (DR) on those issues, using a rodent model. To cells were observed. The stepwise mechanism of corneal regenera-
achieve this, 3 groups of 12 mice were fed with 3 different diets tion was characterized. It involves epithelium and endothelium
(CTRL – standard Mucedola; HFD – high-fat diet and HFDt – regrowth and activation of healthy keratocytes. Regeneration
high-fat diet for 60 days, then replaced by standard Mucedola). takes place simultaneously in all corneal layers and it becomes
At 200 days, mice were subjected to Intraperitoneal Glucose Tol- completed by day 7. It was emphasized that multiple apoptosis
erance Test (IGPTT) and to Intraperitoneal Insulin Tolerance of stromal keratocytes can be associated with UV-induced oxida-
Test (IPITT), to assess the onset of MD. Mice weight was moni- tive stress, which manifested in the cornea as elevation of MDA
tored throughout the experiment. After sacrifice, several tissues content. The feasibility of SkQ1 based antioxidant therapy for
were collected and weighted. Testicular tissue was used for meta- enhancement of corneal regeneration after UV irradiation was
bolomics analysis by H1-NMR. Sperm was obtained from epi- demonstrated. The therapy prevents the loss of stromal kerato-
didymis, and fertility parameters were determined. Our results cytes and other corneal cells, facilitates re-epithelialization of
show that high-fat diet significantly increases mice weight, and its damaged tissue area and enhances stromal remodeling. In gen-
withdrawal quickly normalizes it. CTRL and HFDt mice showed eral, clinical and histological data indicate that the treatment
significant lower glycaemia during IGPTT than HFD suggesting accelerates corneal healing by 3 days. According to biochemical
that DR improves the metabolic state of the mice. Nevertheless, studies, the instillations of SkQ1 also suppresses oxidative stress
DR was unable to restore all fertility parameters since HFD and in the cornea. In conclusion, our data suggest that the mitochon-
HFDt mice had significantly lower sperm viability and motility dria-targeted antioxidant SkQ1 is effective in improving regenera-
than CTRL mice. Metabolomics analysis revealed an altered lipid tive phases of corneal healing after UV damage. This study was
and energy metabolism in HFD mice, coupled to increased supported by the Russian Science Foundation (Project no. 16-15-
oxidative stress. HFDt normalized lipid metabolism illustrating a 00255).
limited improvement after DR. Overall our data suggest that
high-fat diet compromises male fertility and DR has limited
impact on the affected parameters. Thus, weight gain cause per- P.09-025-Mon
manent damage on male reproductive tract. Study of CD sensitivity of iron(II)
clathrochelates on various globular proteins
M. Kuperman1, S. Vakarov2, E. Gumienna-Kontecka3,
Y. Voloshin4, O. Varzatskii2, V. Kovalska1,5
1
Institute of Molecular Biology and Genetics, NASU, 150
Zabolotnogo St., 03143, Kyiv, Ukraine, 2V.I. Vernadskii Institute
of General and Inorganic Chemistry NASU, 32/34 Palladin Av.,
03080, Kyiv, Ukraine, 3Chemical Faculty, Wroclaw University,
14 F. Joliot-Curie Str., Wroclaw, Poland, 4A.N. Nesmeyanov
Institute of Organoelement Compounds RAS, 28 Vavilova St.,
119991, Moscow, Russia, 5SC Princeton Biomolecular Research
Labs, Saperne pole st., 26A, 01042, Kyiv, Ukraine
Iron (II) clathrochelates are cage complexes with a wide range of
bioactivities. They are able to bind proteins; upon binding to
serum albumins, they induce strong specific circular dichroism
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 215
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
(CD) signal in 350–600 nm range. The most pronounced CD parameters between hybrid and purebred lambs as well as between
responses gain compounds with carboxyphenyl ribbed sub- male and female lambs of the same genotype. This study was sup-
stituents. Here we explored the clathrochelates’ ability to induce ported by Russian science foundation (project No. 14-36-00039).
CD response upon binding to series of proteins of various struc-
ture and functionality: beta-lactoglobulin, lysozyme, trypsin,
lipase and insulin. Dependence of the binding to proteins on num- P.09-027-Wed
ber (one/two/six) and isomery of carboxyphenylsulfide sub- Digestive complex of Tenebrionidae insects
stituents in clathrochelate molecule was analyzed. In case of beta- effectively degrades resistant to proteolysis
lactoglobulin, clathrochelates acquired the strongest CD responses
gliadins
of different shapes and intensity; the most pronounced CD bands
V. Tereshchenkova1, E. Dvoryakova2, I. Goptar1, I. Filippova1,
(90 mdeg) gained by di-ortho clathrochelate. Binding to insulin
E. Elpidina2
resulted in inducing the intensive CD response only by compounds 1
Lomonosov Moscow State University, Chemistry Department,
with six substituents (up to 76 mdeg for hexa-para isomer); CD
Moscow, Russia, 2Belozersky Institute of Physico-Chemical
signals varied by the intensity and shape for different isomers. CD
Biology, Lomonosov Moscow State University, Moscow, Russia
bands induced by lysozyme were less strong (up to 13 mdeg for di-
meta isomer), had similar shape for all clathrochelates. CD bands Gliadins are widespread dietary proteins, which contain 10–30%
of clathrochelate series in presence of trypsin and lipase were Pro and 30–50% Gln residues. Several toxic gliadins peptides,
indistinct (1–4 mdeg). Thus, it is shown that the number and resistant to proteolysis by human digestive enzymes cause autoim-
geometry of ribbed substituents are important for clathrochelate mune Celiac Disease in 1% of the susceptible human population.
binding to proteins; these affect the fitting of the compound to a Search for natural enzymatic systems, capable to hydrolyze glia-
binding site and determine the arrangement of the guest-host dins is an urgent task. Tenebrionidae insects are stored product
assembly. Clathrochelates could serve as prospective scaffolds for pests and predicted hosts of such proteolytic system. Gliadins are
development chiroptical probes; to reflect the protein structural their main food proteins; therefore, tenebrionids should possess
peculiarities by distinctions in CD spectra, clathrochelates could enzymes capable to digest them. We have carried out a bioinfor-
be adjusted by various number and isomery of substituents. The matic search for proline-specific peptidases (PSP) in Tenebrio
project leading to these results has received funding from the molitor larval gut transcriptome and found 11 sequences homolo-
European Union’s Horizon 2020 research and innovation pro- gous to human PSP. Combination of gene expression studies of
gramme under the Marie Skłodowska-Curie grant agreement No the larval gut transcriptome with biochemical localization experi-
778245. ments allowed us to propose the digestive function for the highly
expressed secreted dipeptidyl peptidase 4 (DPP 4) and prolylcar-
boxypeptidase (PRCP), and also for tissue localized prolidase
P.09-026-Tue (XPD). Previously we have shown that cysteine cathepsins (CC)
Effect of gender on blood lipids parameters of are the major post-glutamine cleaving endopeptidases (PGP) in
the Ovis aries and the Ovis ammon Tenebrionidae insects. We studied the effect of individual T. moli-
tor digestive PGP–CC and PSP–DPP 4 and PRCP, and combined
interspecific hybrids
action of these peptidases on gliadins and suggested the hypotheti-
A. Volnin1, S. Zaitsev2, V. Bagirov1, N. Bogolubova1, R. Rykov1,
cal scheme of complete hydrolysis of c- and x-gliadins fragments
N. Zinovieva1
1 with PQQPFPQ repeats. At the first stage, CC can hydrolyze the
L.K. Ernst Federal Science Center for Animal Husbandry,
bond between two Gln residues. Resulting fragments are sub-
Podolsk, Russia, 2Moscow SAVMB, Moscow, Russia
strates for DPP 4, which cleaves dipeptides from their N-terminus.
Hybridization of domestic and wild species is the promising way Remaining tripeptides can be hydrolyzed by PRCP. XPD may
to introduce the new capacities in farm animals. Here, we esti- complete gliadins degradation, cleaving final dipeptides. There-
mated the gender influence on major lipids of lambs blood serum fore, this natural complex provides complete gliadins hydrolysis
of interspecies hybrids of Argali and domestic sheep with differ- and has a high potential as a possible preparation for Celiac Dis-
ent genotypes. Blood samples were collected before feeding from ease enzyme therapy. This work was supported by RFBR grants
female (n = 15) and male (n = 12) lambs at the age of 5 months. 17-54-61008 Egypt_a, 18-04-01221_a; RFBR-National Intellectual
The animals (of each gender) were divided among the three groups Development grant 17-34-80158 mol_ev_a.
according to their genotype: 1) hybrids of 25% Argali and 75% of
Romanov sheep; 2) hybrids of 18.75% Argali and 81.25% of
Romanov sheep; 3) purebred Romanov sheep. The cholesterol and P.09-028-Mon
triglycerides concentrations by automatic analyzer ChemWell DNA-binding ability, topoisomerase I/II and
(Awareness technology, USA) with Spinreact assay kits (Spain) anticancer activity of novel biscoumarin
were measured. Kruskal-Wallis test (comparison of analog groups)
derivatives with different length linkers
and U-test (effect of gender) were used for statistical analyses. The
M. Hudacova1, E. Konkol’ova1, S. Hamul’akova2, J. Vargova3,
female lambs of 2nd group had a higher concentration of triglyc- 3, P. Fedorocko3, M. Kozurkova1
R. Jendzelovsky3, J. Sevc
erides (P < 0.01) in comparison with 1st group (+15.9%) and 3rd 1
Department of Biochemistry, Institute of Chemistry, Faculty of
group (+10.3%). Male lambs of 3rd and 1st groups had a higher arik, Moyzesov
Science, University of P. J. Saf a 11, Kosice,
cholesterol concentration (P < 0.05) comparing to 2nd group at
Slovakia, 22Department of Organic Chemistry, Institute of
4.8% and 6.5%, respectively. We found gender-related differences arik,
Chemistry, Faculty of Science, University of P. J. Saf
in 1st group for cholesterol concentration (P < 0.05), in 2nd group
Moyzesov a 11, Kosice, Slovakia, 33Department of Cellular
– for triglyceride concentration (P < 0.05) and in purebred Roma-
Biology, Institute of Biology and Ecology, Faculty of Science,
nov sheep (3rd group) - for both lipids (P < 0.05). Female lambs arik, Moyzesov
University of P. J. Saf a 11, Kosice, Slovakia
had higher concentrations of lipid metabolites in comparison with
male lambs: +20.3% for cholesterol in the 1st group, +19.7% for Coumarin derivatives exhibit antitumor activities at different
triglycerides in the 2nd group, +14.6% and 20.6% for triglycerides stages of cancer formation through various mechanisms, such as
and cholesterol, respectively, in the 3rd group. The patterns, estab- blocking of the cell cycle, the induction of cell apoptosis or the
lished in this study, indicated the essential differences in lipid inhibition of DNA-associated enzymes. In recent years, the O-
216 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
substituted analogs of 7-hydroxycoumarins and biscoumarins In the presence of AMI, mutant EF-G promotes retention of the
have attracted intense interest due to their anticancer effects. In protein synthesis efficiency, while existence only intact EF-G
this work, we examine the spectral and binding properties of a form considerably decreases it. Thus, AMI inhibits protein trans-
new biscoumarin derivatives based on 7-hydroxycoumarin (a-e), lation by the disruption of initiation complex formation and the
linked together by linkers of different lengths. The interaction of alteration of mRNA-tRNA complex movement upon transloca-
a-e with calf thymus DNA (ctDNA) were investigated using dif- tion. This work is supported by Russian Science Foundation
ferent spectral methods. The fluorescence test at different temper- grant 17-14-01416 and RFBR 17-00-00368.
atures revealed that the quenching mechanism was a static type.
The binding constant K values of biscoumarins a-e with ctDNA
were in range of 5.82 9 102 - 2.57 9 103 M1. Thermodynamic P.09-030-Wed
parameters DH and DS measurements were taken at different Fine tuning of bacterial translation initiation
temperatures and indicated that van der Waal’s forces and by small compounds
hydrogen bonding formation played major roles in the binding D. Vinogradova1,2, E. Maksimova1,3, P. Kasatsky1, V. Zegarra4,
process in the case of compounds b-e. However, the DH and DS A. Paleskava1, P. Milon4, A. Konevega1,3,5
values for derivative a were both positive, a finding which is 1
Petersburg Nuclear Physics Institute named by B.P. Konstantinov
indicative of hydrophobic interaction. The values of DG for each of NRC “Kurchatov Institute”, Gatchina, Russia, 2NanoTemper
compound were negative, and this confirms that the binding pro- Technologies Rus, Saint-Petersburg, Russia, 3Peter the Great St.
cess is enthalpy driven and spontaneous. The results of other Petersburg Polytechnic University, Saint-Petersburg, Russia,
experiments such as iodide inducted quenching, competitive bind- 4
School of Medicine, Faculty of Health Sciences, Universidad
ing assay with ethidium bromide and CD spectral analysis sug- Peruana de Ciencias Aplicadas - UPS, Lima, Peru, 5NRC
gest that compound a-e may bind to ctDNA the groove binding “Kurchatov Institute”, Moscow, Russia
mode. Gel electrophoresis analyses indicated that biscoumarin
derivatives (a-e) did not display DNA nuclease activity. Topoiso- The translation of the genetic information from the messenger
merase I/II inhibition assay were performed. The studies deriva- RNA (mRNA) into proteins starts with the formation of 30S ini-
tives were also analysed against A549 adherent lung tiation complex (30SIC). During the initiation specialized transfer
adenocarcinoma cells. This study was supported by Internal RNA (fMet-tRNAfMet) binds to the initiator mRNA codon in
Grant Programme of University of P.J. Safarik in Kosice VVGS- the P site of the 30S ribosome subunit. The initiation factors IF1,
PF-2018-754. IF2, IF3 are responsible for the accuracy and the efficiency of
the process. As a result of amino acid starvation, lack of iron,
phosphorus, fatty acids and other stress conditions in the cell, a
P.09-029-Tue process called stringent control is started. As a result, the pro-
Amicoumacin A: the molecular mechanism of cesses of protein biosynthesis and cell duplication are slowed
down. Stringent control carries out a global regulation of all
translation inhibition and antibiotic resistance
metabolic processes of the cell through an alarmone (p)ppGpp.
E. Maksimova1,2, D. Vinogradova1,3, A. Paleskava1,
At this work we study the (p)ppGpp-mediated regulation at the
P. Kasatsky1, I. Osterman4, O. Dontsova4, P. Sergiev4,
onset of protein synthesis in the in vitro bacterial system. We
A. Konevega1,2,5
1 apply advanced techniques of fluorescent spectroscopy to study
Petersburg Nuclear Physics Institute named by B.P. Konstantinov
the mechanism of (p)ppGpp interaction with IF2 along the path-
of NRC “Kurchatov Institute”, Gatchina, Russia, 2Peter the Great
way of 30S IC formation. Microscale Thermophoresis (MST)
St. Petersburg Polytechnic University, St. Petersburg, Russia,
3 confirms the cooperative character of 30S initiation complex for-
NanoTemper Technologies Rus, St. Petersburg, Russia,
4 mation, strongly dependent on all ligands, and highlights guano-
Lomonosov Moscow State University, Department of Chemistry
sine nucleotides GTP and (p)ppGpp as key components to
and A.N. Belozersky Institute of Physico-Chemical Biology,
regulate fMet-tRNAfMet and mRNA recruitment. Pre-steady state
Moscow, Russia, 5National Research Centre “Kurchatov Institute”,
kinetic assays show that the alarmone prevents rapid 50S joining
Moscow, Russia
to the 30S subunit, indicating that (p)ppGpp promotes 30S pre-
Amicoumacin A (AMI) is an insufficiently studied antibiotic, IC. Nucleotide competition experiments show that (p)ppGpp
which possesses not only strong antimicrobial, but anti-inflamma- competes better than GDP with GTP. Furthermore, the competi-
tory and antitumor activities. Despite the potential using AMI as tive character of (p)ppGpp appears to be mRNA dependent.
medical agent, molecular mechanism of its action is still Altogether, we provide a novel model for translation initiation
unknown. The crystal structure of bacterial ribosome in complex regulation by (p)ppGpp and suggest that upon stringent response
with AMI revealed that antibiotic binds in the E site of 30S sub- the alarmone regulates translation initiation in an mRNA-depen-
unit, forming contacts with 16S rRNA and mRNA, but does not dent manner by regulating the transition of 30S pre-IC to 30S
interacting with tRNA. Moreover, point mutations (ins544K, IC. This work is supported by Russian Science Foundation grant
G542V and G581A) in the IV domain of EF-G compensated the 17-14-01416 and RFBR 17-00-00368.
AMI inhibitory activity. In our studies, we show that on the initi-
ation step, AMI inhibits binding of initiator tRNAfMet to 30S
subunit and prevents ribosomal subunit association. AMI does
not change the rate of aminoacyl-tRNA binding in the A site
and does not inhibit the peptide bond formation, but changes
conformation of bound tRNA. On the translocation step, it sig-
nificantly reduces the rate of peptidyl-tRNA movement from the
A to P site of the ribosome and changes the position of its accep-
tor end in the peptidyl transferase center. AMI also stimulates
binding of deacylated tRNA to the E site. Substitutions in
domain IV of elongation factor EF-G lead to strong deceleration
of the motion of peptidyl-tRNA D-loop region and but did not
change the motion of tRNA acceptor end during translocation.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 217
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
218 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
components, such as DNA and proteins. Changes in mitochon- cardiac injury. Regression analysis showed the only association
drial biogenesis and OXPHOS activity correlate with many age- with myocardial infarction in patients with DMT2, but not with
related diseases. Aged mitochondria show decline in ATP synthe- other chronic illnesses. MPV values increased with the increasing
sis concomitantly with increase in production of oxidants. number of comorbid conditions with the highest platelet volume
Adjustment of protein synthesis is an essential adaptation of cells indices were observed in patients with myocardial infarction.
to oxidative stress. Dysfunctional mitochondria influence global
protein synthesis. We showed that upon defective protein import
into mitochondria cytosolic translation is inhibited in yeast (Wro- P.09-036-Wed
bel et al., 2015). Downregulation of protein synthesis is also a Novel peptide analogues of antibiotic
feature of aging. However, the signals leading to its modulation chloramphenicol
remain unclear. Cells to maintain protein homeostasis must tightly A. Tereshchenkov1, I. Osterman2, N. Sumbatyan2,
control the process of protein synthesis. Translation can be regu- A. Bogdanov1,2
lated at any step of the process; nevertheless most common in 1
A.N. Belozersky Institute of Physico-Chemical Biology,
eukaryotic cells is the regulation at the level of translation initia- Lomonosov Moscow State University, Moscow, Russia,
tion. At the beginning of the protein synthesis, the key role in its 2
Department of Chemistry, Lomonosov Moscow State University,
regulation plays the eukaryotic initiation factor 2 (eIF2) complex. Moscow, Russia
In response to stress the alpha subunit of eIF2 (eIF2a; Sui2 in S.
cerevisiae) is phosphorylated, which causes translation attenuation. In view of the growing bacterial resistance, the development of
Our aim is to unravel signals and mechanisms, which regulate cel- new antibiotics is an urgent task. The modification of widely used
lular protein synthesis during stress conditions caused by dysfunc- antibacterials with various chemical residues is one of the main
tional mitochondria. approaches for creating new compounds with improved proper-
References ties. The combination in the antibiotic structure several moieties
Wrobel, L. et al. (2015) Mistargeted mitochondrial proteins acti- that act on different active sites will allow to trick the mecha-
vate a proteostatic response in the cytosol. Nature 524, 485–488. nisms of bacterial resistance. Chloramphenicol is a widespread
antibiotic that acts by inhibiting bacterial protein biosynthesis.
Its binding site is located in the A-site of the peptidyl transferase
P.09-035-Tue center of the ribosome. The replacement of the dichloroacetyl
Increased mean platelet volume is associated residue in the chloramphenicol molecule by a peptide will expand
its binding site to the ribosomal tunnel. These chimeric molecules
with acute myocardial infarction in patients
can also be used for studying the interaction of ribosome nucleo-
with diabetes mellitus type 2 tides with the peptide attached to the antibiotic. To find the opti-
E. Spahic1, S. Hasic2, A. Joguncic3, N. Sarajlic3, mal amino acid sequences of the peptides, molecular docking
A. Salihbegovic3, F. Krupic4 based virtual screening of all possible tripeptide chloramphenicol
1
Faculty of Medicine, University of Sarajevo, Sarajevo, Bosnia analogues for their binding to the ribosome was carried out.
and Herzegovina, 2Department of Medical Biochemistry, Faculty Compounds containing peptides FWH, VFR, RAW, AAA, both
of Medicine, University of Sarajevo, Sarajevo, Bosnia and with the free N-terminal amino group and the protected by acetyl
Herzegovina, 3Forensic medicine, Faculty of Medicine, University group, were selected and synthesized. The affinity of the com-
of Sarajevo, Sarajevo, Bosnia and Herzegovina, 4Department of pounds to the ribosome was measured using a competitive dis-
Orthopaedics, Institute of Clinical Sciences, The Sahlgrenska placement assay. It turned out that some of the analogues bind
Academy, University of Gothenburg, Gothenburg, Sweden, to the ribosome 30 times better than the original antibiotic,
Gothenburg, Sweden which is consistent with the virtual screening results. The use of
Overdose of more chronic illnesses is increasingly a common 23S RNA chemical probing made it possible to determine the
health problem in the general population. The association of dia- nucleotides involved in the interactions with the peptide moiety
betes mellitus and acute myocardial infarction is greater every of the molecules and suggest their binding mechanism. The abil-
day. The mean platelet volume (MPV), which is the determinant ity of some compounds to inhibit protein biosynthesis in vitro
of platelet function, is an independent risk factor for cardiovas- was comparable to that of chloramphenicol, making the obtained
cular disease. Available research data show that increased MPV analogues promising for further antibacterial activity studies.
is linked to the presence of risk factors for cardiovascular disease This work was supported by the grants RFBR (16-04-00709) and
including others: diabetes mellitus, hyperlipidemia, hypothy- RSF (14-24-00061-P).
roidism and other autoimmune disease. The aim of this study
was to investigate the effect of each disease (hypothyroidism,
hypertension, myocardial infarction) individually on MPV in dia-
P.09-037-Mon
betic patients. The cross-sectional study enrolled 102 diabetes Organization of respiratory chain in human
mellitus type 2 (DMT2) patients, both sexes (46 females, 56 fibroblasts with cytochrome c oxidase
males), with a mean value of age 58.91 (SD = 12.93) treated at deficiency caused by loss of COX8A subunit
the Primary Health Centre in Zenica from May to July 2017. All D. Rotko1, B. Kulawiak1, W. Kunz2, A. Szewczyk1
respondents had diabetes mellitus, the disease lasted for almost 1
Nencki Institute of Experimental Biology, Polish Academy of
10 years in both sexes. Mean platelet volume was significantly Sciences, Warsaw, Poland, 2Department of Epileptology and Life
higher in patients with myocardial infarction than in those with- and Brain Centre, University of Bonn, Bonn, Germany
out myocardial infarction (11.04 1.43 vs. 9.409 1.53;
P = 0.001). Regression analysis showed that the prevalence of Biogenesis of cytochrome c oxidase (COX) protein complex is
myocardial infarction had the highest predictive significance for executed under dual genetic control. While COX deficiencies are
MPV values, (predictor importance 0.49; coefficient 1.275, some of the most frequent causes of respiratory chain disorders
P < 0.001). Mean platelet volume was significantly higher in in humans, they are largely associated with aberrations in trans-
patients with diabetes mellitus and myocardial infarction than in lation of mitochondrial-encoded COX subunits and disruptions
DM patients, without myocardial infarction. We propose that of the tightly regulated process of COX assembly. Mutations in
MPV might be an important predictive factor for damage after the nuclear genes of structural subunits of COX are very rare
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 219
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
and few clinical cases have been reported to date. One of them, health problems. The toxic effects of environmental pollutants on
manifested in the patient with the Leigh-like syndrome with apoptotic pathways at different stages of organisms on the food
leukodystrophy and severe epilepsy, was ascribed to the mutation web provide basic data to understand and estimate the effects on
in COX8A leading to aberrant splicing and decreased transcript the human beings.
amount. It resulted in the loss of wild type COX8A protein – the
smallest nuclear-encoded structural subunit of COX, which is
indispensable for stability and activity of the COX complex. To P.09-039-Wed
further characterize organization of the respiratory chain in the Carbohydrate source effects on the
patient’s fibroblasts, we studied the supramolecular complexes transcriptome of Streptococcus mutans
containing COX and its pattern of structures at different stages M. Zapletalova, K. Paskova, J. Kucera, J. Lochman, P. Borilova
of assembly by blue native polyacrylamide gel electrophoresis Linhartova
(PAGE). We identified that a decreased amount of COX was lar- Masaryk University, Brno, Czech Republic
gely recruited to the supercomplexes together with complexes I
and III. Furthermore, to assess adaptive changes in the observed Dental caries is one of the most common chronic multifactorial
phenotype, we analyzed steady state levels of selected respiratory disease. The plaque ecological balance is considered to be the key
chain components with SDS PAGE and Western blot. Addition- factor in aetiology of caries. For this balance is crucial the role
ally, amount of transcripts encoding mtDNA maintenance and of dietary carbohydrates. The primary etiologic agent of human
translation machinery, alongside with the variation in the gene dental is an acidophilic bacteria Streptococcus mutans. By
expression for selected structural subunits of respiratory chain employing deep sequencing of RNA (RNA-Seq) technology we
complexes, were assessed by quantitative reverse-transcription have established the effects of various sugars on the regulation of
polymerase chain reaction. From our findings, supercomplex gene expression in bacterium S. mutans growth on tryptic soy.
assembly emerges as a crucial step for stabilizing residual COX Bacterium S. mutans was grown in a 5% CO2-aerobic atmo-
activity. This project was supported by the Marie Sklodowska- sphere at 37°C on solid and liquid tryptic soy broth with cario-
Curie COFUND grant No. 665735 and Polish National Science genic sugars represented by sucrose, fructose, glucose, galactose,
Center grant No. 2015/18/E/NZ1/00737. lactose and non-cariogenic sugar xylitol. Gene expression was
monitored by Illumina sequencing technology after a 12-hour
growth of S. mutans on cariogenic or non-cariogenic sugars and
P.09-038-Tue compared to control culture grown without carbohydrates when
The evaluation of endocrine disruptor effects results were considered as significant at padj < 0.01. Bacteria
grown on xylitol, galactose or lactose showed only minor changes
of environmental pollutant zinc and copper
in gene expression when there was mainly downregulation of
pyrithione via apoptosis pathways by expression compared with control bacteria grown on tryptic soy
histopathological and biochemical analyses broth. Another group of sugars was represented by fructose and
R. Semsi €ozkisa1, R. Tural2, A. Ç. G€
unal3, A. Sepici Dinçel1 glucose where genes involved in glycolysis and butanoate meta-
1
Department of Medical Biochemistry, Faculty of Medicine, bolism were strongly upregulated. In agreement with previous
University of Gazi, Ankara, Turkey, 2Vocational School of Health studies, the largest changes in gene expression were observed in
Services, University of Sinop, Sinop, Turkey, 3Gazi Education bacteria grown on medium with sucrose. The obtained data helps
Faculty Biology Education, University of Gazi, Ankara, Turkey to understand the impact of individual sugars on S. mutans meta-
Growing living organisms can accumulate in large numbers on bolism involved in one of the most common chronic diseases
surfaces like pipes, tanks, and ship hulls, resulting in corrosion, affecting the human population.
clogging and contamination, known as biocides. The antifouling
paint biocides copper pyrithione (CuPT) and zinc pyrithione P.09-040-Mon
(ZnPT) are environmental pollutants especially effect the non-tar-
get organisms. Zebrafish (Danio rerio) is a model organism of Novel N-alkynyl and N-indolylethyl amino
biomedical researches for disease modelling and functional neuro- steroids as inhibitors of CYP17A1 activity
science. The aim of the present study is to determine the effects J. Panada1,2, Y. Faletrov1,2, N. Frolova1, E. Rudaya1,
of these environmental pollutants (Cu-ZnPT) on aquatic toxicol- V. Shkumatov1,2
1
ogy and the possible effects on humans by evaluating apoptotic Research Institute for Physical Chemical Problems of the
pathways and endocrine disrupting biomarker, vitellogenin. To Belarusian State University, Minsk, Belarus, 2Belarusian State
generate tissue damage four groups of zebrafish (length 3– University, Minsk, Belarus
4.5 cm) were exposed to 0.02 lg/L CuPT, 0.1 lg/L CuPT, Cytochrome P450 17A1 is a core enzyme in the androgen biosyn-
0.1+1 lg/L CuPT+ZnPT and 1 lg/L ZnPT for the planned time thesis and the molecular target of abiraterone, a heterocyclic ster-
intervals as 24 and 96 h. Several biochemical assays including oid used for post-surgical treatment of prostate cancer. A major
hematoxylin-eosin staining, immunohistochemistry, ELISA for class of mechanism-based inactivators and probes for cytochrome
whole body vitellogenin and Western blot were performed to elu- P450 is based around a terminal alkyne group. In this work, the
cidate the mechanism of action of Cu-ZnPT. The gill tissues were synthesis and evaluation of alkyne- and indole-containing amino
damaged in both Zn and Cu pyrithione exposed groups. Hyper- steroids as inhibitors of CYP17A1 is reported. The target com-
emia, epithelial lifting and hyperplasia were observed in all Cu- pounds were obtained by reductive amination of 17- or 20-ketos-
ZnPT concentrations. The lesions were more severe in CuPT. teroids with propyneamine/butyneamine and tryptamine in 60–
Western blot and immunohistochemical analysis revealed signifi- 80% and 20% yield, respectively. All structures were confirmed
cant alterations of proapoptotic Bax protein expression and by mass-spectrometry, IR and NMR spectroscopy. Docking sim-
Bcl-2 protein expression (P < 0.05) among the groups. Besides ulations predicted the steroids to localize in the binding site in a
vitellogenin levels were significantly altered compared to time manner that resembled that of native substrate pregnenolone or
control groups (P < 0.05). As marine pollution and deterioration favoured the coordination of indole side chain with the heme.
of ecosystems are directly affect the humans, the results presents The substances were tested for their capability to inhibit proges-
awareness among environmental pollution, marine pollution and terone 17-hydroxylation by engineered Yarrowia lipolytica yeast.
220 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 221
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
222 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
cleavage that releases the second HCV protease NS3, without this P.09-048-Wed
cleavage the virus cannot replicate any further. The lack of an
Oxadiazon-induced toxicity in HCT116 cells:
automatable screening assay has been a major problem in identify-
ing chemicals against HCV NS2. Herein, a cell-based assay for involvement of oxidative stress and apoptosis
HCV NS2 cis-protease activity was validated, miniaturized and I. Graiet1, I. ben salem2, S. Abid Essefi2
1
automatized for 384 well plate for high throughput screening Laboratory of Research on Biologically Compatible Compounds,
(HTS). A targeted library of 2000 cysteine protease inhibitors were Faculty of Dentistry, Monastir, Tunisia., Sousse, Tunisia,
2
tested against the HCV NS2 protease activity. A counterscreen was Laboratory of Research on Biologically Compatible Compounds,
developed and used to filter out the false positive hits. The remain- Faculty of Dentistry, Monastir, Tunisia., Monastir, Tunisia
ing hits were validated in a secondary screen using the HCV cell Pesticides are chemical compounds used to control organisms
culture system with full length and subgenomic replicons. Using considered to be harmful in agriculture and also in other sectors.
bioinformatics techniques, a series of structure – activity relation- They are ubiquitous in the environment and exposure to these
ship (SAR) were tested further. We identified chemical scaffolds compounds is suspected to increase the incidence of certain
with potential different modes of action due to their different effect pathologies especially, cancer. At present, exposure to pesticides
on the NS2-NS3 site processing. In conclusion, it is presented the through food is attracting increasing interest. In this study, we
first cell based HTS assay for HCV NS2 cysteine protease activity were interested in an Oxadiazole family herbicide, Oxadiazon.
which identified new chemicals confirming HCV NS2 as a drug- Although many studies are available regarding its environmental
gable target. and ecological impact, very little is known about its toxicity in
the mammalian system. The aim of this study was to investigate
the toxic effect of Oxadiazon on intestine using an in vitro model
P.09-047-Tue (HCT116). Therefore, we evaluated the cell viability, elucidated
Moderate physical activity alters cardiac lipid the generation of free radicals, measured the mitochondrial mem-
metabolism of male rats on high fructose diet brane potential, and valued DNA fragmentation. Our results
M. Kostic, G. Koricanac, S. Tepavcevic, T. Culafi
c, S. Romic, showed that Oxadiazon is cytotoxic to HCT116. It causes dam-
J. Stanisic, M. Pantelic, M. Stojiljkovic age through the generation of free radicals and induces lipid per-
Laboratory for Molecular Biology and Endocrinology, Vinca oxidation and DNA damage. We also demonstrated that such
Institute of Nuclear Sciences, University of Belgrade, Belgrade, effects can be responsible for Oxadiazon-induced apoptosis.
Serbia
Metabolic syndrome is a growing health problem worldwide P.09-049-Mon
associated with modern day diet, rich in saturated fat and sugar.
Investigation of the nucleotide metabolism
Increase in fructose consumption contributes to ectopic lipid stor-
age which in turn is a risk factor for development of type 2 dia- and growth of Mycobacterium smegmatis
betes and cardiovascular disease. Defective substrate utilization under different genotoxic stress conditions
in the heart caused by systemic derangements in metabolic syn- E. V. Suranyi1,2, J. E. Szab
o3, B. G. Vertessy3, J. T
oth3
1
drome leads to cardiac dysfunction. Exercise is shown to alleviate Budapest University of Technology and Economics, Faculty of
most of the symptoms related to this metabolic disorder. The Applied Biotechnology and Food Science, Budapest, Hungary,
2
aim of this study was to analyze the impact of low intensity exer- Hungarian Academy of Sciences, RCNS, Institute of Enzymology,
cise on molecular mechanism of cardiac free fatty acid (FFA) Budapest, Hungary, 3RCNS, HAS, Budapest, Hungary
transport and metabolism and serum lipid profile of fructose fed The fidelity of replication is essential for every organism to main-
male rats. Male Wistar rats were divided into control group and tain their genome integrity. The fine-tuned pool of deoxyribonu-
two groups that received 10% fructose for 9 weeks, one of which cleoside 5’-triphosphates (deoxynucleotides, or dNTPs) is a
was additionally exposed to low intensity exercise. Concentration critical factor that contributes to the accurate DNA synthesis. It
of circulating FFA, triacylglycerol (TAG), total cholesterol, high- has been shown that perturbation of the well conserved balance
density lipoprotein (HDL) and low-density lipoprotein (LDL) of dNTPs results in elevated mutation rate. Interestingly, the
cholesterol were measured as well as TAG content in heart tissue. pathogenic Mycobacterium tuberculosis (M. tuberculosis) has an
Protein content of fatty acid translocase CD36, carnitine palmi- especially low in vitro mutation rate although it lacks the mis-
toyl transferase 1 (CPT1) and Lipin1 in cardiac lysate and CD36 match repair enzymes. However, in vivo samples from patients
content in plasma membrane were evaluated. High fructose diet often exhibit resistance to more than one drugs, caused exclu-
elevated serum TAG and showed a trend of increase of total sively by various single-nucleotide mutations. Based on these
cholesterol (P = 0.066). Exercise didn’t change TAG and total observations, our aim is to establish the relationship between dif-
cholesterol, but FFA were decreased compared to control group. ferent types of genotoxic stresses and the deoxynucleotide pool
Although there was no change in protein expression of CD36 size and balance in Mycobacterium smegmatis (M. smegmatis).
transporter, exercise raised the level of CD36 in plasma mem- As a valid model of M. tuberculosis, we used the non-pathogenic
brane compared to fructose group. Exercise increased protein M. smegmatis, since they share the same DNA metabolic and
expression of both CPT1, mitochondrial transporter of FFA, and repair routes. Several stresses are important in the lifecycle of M.
Lipin1, enzyme involved in TAG synthesis, compared to control tuberculosis: these are oxidative stress, ionizing radiation, nutrient
group. High fructose consumption changed serum lipid levels, starvation, alkylating agents, hypoxia and currently used antimy-
whereas low intensity exercise had greater influence on increasing cobacterial drugs. We defined appropriate treatment period based
the transport and metabolism of FFA in the heart. on the growth curve of M. smegmatis. The concentration of
chemical agents used for the treatment were specified to inhibit
cell growth. dNTP concentration following genotoxic treatments
were measured applying a fluorescent-based DNA-polymerization
method. Our results showed that different environmental stresses
cause changes in the dNTP pool size: for antimycobacterial
agents there are several orders of magnitude increase in the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 223
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
deoxynucleotide pool size, while exposing cells to nutrient starva- downregulation of IL-1b and IL-6 and a an upregulation in IL-10
tion caused reduced dNTP amounts. We are now in the process in response to lower concentrations (0.1–1 ng/mL) of RvE1 on
of further investigating this phenomenon by determining the days 1 and 6 compared to control/untreated cells (P < 0.01). The
mutation rates and mutational pattern arising from the applied elevated IL-1b/IL-10 and IL-6/IL-10 ratios indicated increased
genotoxic stress. activity of a resolution of the inflammatory process in the early
stage of the experiment. Higher concentrations (10–1,000 ng/mL)
of RvE1 failed to stimulate the proliferation and expression of
P.09-050-Tue tested molecular targets. In contrast, expression of IL-1b, IL-6,
Low intensity exercise prevents disturbances IL-8 was significantly increased in response to higher doses of
in insulin regulation of a subunits of Na+/K+- RvE1 while IL-10 expression was reduced (P < 0.01). We deter-
ATPase in the heart of fructose-fed female rats mined that the increase of IL-1b/IL-10, IL-6/IL-10, IL-8/IL-10
ratios with higher doses of RvE1 on days 1 and 6. RvE1 dose-
J. Stanisic, G. Koricanac, M. Stojiljkovic, T. Culafi
c, M. Kostic,
dependently induced the expression of SOD and GPX in
S. Romic, M. Pantelic, S. Tepavcevic
OCCM.30 (P < 0.01). These findings suggested that cementoblast
Laboratory for Molecular Biology and Endocrinology, Vinca
function was regulated by the RvE1 where lower doses stimulate a
Institute of Nuclear Sciences, University of Belgrade, Belgrade,
resolution of inflammatory process in parallel with an increased
Serbia
enzymatic activity, which would favor a regenerative process and
Na+/K+-ATPase is an enzyme essential for regular functioning higher doses favor an activation of inflammation, which would be
of the heart, regulated by insulin. Since estrogen deficiency com- key for the resorptive role for the cementoblast-induced tissue
bined with fructose rich diet provokes cardiac insulin resistance turnover during the periodontal regeneration (Selcuk University
we hypothesized that sodium/potassium transport would be dete- Scientific Research Projects BAP-17401164).
riorated accordingly and exercise training can prevent this distur-
bance. To test our hypothesis, we used fructose rich diet as animal
model of insulin resistance in ovariectomized (OVX) female rats. P.09-052-Mon
OVX female Wistar rats were divided into three groups: sedentary The anti-neuroinflammation of erinacine A on
control and sedentary and exercise groups submitted to fructose LPS-induced Parkinson’s disease in vivo and
diet (10% fructose for 9 weeks). We analyzed biochemical param-
in vitro
eters relevant for insulin action. Expression, phosphorylation and/
T. Y. Chin1, S. L. Lee2, J. Y. Hsu1, T. C. Chen1
or subcellular localization of cardiac insulin receptor (IR), insulin 1
Department of Bioscience Technology, Chung Yuan Christian
receptor substrate 1 (IRS1), protein kinase B (Akt) and Na+/K+-
University, Taoyuan, Taiwan, 2Department of Biological Science
ATPase in basal and insulin-stimulated conditions were evaluated.
and Technology, China Medical University, Taichung, Taiwan
Fructose diet did not change blood glucose level, but it increased
plasma insulin level as well as homeostasis model assessment Hericium erinaceus (H.E.) is a kind of medicinal mushrooms in
index, indicating insulin resistance. Exercise reversed these param- Asia, and has anti-oxidative, anti-diabetics and anti-hypertensive
eters to the control level. Fructose diet didn’t have effects on car- effects. Erinacine A (EA), the main active compound of H.E., is
diac IR, Akt (Thr308) and IRS1 (Tyr632) phosphorylation but it demonstrated that it could inhibit the inflammatory cytokine
reduced Akt (Ser473), increased inhibitory IRS1 (Ser307) phos- expression in the model of stroke rats. However, few of
phorylation and increased expression and plasma membrane level researches discussed the preventive potencies of EA in neurotoxi-
of a1 and a2 subunits of Na+/K+-ATPase. Exercise returned Akt city which is induced by inflammatory response. The previous
phosphorylation at Ser473 and increased at Thr308, without effect studies found that neuroinflammatory response is induced by
on IRS1 phosphorylation (Ser307), but returned total and plasma activated microglia which cause neurotoxicity and promote dete-
membrane level of a1 and a2 subunits of Na+/K+-ATPase. In riorating of Parkinson’s syndrome. This study aimed to investi-
conclusion, exercise prevents disturbances in cardiac sodium/ gate the neuroprotective effects of EA on LPS-induced
potassium transport in fructose-fed OVX rats suggesting that low neuroinflammation in the microglia cell, astrocyte, differentiated
intensity physical activity might be important nonpharmacological neuro-2a and animal model of semi-Parkinson’s disease. Results
treatment for cardiac insulin resistance. demonstrated EA and H.E. could improve the physical coordina-
tion in the rotation test and dramatically decrease the expression
of inflammatory cytokines, TNF-a, iNOS and IL-1b, in vivo. In
P.09-051-Wed addition, pretreated EA significantly suppressed iNOS expression
Resolvin E1 regulates inflammatory mediators and NO production in LPS-induced activated BV-2 cells. More-
and oxidative stress enzymes of the over, pretreated EA could decrease TNF-a mRNA expression in
cementoblasts astrocyte which was stimulated by LPS. Additionally, EA and
H.E. could increase TH but decrease p-NF-kB, p-JNK expression
S. B. Bozkurt1, S. Sezgin Hakki1, A. Kantarci2
1 on BV-2 conditioned medium in differentiated neuro-2a. Those
Selcuk University, Faculty of Dentistry, Research Center, Konya,
results show that, H.E. and EA could ameliorate LPS-induced
Turkey, Konya, Turkey, 2The Forsyth Institute, Boston, MA,
the semi-Parkinson’s syndrome, inhibit the iNOS expression in
USA., Boston, United States of America
activated BV-2 cells and also prevented the cell death from BV-2
Resolvin E1 (RvE1) has been effective in treatment of periodonti- conditioned medium in differentiated neuro-2a. Therefore, H.E.
tis and regenerated lost periodontal apparatus including new and EA may possess potent anti-inflammatory activity and
cementum formation in animals. The aim of this study was to improvement of Parkinson’s disease.
investigate the action of RvE1 on the gene expression of inflam-
matory mediators and oxidative stress enzymes in cementoblasts.
Murine immortalized cementoblasts (OCCM.30) were treated with
different concentrations of RvE1 (0–1,000 ng/mL). The impact of
RvE1 on OCCM.30 proliferation was determined for 360 h. RvE1
had a dose-dependent and bimodal impact on the expression of
cytokines and enzymes. There was a statistically significant
224 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
P.09-053-Tue who developed the first SBP incident during follow-up showed
significantly higher levels of endocan than SBP-free patients. The
The effects of high ferritin level on bone
best cut-off of endocan levels was 2.07 ng/mL. At this cut-off, v²
turnover biomarkers in beta thalassemia major analysis revealed that endocan level was significantly different
patients living in the Turkish Republic of between the SBP-free compensated cirrhotic patients with DM
Northern Cyprus and cirrhotic patients with DM who developed SBP in follow-up.
Z. Salman1, T. yilmaz2 While we could confirm traditional risk factors for SBP develop-
1
Near East University, Nicosia, Mersin 10, Turkey, 2Near East ment, such as the presence of hyponatremia or MELD, we were
University, Faculty of Dentistry, Mersin 10, Turkey also able to identify a moderately elevated endocan level as pre-
dictor of SBP incident development in compensated cirrhotic
Beta thalassemia Major (BTM) is a severe life threatening genetic
patients with diabetes.
blood disorder. BTM patients need regular and adequate blood
transfusion to survive, resulting iron overload, toxicity and other
complications. Osteoporosis and osteopenia cause serious health P.09-055-Mon
problems such as fractures, cracks and deformations in the
Strain-specific effect of AMP-activated protein
bones. In the study, 50 patients (25 male, 25 female) with BTM
and 30 normal volunteers as a control group (15 male, 15 female) kinase on glucose oxidation in mice
between the ages of 20 and 50 were evaluated. Blood samples I. Irodenko, K. Bardova, O. Horakova, J. Hansikova,
were taken in the morning on an empty stomach. Calcium, phos- M. Rossmeisl, J. Kopecky
phorus, ALP, PTH, Osteocalcin, 25OH D3 and Ferritin were Institute of Physiology, The Czech Academy of Sciences, Prague,
studied for all participants. There was no significant difference Czech Republic
between the BTM patient group and the control group in terms Differences in susceptibility to obesity and glucose intolerance in
of total calcium, phosphorus, ALP and Parathormon (P > 0.05). various mouse models offer a possibility to characterize novel
The values in these parameters were determined within normal targets for the treatment of obesity and related diseases, such as
limits in both groups. Ferritin levels of patients with BTM were type-2 diabetes. AMP-activated protein kinase (AMPK) is
significantly higher than those of the control group (P < 0.001). a heterotrimeric protein complex that plays a key role as an
Especially Ferritin levels of BTM women were found to be signif- energy sensor regulating energy homeostasis in cells. Once acti-
icantly higher than BTM men (P < 0.05). When we look at the vated, it stimulates ATP-producing pathways like glucose uptake
results of 25OH D3, the mean values of the BTM patient group and b-oxidation and inhibits lipolysis and lipogenesis. We aimed
was low <20 ng/mL and for the control group was found to be to detect possible differences in glucose tolerance and metabolic
inadequate <30 ng/mL. There was a significant difference flexibility between A/J and C57BL/6J (B6) murine strains, resis-
between the two groups (P < 0.001). Osteocalcin levels were sig- tant or prone to a high-fat induced obesity, respectively, and
nificantly lower in the BTM group than in the control group mice with a whole-body deletion of AMPK a2 subunit
(P < 0.001). A low negative correlation (-.196) was found (AMPKa2-KO) mice on A/J or B6 background. Mice were char-
between Ferritin and 25OH D3 levels in patients with BTM. It acterized at 4 weeks of age, e.g. at a time of weaning onto a
has been shown that, the 25OH D3 levels in the thalassemia high-carbohydrate diet. Glucose tolerance test was performed
patients were fairly low and insufficient even in the control using a dose of 1 mg of glucose per g of body weight (mg/g),
group. It is possible to link extremely low levels of 25OH D3 in administered either intraperitoneally or orally. For assessment of
patients with BTM to extremely high levels of ferritin. It is neces- a whole-body metabolic flexibility, we introduced an indirect
sary to add the 25OH D3 test to the routine check-ups of healthy calorimetry (INCA) protocol at thermoneutral temperature
persons as well as the routine tests applied to BTM patients. (34°C) including glucose delivery at 7.5 mg/g via gastric gavage.
A/J mice showed lower mean glucose levels and lower glucose
tolerance as well as faster increase in respiratory quotient (RQ)
P.09-054-Wed in response to glucose than B6 mice. AMPKa2-KO A/J mice dis-
Risk of a spontaneous bacterial peritonitis in played higher RQ after a glucose gavage suggesting higher de-
cirrhotic patients with diabetes, with high novo lipogenesis on whole-body level as compared to their wild
serum endocan levels type littermates, while this effect was not detected in AMPKa2-
J. Zuwala-Jagiello1, K. Simon2, E. Grzebyk1, M. Pazgan-Simon2 KO B6 mice. We have detected higher glucose tolerance and
1
Department of Pharmaceutical Biochemistry, Wroclaw Medical metabolic flexibility in obesity-resistant A/J mice as compared
University, Wroclaw, Poland, 2Clinic of Infectious Diseases, Liver with obesity-prone B6 animals. Deletion of AMPK activity had
Diseases and Acquired Immune Deficiency, Wroclaw Medical an expected effect on de-novo lipogenesis on A/J background
University, Wroclaw, Poland only, suggesting the key role of AMPK in the lean phenotype.
Funding: Czech Science Foundation (14-36804G).
The presence of diabetes mellitus (DM) in patients with cirrhosis
is associated with an increased risk of spontaneous bacterial peri-
tonitis (SBP), which may represent an increased susceptibility to P.09-056-Tue
infections. We aimed to identify the endocan as risk factor for The characteristics of Von Willebrand domain-
development of decompensation event (i.e. SBP) to optimize
containing protein 8
stratification for primary prophylaxis and therapeutic strategies
L. Alan1,2,3, L. Scorrano1,3
to improve survival. 84 diabetic subjects with compensated and 1
Department of Biology, Padova, Italy, 2Institute of Physiology,
decompensated liver cirrhosis were recruited. At the time of
The Czech Academy of Sciences, Prague, Czech Republic,
enrolment, the first SBP incident was diagnosed in 20 decompen- 3
Venetian Institute of Molecular medicine (VIMM), Padova, Italy
sated cirrhotic patients with diabetes. The 64 compensated cir-
rhotic patients with DM were followed up for the occurrence of Von Willebrand Domain-containing Protein 8 (Vwa8) has been
SBP. Median follow-up was 7.7 (interquartile range, 3.6–9.2) recently described to be a mitochondrial protein. The protein
years. Endocan levels were determined by ELISA analyses possesses a mitochondrial targeting sequence, AAA+ ATPase
(JDIEK H1) (Lunginnov SAS, Lille, France). Diabetic patients dynein related domain, P-loop containing nucleotide triphosphate
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 225
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
226 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 227
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
12 was used as a model cell culture. Dead cells in the population P.09-064-Mon
were stained by propidium iodide and the percentage of dead
Fatty acid synthesis activation protects brain
cells was measured by flow cytometry. Incubation of cells in the
presence of 5 mM H2O2 for 2 h showed their sensitivity to tissue from ischemic stroke
H2O2; the maximum level of ROS in cells was observed after M. Golovko, S. Golovko
15 min, while the maximum cell death - only after 2 h. The University of North Dakota, Grand Forks, United States of
appropriate concentrations of LO for investigation of ROS role America
were determined. Incubation of cells in the presence of LO Brain hypoxia is one of the major factors for loss of neuronal
(4.2 9 10–4 and 8.4 9 10–4 lM) for 15 min increased the ROS functions and viability in many disorders, including ischemic
level 3 and 8 times compared to the control and then continued stroke and heart disease - the leading causes of death and long-
to grow, after 1 h incubation maximum amounts of ROS were term disability. Our goal is to understand the mechanism under-
observed, exceeding the control 6.5 and 15.3 times; later ROS lying neuron adaptation to hypoxia to outline potentially new
concentrations gradually decreased. The dependence of cell death therapeutic targets and approaches for treatment of brain
on LO concentration was demonstrated. The increase in intracel- hypoxic conditions. To this end, we have identified in cultured
lular reactive oxygen species detected by the 2,7-dichlorodihydro- cell lines and primary cells a novel, neuron-specific protective
fluorescein coincides in time with the death of cells. This result response to hypoxia through a dramatic activation of fatty acid
suggests that the oxidative pathway is one of mechanisms under- (FA) synthesis from glutamate (Glu) and glutamine (Gln). This
lying the cytotoxic LO action. The publication was prepared with suggests that activation of this mechanism under hypoxia sup-
the support of the “RUDN University Program 5-100”. ports neuronal anaerobic metabolism by attenuating the level of
Glu and its downstream excitotoxicity, while also balancing cellu-
lar reduction potential. To validate these findings in vivo, we uti-
P.09-063-Wed lized an animal model for ischemic stroke. Fatty acid synthase
Stabilization of papain by immobilization on inhibitors significantly increased basal brain glutamate levels.
chitosan Upon fatty acid inhibitor treatment, infarct volume, edema, neu-
V. Koroleva1, M. Holyavka1, S. Olshannikova1, S. Pankova1, rological deficiency, and reductive potential were significantly
A. Belenova1, M. Kondratyev2, A. Samchenko2, A. Kabanov2, increased upon middle cerebral artery occlusion, but had no
V. Artyukhov1 effect in the sham control animals. These results are consistent
1
Voronezh State University, Voronezh, Russia, 2Institute of Cell with our previously published in vitro data and indicate a novel
Biophysics of Russian Academy of Sciences, Pushchino, Moscow protective role for fatty acid synthesis under hypoxia through
Region, Russia attenuation glutamate excitotoxicity, normalization reductive
potential, thus supporting anaerobic energy metabolism.
Enzymes of medical appointment become growingly important in
the last decades in view of increasing resistance of pathogenic
microorganisms to antibiotics of various classes. Enzymes can be P.09-065-Tue
used not only as antimicrobial agents, but also as anti-inflamma-
Inhibition of histone deacetylase attenuates
tory and antiedematous means. Water-soluble enzymes have a
number of essential disadvantages. Free enzymes are subject to development of type I diabetes through
microbial and fungal degradation, they are quickly inactivated at reduction of oxidant stress
high temperatures and extremely acidic or alkaline environments, H. Lee, I. Kim
and can be hydrolyzed by proteases of the patient organism. Sim- Kyungpook National University, Daegu, South Korea
ilar disadvantages are removed by a biocatalyst immobilization Pancreas is vulnerable tissue to oxidative stress because of its
on the insoluble carrier. Chitosan perfectly proved itself in the lower expression level of antioxidant enzymes than other tissues.
field of medicine and veterinary science due to antimicrobial, Thus, pancreas is weak to Streptozotocin (STZ) which increases
antitumor and immunomodulatory action. The aim of this study reactive oxygen species (ROS) through several pathways such as
is to produce a series of papain preparations immobilized on a damage to DNA and mitochondria, excess nitric oxide generation
various type of chitosan for enzyme stabilization during storage and xanthine oxidase. We hypothesized that HDAC11 inhibition
and application. A virtual screening of high-affinity carriers for induces antioxidant enzymes such as catalase, superoxide dismu-
immobilization has been performed using computer modelling. tase (SOD), and glutathione reductase (GR) which results in
Based on the comparative analysis of the total energy and the resistance to oxidative stress in the pancreas. Change of cell mor-
localization of the ligand binding sites, some assumptions about phology and viability was investigated by IncuCyte and MTT
the enzyme interactions with suggested carriers were made. A assay respectively. Expression of antioxidant enzymes was ana-
number of heterogeneous enzyme preparations have been offered lyzed by qRT-PCR. To establish type I diabetes animal model,
and the structural features of these complexes were predicted. STZ (40 mg/kg) was injected intraperitoneally. MGCD0103
The papain adsorption on medium-molecular (200 kDa) and (2 mg/week) was infused with subcutaneous osmotic mini-pump
high-molecular (350 kDa) chitosans allowed preserving up to for one week. STZ treatment decreased cell viability in a dose-
70% of the catalytic activity. The most suitable buffer system for dependent manner in several cell lines. INS-1E was the most sen-
immobilizing the enzyme on acid-soluble chitosans is a glycine sitive to STZ compared with THP-1, HepG2, and HEK293.
buffer in the pH range of 8.6–10.0. Being immobilized on chi- MGCD0103 treatment protected from STZ-induced INS-1E cell
tosan, the enzyme stability was increased by 10 times as com- death, disruption of mitochondrial membrane potential, and
pared with the soluble protein. The papain, sorbed on the intracellular ROS generation. MGCD0103 increased the expres-
chitosan matrix, was more thermally stable at 70 °C than its sol- sion of antioxidant enzymes such as catalase (Cat), glutathione
uble form. We acknowledge the support from Russian Ministry peroxidase (Gpx), glutathione reductase (GlutR), and superoxide
of Education and Science (Grant No. 3.1761.2017/4.6). dismutase (SOD) in INS-1E cells. STZ injection increased the
blood glucose level which was inhibited by MGCD0103. STZ
destroyed the structure of pancreatic islets, which was partially
restored by MGCD0103 infusion. MGCD0103 infusion induced
228 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
expression of SOD1, 2, and 3 in the pancreas. The present study extract of Viscum album was injected intraperitoneally (i.p.) into
demonstrated that HDAC11 inhibition induces antioxidant 15 Wistar albino rats as 10 mg per kg of body weight for nine
enzymes which results in resistance to oxidative stress in the pan- consecutive days, while 15 rats were used as a control. Then, fol-
creas. lowing the decapitation of the rats, the livers were removed, and
microsomal fractions were prepared. Cytochrome P450 activities
were determined using aminopyrene (CYP2C9), benzyloxyresorufin
P.09-066-Wed (CYP2B), caffeine (CYP1A2), dibenzylfluorescein (CYP19), ery-
Influence of passive smoking on some thromycin (CYP3A4), ethoxyresorufin (CYP1A1), methoxyre-
biochemical characteristics of amniotic fluid sorufin (CYP1A2), pentoxyresorufin (CYP2B) as substrates. The
S. Notova1, L. Lizurchik2, E. Kiyaeva1, I. Larjushina2, results showed that the extract of Viscum album decreased all the
O. Marshinskaya2, T. Kazakova2 tested CYP activities significantly at varying degrees between 20–
1
Federal Research Centre of Biological Systems and Agro- 40% except the CYP2C9 (aminopyrine) activity, which was
technologies of the Russian Academy of Sciences, Orenburg, increased about 2-fold with respect to control. These are the major
Russia, 2Orenburg State University, Orenburg, Russia contributing CYP isoforms for the biotransformation of the drugs
in the liver; hence, the therapeutic index and the pharmacokinetics
The aim of the research was to study the effect of passive smok- of many prescribed medicine would be changed when the mistletoe
ing on biochemical characteristics of amniotic fluid of laboratory is combined with other anti-cancer drugs. In conclusion, clinically
animals. The study was carried out on pregnant female Wistar critical herb-drug interactions are expected with Viscum album.
white rats (n = 40), weighed 130 10 g at the beginning of the This work was supported by a grant from TUBITAK 109R012.
experiment. Animals were divided into two groups: experimental
and control. The animals of the experimental group underwent
passive smoking in the seed chamber for 30 min 2 times a day P.09-068-Tue
throughout the pregnancy. Each animal received 0.048 mg of Effects of radiation on activity of metabolism
nicotine per day. The control group consisted of intact animals.
enzymes of purine nucleotides in the
The obtained data indicated that exposure to tobacco smoke led
to changes in the biochemical composition of amniotic fluid of experiment
rats. Alkaline phosphatase significantly increased by 33% in G. Ilderbayeva1, A. Argynbekova1, O. Ilderbayev2,
experimental group. Such increase could be observed during pla- L. Chulenbayeva2, Z. Taldykbayev2
1
cental insufficiency, fetal hypoxia, gestosis. In addition, in the Semey State Medical University, Semey, Kazakhstan, 2L.N.
experimental group, the level of creatinine was significantly, by Gumilyov Eurasian National University, Astana, Kazakhstan
28.1% higher than in the control group. A high content of crea- The change of metabolism of purine nucleotides in immunocom-
tinine in the amniotic fluid could be accompanied by intrauterine petent cells determines its functional activity state. Researches of
growth restriction, acute and chronic fetal hypoxia. Also, a sig- purine exchange ferments provide new opportunities for the bio-
nificant increase in urea by 41.5% and glucose by 38.9% was chemical approach to treatment of patients with deficiency of
detected in the group of animals who underwent passive smok- enzymes, which will be facilitated by the studying of its function-
ing. A high urea concentration could be observed in chronic fetal ing on a molecular level after an organism has had radiation poi-
hypoxia and intrauterine growth restriction. Concentration of soning. To understand mechanisms of adaptation reaction,
iron in the amniotic fluid of experimental animals was signifi- researching of enzymes of purine exchange is of particular inter-
cantly lower by 74.2%. This microelement is necessary for the est. The aim of our research was to study the effects of radiation
construction of hemoglobin and myoglobin, is an integral part of on enzyme activity of the purine nucleotides metabolism – 5’-
cytochromes and oxidation-reduction enzymes. Thus, our analy- nucleotidase (5’-NT), adenosinedeaminase (ADA), adenylat-
sis of the biochemical characteristics of the amniotic fluid testifies edeaminase (AMP-DA) in different organs and tissues within the
the toxic effect of the studied factor, which can lead to placental experiment. To attain the goals established 2 series of tests were
insufficiency and fetal distress, accompanied by the development conducted on laboratory rats: inactive (gr.I) and exposed to radi-
of hypotrophy and hypoxia. ation (gr.II). Animals of group II were exposed to ionizing radia-
tion using Teragam radiotherapy unit in a dose of 6 Gy. During
the research it was identified that the activity of 50 -NT in irradi-
P.09-067-Mon ated animals’ spleen was lowered by 86.3%, ADA was lowered
Alteration of drug pharmacokinetics with by 50.9%, AMP-DA tended to decrease. 5’-NT activity in lymph
medicinal plant Viscum album L. by rendering nodes of small intestine among animals of group II was lowered
major cytochrome P450 activities by 39.1%, ADA was lowered by 34.3%, AMP-DA – by 20.1%.
A. Sen1, G. Celik-Turgut1, A. M. G. Gencler-Ozkan2, O. Adali3 5’-NT activity in thymus of irradiated animals was lowered by
1
Pamukkale University, Faculty of Sciences, Department of 40.41%, a ADA - by 34.2% and AMP-DA – by 20.1%. The
Biology, Denizli, Turkey, 2Ankara University, Faculty of results of research showed that 50 -NT and ADA enzymes activity
Pharmacy, Department of Pharmaceutical Botany, Ankara, in liver did not change in comparison with the inactive group,
Turkey, 3Middle East Technical University, Department of and the activity of AMP-DA was increasing by 80.1%. Exposure
Biological Sciences, Ankara, Turkey to radiation causes significant dysfunctions of purine exchange
enzymes, which characterizes the exertion of an organism’s com-
Medicinal plant mistletoe (Viscum album L.) have been tradi- pensatory/adaptive mechanisms. 50 -NT is a marker enzyme of
tionally used as anti-hypertension, coagulant, analgesic, car- plasma membrane, when its activity lowers adenosine is taken up
diotonic, and anti-cancer agent in Anatolia, Turkey. In addition, in insufficient amount, which leads to cell’s dwindling energy
extracts of Viscum album have been used as prescription drugs in resources.
Europe while considered as a dietary supplement in the US.
Thus, potential drug interactions have turned into a noteworthy
concern in mistletoe treatment. The present study was under-
taken to study the in vivo effects of Viscum album L. on crucial
drug-metabolizing P450 enzyme activities in rat liver. The water
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 229
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
230 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
E. coli cells, retaining more than half of the initial enzyme activ- Aldosterone injection was increased the blood pressure from
ity. The proposed approach of searching for lactonase genes in 118 9 mmHg to 164 2 mmHg (P < 0.0001). 68 miRNAs’
the used probiotic strains will make it possible to create pharma- expression and 2705 mRNAs’ expression were altered. Aldos-
cological preparations with a new principle of action on their terone biosynthesis related mRNA and protein expressions of
basis, they will block intercellular communication. The research CYP11B1, CYP17A1, CYP11A1, CYP21A2, AGTR1, AGTR2,
was supported by a grant from the Russian Science Foundation NR5A2, NR3C2, MC2R HSD3B2, THOP1, ANG1, MAPK3,
(project No. 16-16-10048). HSD11B1, ERK, ELK, and LNPEP were measured by qRT-
PCR, western blot/immunohistochemically. Especially, CYP11A1
(3-fold), CYP11B2 (3.3-fold) and CYP21A2 (1.6-fold) mRNA
P.09-074-Tue expressions were upregulated, CYP11B1 (36%) and CYP17A1
HDAC8 regulates neural differentiation (47%) mRNA expressions were downregulated in NCI-H295r
through embryoid body formation in P19 cells cells. Inhibition of hsa-miR-187-3p expression with miRNA
S. Katayama1, A. Morii1, J. Makanga1, T. Suzuki2,3, N. Miyata4, mimic was increased expression of CYP11A1 (18-fold), and
T. Inazu1 CYP17A1 (4.5-fold). CYP21A2 mRNA and protein expressions
1
Ritsumeikan Univ., Shiga, Japan, 2Kyoto Prefectural University were increased 12-fold after transfected with hsa-miR-128-1-5p
of Medicine, Kyoto, Japan, 3Japan Science and Technology inhibitor (P < 0.0001). This study clarifies the molecular mecha-
Agency (JST), Saitama, Japan, 4Nagoya City University, Aichi, nism of aldosterone synthesis and potential marker in the regula-
Japan tion of hypertension. TUBITAK supported (114Z734).
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 231
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
In this regard, it is necessary to know not only the acid content, After 20-min incubation, there was no difference in the effects
but also the ratio of the molecular and ionic form of the acid. on leukocytes between mucin-treated and non-treated E. coli.
We have developed a methodic of potentiometric titration to As shown by LCL, both Crohn’s patients’ (n = 5) and healthy
determine the content of organic acids. This method has a num- persons’ (n = 5) E. coli isolates significantly varied in the
ber of advantages, since it does not require dilution of intensely extent of mucin binding-mediated effects on leukocytes activa-
colored extracts and excludes random errors associated with tion. Probably, in inflammatory conditions mucin adsorbed
visual fixation of the equivalence point, as when using the indica- onto bacteria could play role of endogenous immunomodula-
tor. The methodic was tested and validated for a number of her- tor. The research was supported by Russian Science Founda-
bal medicines: rose hips, guelder rose, mountain ash, black tion (16-15-00258).
currant, syrups, infusions and tinctures based on them et el. It
has been found that the potentiometric titration methodic show
well reproducible and accurate data. Direct potentiometric titra- P.09-079-Mon
tion is possible when the pH value of the sample does not exceed Glucagon-like peptide-1 (GLP-1) signaling
5.5. In opposite cases, reverse titration can be used. To derive an plays a neuroprotective role against diabetes-
equation describing the curve of potentiometric titration of a related Ab-induced neurotoxicity via gut-brain
solution containing a mixture of several single- and dibasic weak
acids, the following regularities were used: A)Equations of chemi- axis
cal equilibria existing in solution; B)Equations of material bal- C. Lin1, H. Li1, C. Huang2, Y. Yang2, E. Kornelius2
1
ance according to the forms of acid that make up the system; C) Institute of Medicine, Chung Shan Medical University, Taichung,
Equation expressing the electroneutrality of the solution. As a Taiwan, 2Division of Endocrinology and Metabolism, Chung Shan
result, an expression was obtained in which the parameters deter- Medical University Hospital, Taichung, Taiwan
mined as a result of titration (the concentration of acids and Growing evidence suggests a pathophysiological connection
cations in the initial solution) linearly enter. The statistical esti- between type 2 diabetes (T2D) and Alzheimer’s disease (AD). In
mation of these parameters from the experimental data can be particular, insulin signaling blockade represents a common
obtained by the method of least squares, which greatly simplifies molecular link between these two diseases, suggesting AD may
the calculations. A computer program was written based on the be a brain-specific form of T2D. Interestingly, our previous stud-
mathematical model. This publication was supported by the Min- ies have demonstrated that glucagon-like peptide-1 (GLP-1) can
istry of Education and Science of the Russian federation (the exert a neuroprotective role by against Ab-induced toxicity.
Agreement № 02.A03.21.0008). Because of the key role of Ab in AD pathogenesis, it can be
accepted that reducing Ab-mediated insulin resistance may have
promising therapeutic potential. However, the mechanism how
P.09-078-Wed insulin signaling influences the onset and progression of AD
Modulatory role of mucin in activation of remains incompletely understood. As GLP-1 is targeted on both
blood neutrophils by Escherichia coli in vitro central and peripheral tissues, we postulate that GLP-1 may exert
E. Mikhalchik1, D. Rakitina1, N. Balabushevich2, Z. Kharaeva3, its neuroprotective effects by maintaining insulin signaling and
S. Gusev1, A. Gusev1, A. Sokolov4, T. Vakhrusheva1, metabolic homeostasis in gut-brain axis. To examine how Ab
O. Pobeguts1, J. Baykova1, P. Scherbakov1, V. Govorun1 modulates neuronal insulin signaling and evaluate the potential
1 benefits of GLP-1, we investigated the correlation between insulin
Federal Research and Clinical Center of Physical-Chemical
Medicine, Moscow, Russia, 2Lomonosov Moscow State University, signaling and Ab-induced neurotoxicity by both in vitro and
Department of Chemical Enzymology, Moscow, Russia, in vivo models. Our results demonstrated that GLP-1 significantly
3
Kabardino-Balkarian State University, Nalchik, Russia, 4Institute attenuated Ab-induced brain insulin resistance and oxidative
of Experimental Medicine, St.-Petersburg, Russia stress, and the GLP-1-activated gut-brain axis insulin signaling
may be involved in against Ab-mediated neurotoxicity in the
Previously we showed that E. coli isolates obtained from the brain. Moreover, this protective effect is partly mediated by acti-
intestinal mucosa of Crohn’s disease patients and feces of healthy
vation of AMP-activated protein kinase (AMPK), which restores
volunteers as well as the laboratory E. coli DH5alpha strain neuronal insulin signaling and leads to upregulation of nuclear
adsorbed mucin from dilute solution (0.1 mg/mL). Role of mucin factor erythroid 2-related factor 2 (NRF2)/heme oxygenase 1
in the interaction between E. coli and immune cells is still (HO-1) antioxidant pathway. In conclusion, restoration of brain
unknown. Our aim was to investigate whether bacterium-bound
insulin signaling by targeting GLP-1 may demonstrate important
mucin influences bacteria-induced activation of normal human implications to develop novel diagnostic or therapeutic strategies
blood leukocytes in vitro. One of the patients’ isolates was trea- for slowing disease progression of diabetes-related AD.
ted with FITC-labeled mucin, and mucin adsorption onto bacte-
rial surface was evidenced by confocal microscopy. Mucin-
treated E. coli or non-treated E. coli (control) were added to nor-
mal human blood and incubated for 5 and 20 min at room tem-
perature. Leukocyte activation was assessed by luminol
chemiluminescence (LCL) of whole blood, morphologic analysis
of blood smears, and the amount of myeloperoxidase (MPO) and
lactoferrin (LF) was measured in blood plasma by ELISA. Blood
LCL after 5-min incubation with mucin-treated E. coli was 80%
in respect to probes with control bacteria. 58 11% of neu-
trophils appeared morphologically activated in blood incubated
with mucin-treated E. coli unlike 86 7% in control probes.
Plasma MPO and LF concentrations after incubation with
mucin-treated E. coli were less by 15% and 25% respectively vs
probes with control E. coli. Mucin solution per se did not acti-
vate leukocytes, at least under experimental conditions used.
232 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 233
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
234 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 235
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
236 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 237
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
in all fields of vision on day 3 (0.04 vol.%) and were absolutely P.09-096-Wed
absent on day 5 (0 vol.%). It is concluded that in case of diffuse
Selective ligands of membrane progesterone
diphtheria toxic injury of the myocardium, BECN1-associated
autophagy is inhibited due to depletion of the responsible molec- receptors and progesterone are predominantly
ular mechanisms. The publication was prepared with the support proinflammatory immunomodulators in
of the “RUDN University Program 5-100”. human peripheral blood mononuclear cells
A. Polikarpova1, I. Levina2, L. Kulikova2, I. Morozov3,
P. Rubtsov3, I. Zavarzin2, A. Guseva1, O. Smirnova1,
P.09-095-Tue T. Shchelkunova1
Transition metals catalyzed reactions – a new 1
Lomonosov Moscow State University, Faculty of Biology,
approach to the aryl-, azolyl- and Moscow, Russia, 2Zelinsky Institute of Organic Chemistry,
triazolylsubstituted steroids –active Russian Academy of Sciences, Moscow, Russia, 3Engelhardt
Institute of Molecular Biology, Russian Academy of Sciences,
compounds against human MCF7 breast and Moscow, Russia
SCOV3 ovarian carcinoma cells
E. Lukasheva1, Y. Kotovshchikov2, G. Latyshev2, E. Kalinina1, The immunomodulatory role of progesterone (P4) is highly
A. Shtil3, E. Kolotova3, N. Lukashev2 important for successful ovulation, implantation, maintenance of
1
Peoples’ Friendship University of Russia (RUDN University), pregnancy and initiation of labor. The functions of progestins
Moscow, Russia, 2Chemistry Department, M.V. Lomonosov are mediated both by nuclear (nPRs) and membrane (mPRs)
Moscow State University, Moscow, Russia, 3N.N. Blokhin receptors of adipoQ receptor family. Human peripheral blood
National Medical Research Center of Oncology, Moscow, Russia mononuclear cells (PBMC) comprise of T and B lymphocytes,
monocytes and other types of immune cells, expressing mPRa
Steroids possess a unique position among other natural com- and mPRb. Among synthesized progesterone derivatives we
pounds due to their high biological activity and involvement into found two selective ligands of mPRs which do not interact with
the most important processes in living organisms. One of impor- nPRs: 19-hydroxypregn-4-en-20-one and 19-hydroxypregn-3-ene-
tant directions of synthetic organic chemistry is creation of new 20-one. The aim of this study was to assess the effects of these
drugs by modification of steroids. During 20-th century methods selective ligands and P4 on gene expression and the secretion of
of classical organic chemistry were used to transform steroids. cytokines (IL-1b, IL-6, IL-10, IL-2, TNFa, TGFb and IFNc) in
Presently the reactions, catalyzed by the complexes of transition PBMC using qRT-PCR and ELISA. PBMC were isolated from
metals, primarily by complexes of palladium, nickel and copper, blood of 8 men and 8 women, stimulated with lipopolysaccharide
are intensively applied for this purpose. The development of new and incubated with different concentration of hormones for 48 h.
efficient protocols for steroid functionalization is an important P4 and both selective ligands significantly increased gene expres-
synthetic problem. On the base of computer docking experiments sion and protein secretion of IL-1b, IL-6 and TNFa and gene
we prepared a big series of 4- and 6-arylsubstituted steroids by expression of TGFb and IFNc. IL-10 secretion was reduced sig-
Pd-catalyzed cross-coupling 6-halogen steroids with arylboronic nificantly after exposure with steroids, whereas mRNA level was
acids. Many of these compounds demonstrated high inhibition unchanged. These compounds also significantly reduced IL-2
activity against MCF7 cancer cells. As well we have developed a mRNA level. Sexual differences in cytokines levels were not
highly efficient procedure for the synthesis of azolyl-substituted detected. The selective ligands of mPRs in some cases acted at
steroids utilizing a cheaper catalyst system based on Cu-catalyzed lower concentrations on PBMCs than P4 and can successfully
reactions. A number of nitrogen heterocycles was involved into replace it without having a side effect on the nPR-positive cells.
the vinylation of halogen containing steroids affording the corre- Thus, P4 and selective analogues of mPRs have both pro-inflam-
sponding coupling products in good to excellent yields. In addi- matory and anti-inflammatory effects on PBMC in men and non-
tion a series of triazolyl substituted steroids and triazole pregnant women with mainly pro-inflammatory action. Thus, the
annelated non-steroidal derivatives was prepared and tested concept that progestins have predominantly immunosuppressive
against ovarian carcinoma SCOV3 and human breast cancer effects is oversimplistic. In summary a complex and diverse
MCF7 cell lines to investigate the antiproliferative activity. The action of these hormones depending on the type of immune cells,
cytotoxic effect was detected by MTT-test. Using the results on tissue and hormonal context was proposed.
the cytotoxicity, survival curves were constructed and IC50 were
calculated. The values of IC50 were in the range 3–22 lmol/L.
These low values of IC50 indicate the prospectivity of the further P.09-097-Mon
study of prepared substances as antitumor agents. The publica- Alkaline phosphatase regulates the hair-
tion was prepared with the support of the “RUDN University
inductive capacity of 3D-cultured human DP
Program 5-100” and RFBR grant 16-03-00390a.
cells
Y. Sung, M. Kwack, M. Kim, J. Kim
Kyungpook National University, Daegu, South Korea
Recent studies showed that sphere formation enhances the ability
of cultured dermal papilla (DP) cells to induce new hair follicles.
Alkaline phosphatase (ALP) activity is known to be correlated
with the hair-inducing capacity (trichogenicity) of DP cells and
expression of ALPL (ALP, liver/bone/kidney) transcript is
restored in DP spheres. We investigated whether restoration of
ALPL expression by sphere formation plays a critical role in
hair-inducing capacity of DP spheres and, if so, to investigate the
mechanism. We employed ALPL siRNA-mediated gene knock-
down and expression vector-mediated ALPL overexpression in
combination with a hair reconstitution assay. Knockdown or
238 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 239
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
et al., 2017); however, the precise mode of action of this com- P.09-102-Wed
pound remained unclear. As the chemical structure of TP053 is
Effect of electric pulse stimulation on
characterized by the presence of NO2 group, reduction of a
nitroaromatic moiety of TP053 by Mrx2 was hypothesized to transcriptome and [Na+]i/[K+]i ratio in C2C12
result in NO release and toxicity of the activated compound. myotubes: a comparative analysis
Analysis of the products of enzymatic activation of TP053 by E. Klimanova1,2, S. Sidorenko1,2, K. Milovanova2,
Mrx2 by the Griess reagent clearly confirmed production of nitric L. Kapilevich2, S. Orlov1,2
1
oxide in a time-dependent manner. Mass-spectra of cell lysates of M.V. Lomonosov Moscow State University, Faculty of Biology,
TP053-treated M. tuberculosis bacilli demonstrated transforma- Moscow, Russia, 2National Research Tomsk State University,
tion of TP053 to its non-active metabolite with Mw = 261 that Tomsk, Russia
corresponds the scenario of NO release accompanied by the Skeletal muscle typify as an endocrine organ producing peptides
destruction of the reduced thiophene ring with formation a and other bioactive substances termed myokines. Numerous
highly reactive mercapto group. The multitarget mechanism of researches demonstrated that myokine transcription, expression
NO toxicity for bacteria is known to include DNA damage, and and secretion are controlled by physical activity and provide a
biosynthesis proteins and lipids. Thus, TP-053 drug-like scaffolds beneficial effect on the function of skeletal muscles and neigh-
prospective for further development of novel anti-TB drug. This bouring tissues. In our study, we employed mice skeletal muscle
work was financially supported by the Ministry of Education and C2C12 myotubes subjected to electric pulse stimulation (EPS,
Science of the Russian Federation (Agreement No 14.616.21. 2 hr, 40 V, 1 Hz, 10 ms) as in vitro model of muscle contraction.
0065; unique identifier RFMEFI61616X0065). Using the Affymetrix GeneChip technology we found that EPS
resulted in differential expression of dozen of myokines including
statistically significant (P < 0.05) by more than 20% up-regula-
P.09-101-Tue
tion of Angptl4, APLN, CCL2, CXCL1, FGFBP1, IL6 and
Recombinant destabilase, a potential down-regulation of Adipoq, Bmp4, LRP4, Tgfb2. Both EPS and
thrombolytic, cleaves isopeptide bonds in inhibition of the Na,K-ATPase by 30 mkM ouabain led to dissi-
stabilized fibrin pation of transmembrane Na+ and K+ gradient resulted in ele-
I. Baskova1, S. Kalabushev1, D. Akhaev1, V. Manuvera2,3, vation of the [Na+]i/[K+]i ratio by 50%. With an exception of
V. Lazarev2,3, P. Bobrovsky2 FGFBP1, ouabain didn’t affect transcription of any EPS-sensitive
1
Lomonosov MSU, Moscow, Russia, 2Federal Research and myokines. Inhibition of L-type voltage-gated Ca2+-channels by
Clinical Center of Physical-Chemical Medicine of Federal Medical 10 mkM nicardipine completely abolished [Ca2+]i oscillations
Biological Agency, Moscow, Russia, 3Moscow Institute of Physics triggered by EPS in fura-2-loaded cells as well as differential
and Technology (State University), Dolgoprudny, Moscow Region, expression of Angptl4, APLN, CXCL1, FGFBP1, IL6, Adipoq,
Russia LRP4 and Tgfb2 indicating Ca2+i-mediated mechanisms of exci-
tation-transcription coupling. In contrast, altered expression of
Thrombolytic drugs that are currently in clinical use consist of
Bmp4 and CCL2 was preserved in the presence of nicardipine
various modified serine proteases (metalloproteases) involved in
thus suggesting Ca2+-independent signaling. In conclusion, our
the fibrinolytic system of the blood. Their mechanisms of action
results demonstrate that both Ca2+- mediated and –independent
involve the proteolytic degradation of polypeptide bonds in fibrin
signaling contributes to transcriptomic changes seen in EPS-trea-
polymers, a core component of thrombus. In thrombi, fibrin
ted myotubes. Intermediates of Ca2+-independent signaling as
molecules are cross-linked by the isopeptide bonds. Formation of
well as the role of elevation of the [Na+]i/[K+]i ratio in aug-
the isopeptide bonds is mediated by factor XIIIa. In this work,
mented expression of FGFBP1 should be examined further. This
we describe a fundamentally new mechanism of thrombolysis
work was supported by the Russian Science Foundation grant
caused by recombinant destabilase action and called ‘thromboly-
#16-15-10026.
sis-isopeptidolysis’. Destabilase is an enzyme secreted by the
medicinal leech salivary cells possessing endo-e-(c-Glu)–Lys-iso-
peptidase activity that is responsible for isopeptide bond cleav- P.09-103-Mon
age. We induced venous and arterial thrombosis in rats. Rats
Prohaptoglobin stimulates angiogenesis by
received intravenous injections of recombinant destabilase pro-
duced in Escherichia coli cells, a commercial streptokinase prepa- upregulating placental growth factor
ration or both. 24 h later, we analyzed weight of the thrombi expression via TGF-b/Smad1,5 signalling
and degree of cross-linking in thrombi. We found that destabilase M. K. Oh, I. S. Kim
alone or destabilase co-injected with streptokinase causes a Department of Medical Lifescience, College of Medicine, The
47.6% or 74.6% decrease of weight of venous and arterial Catholic University, Seoul, South Korea
thrombi respectively. The combined administration of destabilase Our previous study demonstrated that prohaptoglobin (proHp), a
and streptokinase has a greater effect than the injection of indi- precursor haptoglobin, enhances angiogenesis by increasing the
vidual enzymes. We showed that destabilase also reduces fibrin expressions of vascular endothelial growth factor-A (VEGF-A)
stabilization in thrombi. Thus, we found that the medicinal leech and VEGF receptor 2 (VEGFR2) in endothelial cells. However,
destabilase is an efficient thrombolytic agent for dissolving the underlying mechanisms of proHp on angiogenesis are still
thrombi. We believe that treatment with destabilase will increase unclear. Here, we investigated the angiogenic factors involved in
the overall effectiveness of conventional thrombolytic drugs. This upstream signals in proHp-treated HUVECs. ProHp enhanced the
work was supported by the Russian Science Foundation (project expressions of placental growth factor (PlGF) and VEGFR1 as
No. 17-75-20099). well as VEGF-A and VEGFR2. The tyrosine-phosphorylations of
VEGFR1 and 2 were also enhanced by proHp treatment. Interest-
ingly, PlGF knockdown attenuated the proHp-induced angiogenic
events - VEGF-A expression, VEGFR1/2 phosphorylation, cell
migration and tube formation on Matrigel. Furthermore, tran-
scription factor profiling assay indicated that Smad is involved in
240 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
the proHp-stimulated PlGF expression. Transforming growth fac- SRD5A3, DOLK, NgBR genes), while other defects are closely
tor-b (TGF-b) expression and Smad1/5 phosphorylation were also associated with its metabolism (genes MDPU1, DPM1-3). The aim
induced by proHp. Blockade of TGF-b signalling by TGF-b of study was to analyze dol content in urine and tissues of patients
receptor kinase inhibitor LY2109761 or Smad1/5 siRNA reduced with suspect deficiency in dol biosynthesis by mass spectrometry
the proHp-enhanced expressions of PlGF and VEGF-A, and with purpose to extend screening methods for CDG. Biological
in vitro tubular network formation. These findings suggest that material for this study consisted of urine samples from 76 controls
the angiogenic effects of proHp were dependent to PlGF and in age 2 months to 82 years, 6 patients with CDG (1xNgBR-CDG,
mediated via TGF-b/Smad1,5/PlGF/VEGF-A/VEGFR1,2 sig- 1xSRD5A3-CDG, 2xPMM2-CDG, 1xDPAGT1-CDG, 1xPGM1-
nalling pathway. CDG) and 43 patients with suspicion of CDG syndrome; samples
of frontal cortex, liver, muscle and heart tissues from 2 NgBR-
CDG patients and controls. Urine samples were stored in -20°C
P.09-104-Tue and tissue homogenates were stored in -80°C until used. Lipid con-
A draft of the Hirudo medicinalis genome tent after extraction was separated by Agilent 1290 Infinity LC Sys-
provides information about potential tem. Dols were analyzed by API 4000 LC-MS/MS System Sciex.
anticoagulant and thrombolytic proteins Peaks of dols with 17, 18, 19 and 20 isoprenoid units were captured
and the ratio of Dol-18/Dol-19 was calculated. In group of con-
D. Kharlampieva1, V. Babenko1, V. Manuvera1,2, E. Grafskaia1,2,
trols, significant correlation between Dol-18/Dol-19 ratio and age
N. Polina1, O. Podgorny1,3, P. Bobrovsky1, A. Belova1,
in urine was found (P < 0.005). Reference range of controls in
O. Miroshina1,2, D. Shirokov1,4, V. Lazarev1,2
1 urine was evaluated. There were not detected differences between
Federal Research and Clinical Center of Physical-Chemical
genders. Ratio of Dol-18/Dol-19 was significantly increased in
Medicine of Federal Medical Biological Agency, Moscow, Russia,
2 NgBR-CDG urine and tissues in comparison with control. Our
Moscow Institute of Physics and Technology (State University),
results showed a new possibility for diagnosis of patients with rare
Dolgoprudny, Moscow Region, Russia, 3Koltzov Institute of
CDG, who cannot be identified by usual screening methods. Sup-
Developmental Biology of Russian Academy of Sciences, Moscow,
ported by: AZV-16-31932A, RVO-VFN64165, SVV-UK 260367/
Russia, 4K.I. Skryabin Moscow State Academy of Veterinary
2017.
Medicine and Biotechnology, Moscow, Russia
Hirudotherapy is used in medicine since ancient times. The salivary
cell secretion (SCS) of medicinal leech contains a lot of biological P.09-106-Mon
active compounds that suppress blood clotting, decrease pain sensi- SGBS preadipocyte cell line can serve for
tivity and enhance local blood microcirculation. However, protein human beige type of thermogenic browning
and peptide composition of SCS is not fully described, and struc-
adipocyte differentiation
ture and properties of many components remain unknown. In our
A. Klusoczki1, E. Kristof1, P. Fischer-Posovszky2, M. Wabitsch2,
work we have generated a draft of the H. medicinalis genome. We
Z. Bacso3, L. Fes€us1
identified earlier unknown homologs for the genes encoding leech 1
Department of Biochemistry and Molecular Biology, University of
anticoagulants in a draft of medicinal leech genome. There were
Debrecen, Debrecen, Hungary, 2Division of Pediatric
homologs of serine proteinase inhibitors (bdellin A, bdellin B3,
Endocrinology and Diabetes University Medical Center Ulm, Ulm,
antistasin, eglin C, hirustasin) among identified sequences. We also
Germany, 3Department of Biophysics and Cell Biology, Debrecen,
determined several homologs of destabilase, a polyfunctional pro-
Hungary
tein. Its isopeptidase activity leads to breakdown cross-links in sta-
bilized fibrin and subsequent thrombolysis. This makes destabilase In contrast to white adipocytes, brown and beige adipocytes con-
to be a potential compound for treatment of thrombosis. Thus, the tain high amount of mitochondria which express uncoupling pro-
draft of the medicinal leech genome provides a database of tein 1 (UCP1) and its main function is thermogenesis and energy
sequences encoding the unique leech proteins for developing new expenditure. However, there is only limited data about regulatory
pharmacological compounds. This work was supported by the networks that drive human brown or beige adipocyte differentia-
Russian Science Foundation (project No. 17-75-20099). tion. Therefore, human cell line models are needed in order to
explore the key molecular targets of novel pharmacological treat-
ments that can enhance browning and help the therapy of
P.09-105-Wed patients suffering from metabolic syndrome. The Simpson-
Analysis of dolichol content in urine and Golabi-Behmel syndrome (SGBS) preadipocyte cell line provides
tissues of patients with congenital disorder of a useful tool for studies of human adipocyte biology. Our aims
glycosylation were to investigate whether brown or beige adipocyte differentia-
tion can be induced in SGBS cells and to clarify the effect of Iri-
a1, N. Ondruskova1, L. Kuchar2, H. Hansıkova1
L. Zdrazilov
1 sin (myokine which stimulates beige differentiation in response to
Laboratory for Study of Mitochondrial Disorders, Department of
exercise) and BMP7 (autocrine mediator which induces classical
Pediatrics and Adolescent Medicine, First Faculty of Medicine,
brown development) treatment during adipocyte differentiation.
Charles University and General University Hospital in Prague,
We aimed to test the involvement of the creatine phosphate sub-
Prague, Czech Republic, 2Research Unit for Rare Diseases;
strate cycle in the heat production of SGBS derived beige adipo-
Department of Pediatrics and Adolescent Medicine, First Faculty
cytes and investigate whether beige differentiation can be reversed
of Medicine, Charles University and General University Hospital in
to white adipocytes or they maintain their beige morphology. We
Prague, Prague, Czech Republic, Prague, Czech Republic
applied white and PPARc-driven browning (including long-term
Dolichol (dol) is membrane lipid, which carries glycans for Rosiglitazone treatment) differentiation cocktails to induce adipo-
N-linked protein glycosylation, O-mannosylation and GPI anchor cyte differentiation. The application of Rosiglitazone could be
biosynthesis ongoing in endoplasmic reticulum. Its structure is successfully used to induce browning of SGBS cells. Irisin treat-
composed by isoprenoid units. Dol is presented in all tissues and ment resulted in up-regulation of UCP1 and TBX1 genes. BMP7
most membrane organelles of eukaryotic cells. Recently some types moderately induced a classical brown phenotype. The browning
of congenital disorders of glycosylation (CDG) were described as protocol or Irisin induced a beige phenotype with high oxygen
consequence of dol biosynthesis defects (mutations in DHDDS, consumption rate for UPC1-dependent and creatine phosphate
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 241
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
futile cycle mediated heat production. Thus, SGBS cells can be P.09-108-Wed
shifted into both white and beige adipocytes. The continuous
ATP6AP1-CDG: Biochemical and molecular-
Rosiglitazone or Irisin treatment could maintain a beige pheno-
type under long-term (28 days) differentiation program. genetic characterization of two cases with
severe phenotype
N. Ondruskova, A. Vondrackova, M. Tesarova, T. Honzik,
P.09-107-Tue H. Hansikova, J. Zeman
Protective effect of alpha lipoic acid on apical Laboratory for Study of Mitochondrial Disorders, Department of
periodontitis-induced cardiac injury Pediatrics and Adolescent Medicine, First Faculty of Medicine,
U. Aksoy1, F. Kermeoglu1, A. O. Sehirli2, G. Savtekin3, Charles University and General University Hospital in Prague,
H. Ozkayalar4, S. Sayiner5 Prague, Czech Republic
1
Department of Endodontics, Faculty of Dentistry, Near East Introduction: Congenital disorders of glycosylation (CDG) are a
University, Nicosia, North Cyprus, Turkey, 2Department of Basic rare, clinically heterogeneous group of >100 metabolic diseases
Medicine Science, Faculty of Dentistry, Near East University, caused by deficiencies of enzymes/proteins participating in glycosy-
Nicosia, North Cyprus, Turkey, 3Department of Oral and lation pathways. Here we describe a case of two CDG-suspected
Maxillofacial Diseases and Surgery, Faculty of Dentistry, Near male siblings (P1 and P2) with hyperbilirubinemia, hepatopathy,
East University, Nicosia, North Cyprus, Turkey, 4Department of splenomegaly and wrinkled skin, who both died due to progressive
Medical Pathology, Faculty of Medicine, Near East University, liver failure at the age of 3 (P1) and 11 (P2) months. Results and
Nicosia, North Cyprus, Turkey, 5Department of Biochemistry, discussion: The finding of hypoglycosylated TF (CDG-II pattern)
Faculty of Veterinary Medicine, Near East University, Nicosia, and ApoC-III in sera from P1 and P2 pointed to a combined defect
North Cyprus, Turkey of N- and O-glycosylation. Altered morphology of Golgi appara-
Oxygen free radicals are involved in pathophysiology of apical tus (GA) and delayed retrograde GA transport, assessed by brefel-
periodontitis. This study was designed to assess the possible pro- din A treatment, was detected by immunocytochemistry in the
tective effect of alpha lipoic acid (ALA) on apical periodontitis cultivated fibroblasts of P1. Moreover, peroxisomal disturbance
(AP)-induced cardiac injury. 200–250 g weighed Wistar albino and increased reactive oxygen species were observed, demonstrat-
male rats were randomized into four groups; control group, ALA ing a complex impact of the defect on the cellular function. Whole-
group, AP group and ALA+AP group. In control and ALA exome sequencing in P1 identified a novel hemizygous mutation
groups, rats were not treated, saline and ALA (100 mg/kg) were c. 221T>C (p.Leu74Pro) in exon 2 of ATP6AP1 gene, and the
administrated. In AP and ALA+AP groups, the left maxillary same mutation was later confirmed in P2 by Sanger sequencing,
first molar teeth of the rats were opened with a round dental bur while the mother was found to be a carrier of the heterozygous
until the pulp chamber was exposed. This application was done variant. ATP6AP1 is an accessory protein of the vacuolar H+-
under 100 mg/kg ketamine and 10 mg/kg xylazine anaesthesia ATPase, a proton pump which participates in acidification of vari-
with using high-speed water cooling. It was then left open for ous intracellular compartments including GA. ATP6AP1-CDG
30 days to induce apical periodontitis. Saline and ALA (100 mg/ was first reported in 2016, and compared to the so far 12 described
kg) was administered intraperitoneally every 24 h during the cases presenting predominantly with immunodeficiency, hepatopa-
experiment. At the end of the experiment, animals were eutha- thy and cognitive impairment, our patients manifested with a more
nized by high dose of ketamine-xylazine combination. Serum severe phenotype. Supported by grants: AZV MZ CR 16-31932A,
ALP, LDH, CK and SOD activities were determined using an RVO-VFN64165, UNCE 204011.
automated biochemical analyser and the structural cardiac injury
was assessed pathologically. Results obtained were then statisti-
P.09-109-Mon
cally analysed using GraphPad Prism 7. Results were compared
by means of one-way analysis of variance (ANOVA). Tukey’s Hippocampal Ras protein through downstream
was used as a further analysis in binary comparisons. Serum effectors - Akt and ERK plays a significant role
ALP, LDH and CK activities were elevated in AP group. in the nongenomic regulation of thyroid
Besides, SOD activities were decreased in the AP group. While disorders
the changed enzyme activities was significantly normalized by N. Jojua1, E. Zhuravliova2,3, N. Sharikadze2, D. Mikeladze2,3
ALA treatment. Since ALA administration alleviated the apical 1
European University, Tbilisi, Georgia, 2Ilia State University,
periodontitis-induced heart injury and improved the cardiac Tbilisi, Georgia, 3I. Beritashvili Center of Experimental
structure and function. It seems likely that ALA with its anti- Biomedicine, Tbilisi, Georgia
inflammatory and antioxidant properties may be of potential
therapeutic value in protecting the cardiac tissue against systemic Thyroid hormones (THs) are important regulators of growth,
oxidative injury due to apical periodontitis. development and metabolism. The central nervous system is an
important target for the THs and hypothyroidism in adulthood
has been clearly linked to neurocognitive dysfunctions. It is also
known that THs exert nongenomic effects on the mitochondrial
energy metabolism. Nongenomic effects of THs can be mediated
by ERK and Akt signaling pathways, and their upstream regula-
tors - Ras proteins. Therefore, we decided to investigate quantita-
tive changes of these signaling molecules in the different
compartments of neural cells in the hippocampus of adult rats in
following groups: euthyroid (control), hypothyroid (methimazole-
treated), nobiletin (nobiletin-treated) and T4-treated hypothyroid
states. It was observed that level of phosphorylated ERK was
slightly increased in the cytoplasm of hypothyroid rats and signif-
icantly decreased in case of nobiletin supplementation. Level of
phosphorylated Akt was increased in the cytoplasm of
242 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
hypothyroid rats’ and was restored to control level for T4-treated P.09-111-Wed
animals. Level of K-Ras was increased on the plasma membrane
Tetracenomycin X novel inhibitor of
and significantly decreased on the endoplasmic reticulum of
hypothyroid rats. These parameters were restored to the control translation
level in both cases: with nobiletin supplementation and under T4 D. Lukianov1, E. Komarova2,3, D. Shiryaev1, Y. Zakalyukina4,
treatment. In addition, level of H-Ras was increased on the endo- M. Biryukov5, D. Skvortsov1, D. Rebrikov1, M. Podlesskaia1,
plasmic reticulum of hypothyroid rats and decreased to control I. Khven3, V. Tashlitsky1, T. Zatsepin1,2, M. Serebryakova1,2,
level for the rats taking nobiletin supplementation. Our results V. Polshakov1, A. Bogdanov1, P. Sergiev1,2, O. Dontsova1,2,
suggest that hippocampal Ras protein through downstream effec- I. Osterman1,2
1
tors - Akt and ERK plays a significant role in the nongenomic Lomonosov Moscow State University, Chemistry Department and
regulation of thyroid disorders. Belozersky Institute of Physical and Chemical Biology, Moscow,
Russia, 2Skolkovo Institute of Science and Technology, Skolkovo,
Moscow, Russia, 3Lomonosov Moscow State University, Faculty of
P.09-110-Tue Bioengineering and Bioinformatics, Moscow, Russia, 4Lomonosov
FcrR/ROS/CK2a is the key inducer of NF-jB Moscow State University, Department of Soil Biology, Department
of Soil Science, Moscow, Russia, 5Lomonosov Moscow State
activation in a murine model of asthma University, Department of Microbiology, Department of Biology,
Y. Im, J. Kim, J. Park, H. Lee, D. Kim
Moscow, Russia
Jeonbuk Univ., Jeonju, South Korea
Last few years people faced a problem that there are a lot new
The transcription factor, nuclear factor (NF)-jB plays a pivotal
species of bacteria which could not been influenced by certain
role in the development of allergic airway inflammation, How-
antibiotics. Also already known pathogenic bacteria gained resis-
ever, the mechanism of NF-jB activation in asthma remains to
tance genes. Antibiotic resistance is one of the biggest threats to
be elucidated. CK2a activation was assessed by CK2a phospho-
global health, according to WHO. By 2050, it is expected that
rylation and protein expression. Airway levels of histamine and
mortality rate caused by bacterial diseases will increase 14 times.
cytokines were determined by ELISA. We have used two (active
Therefore, there is great necessity of novel antibiotics in the
and passive) forms of allergic pulmonary inflammation. models.
whole world. Due to reasons mentioned above we decided to
In active form, animals were immunized with OVA via intraperi-
make screening for novel compounds with antibiotics activity.
toneally, followed by airway challenge with OVA. In passive
Screening was performed using reporter E. coli strain pDualrep2,
form, animals were passively sensitized by intratracheal instilla-
which can detect translation inhibitors and DNA damaging
tion with either anti-OVA IgE or anti-OVA IgG, followed by air-
antibiotics. In this screening we found significant antibiotic activ-
way challenge with OVA. The role of NADPH oxidase (NOX) in
ity for cultural broth sample of bacterium Amycolatopsis sp.
CK2a activation was assessed using NOX2-/- and NOX4-/- mice
which shown inhibition of translation. In further study a com-
because NOX2 and NOX4 contribute to many inflammatory dis-
pound was purified by HPLC chromatography, the structure was
eases. Second airway challenge increased CK2a phosphorylation
determined by combination of NMR and mass spectrometry and
and protein expression in airway epithelial cells as well as the
it turned out, that we re-discovered old antibiotic - Tetraceno-
nuclear translocation of the p50 and p65 subunits of NF-jB, all
mycin X (ThX). However, a mechanism of antibiotic activity has
of which were inhibited by the CK2a inhibitor, 4,5,6,7-tetrabro-
not elucidated yet. Further study showed that in vitro translation
mobenzotriazole and the antioxidant N-acetyl-L-cysteine. CK2a
with this compound shows opportunity to decrease level of trans-
phosphorylation and protein expression were significantly
lation. This result was proved by toe-printing assay, which
impaired in NOX2-/-, but not in NOX4-/-, mice. Induction of pas-
demonstrates a translational inhibition as well. By now we also
sive sensitization using anti-OVA IgE, activated neither CK2a
have found some mutant strains of E. coli which have ThX resis-
nor NF-jB. In contrast, induction of passive sensitization using
tance because of mutations in rRNA, this data is also proved by
anti-OVA IgG activated both CK2a and NF-jB. These data sug-
probing assay. Therefore, it could be reasonably assumed that
gested that Fc gamma receptor (FcgR)/reactive oxygen species
ThX probably provides inhibition of translation on elongation
(ROS)/CK2a is a key inducer of NF-jB activation in airway
stage. In view of all data obtained, Tetracenomycin X is of great
epithelial cells in a murine model of asthma.
medical and scientific interest as a molecule which might be a
basis to development of novel antibiotic drug. Thereby it requires
a further scrutiny. Work was supported by the Russian Science
Foundation (grant 18-44-04005) for I.A.O.
P.09-112-Mon
Lactuca capensis mitigate cognitive
dysfunction on Ab1-42-induced a rat model of
Alzheimer’s disease through regulating BDNF
mRNA and IL1b mRNA expression and
antioxidant system activity
P. A. Postu1, J. Noumedem2, O. Cioanca3, M. Mihasan1,
L. Gorgan1, B. A. Petre1, L. Hritcu1
1
Alexandru Ioan Cuza University of Iasi, Iasi, Romania, 2Institut
ote, Douala, Cameroon, 3University of
Universitaire de la C^
Medicine and Pharmacy “Gr. T. Popa”, Iasi, Romania
We investigated the neuropharmacological effects of the
methanolic extract from Lactuca capensis Thunb. leaves (100 and
200 mg/kg) for 21 days on memory impairment in an
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 243
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
244 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 245
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
246 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 247
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
probucol had significant effect on the percentages of Annexin V- training-induced increase in content of mitochondrial enzymes
FITC positive cells and the increase in multicaspase levels when and transcriptional factors involved in mitochondrial biogenesis
compared with the vehicle group. We found a decreased protein in human skeletal muscle. For this purpose ten young untrained
expression of IjBa, phospho-IjBa, p53 and bcl-2, whereas a sig- males performed the one-legged moderate intensity knee-exten-
nificant increase in bax protein levels. Probucol has also down- sion for 1 h. Biopsies from m. vastus lateralis were taken prior
regulated the expression of angiogenesis-associated proteins such to, and after the exercise. This test was repeated after 8 weeks of
as VEGF, MMP-9 and eNOS. Our results indicate that probucol cycling aerobic training. After 8-wk training, the content of mito-
induces anti-proliferative effects via blocking of cell cycle pro- chondrial proteins at rest (NDUFB8, SDHB, UQCRC2, MT-
gression and represent an antimetastatic potential by inhibiting CO1, ATP5A1) increased (by 35–162%, P < 0.05). There were
mRNA expression of metastatic MMP-9 protein in SW480 cells. no changes in the expression of genes encoding these proteins at
rest and after single exercise (both before and after 8-wk train-
ing). An increase in the content of transcriptional regulators was
P.09-125-Tue found after 8-wk training for CRTC2 (60%, P < 0.05), NR4A3
Synthesis of charged meso-arylporphyrins – (100%, P < 0.05) and TFAM (20%, P < 0.06). The expression of
potential photosensitizers for antimicrobial the NR4A3 and TFAM genes increased (P < 0.05) after single
PDT exercise, both before and after 8-wk training. An increase in the
expression of genes in response to single exercise was also
K. A. Zhdanova, N. A. Bragina
observed for other transcriptional regulators (PGC-1a, ESRRG),
Moscow Technological University, Moscow, Russia
but there was no increase in the content of their proteins after 8-
Infectious diseases are the second leading cause of death world- wk training. The reason for the increase in the content of mito-
wide and it’s directly related to the increasing resistance of many chondrial enzymes could not be linked to the increase in basal or
pathogens to the antibiotics. Antimicrobial photodynamic ther- exercise-induced expression of their genes. On the contrary, the
apy (APDT) resolves these problems. Tetrapyrroles is widely uses training-induced increase in the content of some transcriptional
in APDT due to the ability of these dyes to generate singlet oxy- regulators was associated with the activation of their mRNA
gen 1O2 upon irradiation. In this study, new PS for APDT based expression after each single exercise. This work was supported by
on charged cationic and anionic amphiphilic meso-arylporphyrins the Russian Science Foundation (grant no. 14-15-00768).
was created. First, approaches to the synthesis of charged amphi-
philic meso-arylporphyrins (cationic and anionic) have been
developed and optimized. The main strategy is based on the P.09-127-Mon
preparation of pre-functionalized benzaldehydes, which was Modified GDNF has stimulated the neural
introduced into the reaction of monopyrrolic condensation differentiation of progenitor cells and it may
according to Lindsay’s method. Further, cationic porphyrins
be used in the treatment of Parkinson’s
(pyridinium and ammonium) were obtained by the quaternization
reaction. Target anionic porphyrins were received by alkaline disease and ischemic stroke
hydrolysis of the porphyrin precursors with terminal car- G. Pavlova, D. Shamadykova, N. Kust, D. Panteleev,
bomethoxy groups. We’ve varied degree of lipophilicity, arrange- A. Revishchin
ment of the substituents, charge in porphyrins. Considering the IBG RAS, Moscow, Russia
prospects of synthetic porphyrins using for inactivation of GDNF therapy is effective for treating degeneration of dopamin-
microorganisms, the aggregation of potential PS in aqueous ergic neurons in Parkinson’s disease. It is possible that pre-(a)
microheterogeneous systems imitating the intracellular environ- pro-GDNF is needed for conventional neuron survival, and pre-
ment was investigated. It was shown that the cationic derivatives (b) pro-GDNF serves as SOS system during traumatic injury of
of porphyrins in aqueous microheterogeneous systems forms J- neurons or neurodegenerative diseases. To study ‘pro’ region
and H-type homoaggregates, as well as mixed ionic associates in function during fast transport and factor induction properties
the presence of oppositely charged polyelectrolytes and premicel- several versions of modified GDNF were made. Secretion of
lar aggregates of anionic surfactants. Also, we investigated bind- GDNF into medium has been shown. Then modified GDNF
ing of cationic porphyrins with cholesteric DNA dispersion. It were introduced into HEK293 cells. Condition media after trans-
was experimentally proved the formation of two types of com- genic cell culturing was added into culture medium of rat embry-
plexes of all porphyrins with DNA: intercalated and external onic spinal ganglion explant. Deletion of ‘pro’ region essentially
bedding along a small groove. Received porphyrins are promising increases GDNF effects as neural inductor. Study of culture of
candidates for further antimicrobial studies. This work was sup- dissociated spinal ganglion and calculation of neural sprouts
ported by the Russian Science Foundation, project No. 17-73- yielded the same results. Deletion of both pre- and pro-regions
10470. enhances trophic activity of GDNF (mGDNF). Spinal ganglia
cultured in the presence of medium conditioned by cells trans-
fected with mGDNF exhibited active growth of b-3-tubulin-posi-
P.09-126-Wed tive axons by day 4. Then we also demonstrate neurotrophic
Transcription regulation of contents of some effect of mGDNF for PC12 cells in vitro. Studies were also con-
mitochondrial enzymes and transcription ducted in vivo. A model of Parkinson’s disease was used, which
factors in human skeletal muscle was obtained by subcutaneous injection of MPTP into C57Bl / 6
E. Lysenko1,2, D. Popov1,2, R. Bokov1, N. Kurochkina1, mice. Implantation of cells producing mGDNF in caudatum-
O. Vinogradova1,2 putamen smoothed out symptoms of Parkinson’s disease in
1
SSC RF Institute of Biomedical problems of the RAS, Moscow, motor activity tests. Research is supported by Grant from RNF.
Russia, 2Lomonosov Moscow State University, Moscow, Russia
Skeletal muscle adapts to changes in contractile activity by pro-
duction of specific regulatory and structural proteins, leading to
optimization of the contractile function. The aim of our work
was to study the transcription mechanisms regulating the
248 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
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ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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250 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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POSTERS Biochemistry and medicine
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ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
252 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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POSTERS Biochemistry and medicine
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ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
were also increased by TFG overexpression. Conversely, knock- main aim of our research is to study the major lipid parameters
down of TFG using microRNA resulted in the decrease of of the cow blood serum, as well as the application of a regression
SREBP1 and SCD. These findings suggest that TFG is a novel model for the estimation of the triglycerides (Tg) and cholesterol
regulator for lipid production in sebocytes, which can be a new (Ch) content in the cow blood serum using the measured DST
target for acne treatment. parameters at the liquid/air interface. Blood from experimental
animals was taken before morning feeding (on an empty stom-
ach) from the vein. All procedures were carried out with the
P.09-144-Wed approval of the Animal Care Committee of the Moscow SAVMB
Regulation of biofilm formation in bacilli and internationally recognized guidelines. Six DST parameters of
I. Danilova, L. Dinh, N. Rudakova, M. Sharipova the cow blood serum were obtained at different times of surface
Kazan Federal University, Kazan, Russia existence (sigma 0–3: s0, s1, s2, s3, and lambda 1–2: k1, k2). The
biochemical composition was characterized by Tg and Ch con-
Biofilms are a factor of pathogenicity, have multiple resistance,
tent. The regression coefficients obtained as the result of the
cannot be treated by traditional antibiotic methods and are the
regression model provided the following regression equation for
basis for the development of chronic processes in the body. Until
the Tg content in cow blood serum: [Tg] = – 0.11 r1 + 0.14 r2 –
now, there are no reliable ways to combat biofilms, this problem
0.1 r3 – 0.09 k1 + 5.79. The regression equation for the Ch con-
requires further development. Bacteria of the Bacillus genus are
tent is the following: [Ch] = 0.25 r1 – 0.22 r2 – 0.1 k0 + 0.12 k1
convenient model microorganisms for studying the architecture
+ 0.71. The proposed model has predictive value and can be used
of biofilms and the action of various biological agents. In our
to estimate Tg and Ch content in cow blood serum by the
laboratory we selected a culture medium for the formation of
obtained DST parameters. The regression correlation analysis
biofilms by B. subtilis strains. There are differences in the molec-
can contribute to better understanding of the influence of the
ular mechanisms of formation and regulation of biofilms in dif-
major individual components of biological liquids and for the
ferent species of bacilli. Common is the process of repression of
diagnostics purposes in animal medicine and zootechnology. This
the biofilm matrix formation by the SinR protein, which inhibits
work was supported by the Russian Foundation for Basic
the transcription of the operon from 15 genes, the eps-operon,
Research (grant 18-016-00207).
whose function is the biosynthesis of extracellular matrix polysac-
charides. In our laboratory comparative analysis of sinR genes
was performed in different representatives of bacilli, including P.09-146-Tue
pathogenic species - B. cereus, B. thuringiensis, B. anthracis and
Enzymatic production of anticancer
saprophytic - B. subtilis 168, using the Clustal Omega program.
It was found that the sequence of sinR genes in pathogenic thiosulfinates by targeted to ovarian cancer
strains is almost 100% identical. However, the similarity of the cells methionine c-lyase
gene sequences of sinR pathogenic species and B. subtilis is only E. Morozova, V. Kulikova, V. Koval, S. Revtovich,
72%. This probably explains the formation of biofilms with dif- T. Demidkina
ferent architectures in B. subtilis and pathogenic. In the longer Engelhardt Institute of Molecular Biology of the Russian Academy
term, we plan to construct mutant strains of B. subtilis 168 with of Sciences, Moscow, Russia
the inactivated sinR gene, as well as bearing point mutations in
Pyridoxal 5’-phosphate-dependent methionine c-lyase (EC
this gene. This will allow us to evaluate the role of mutations in 4.4.1.11, MGL) is involved in the metabolism of sulfur-contain-
the gene sinR in the formation of various phenotypes of biofilms ing amino acids of bacteria, unicellular eukaryotes and fungus.
in bacilli. Understanding these mechanisms is important for find- We have shown that MGL effectively catalyzes the b-elimination
ing new strategies in creating agents to combat biofilms. The
reaction of S-alk(en)yl-L-cysteine sulfoxides (alliin, methiin, pro-
work is performed according to the Russian Government Pro- piin) to yield thiosulfinates which are capable of killing cancer
gram of Competitive Growth of Kazan Federal University. cells. A variety of malignant cells contain specific markers. The
possibility to use carboxy derivatives of isoflavones as carriers for
P.09-145-Mon affinity targeting of drugs to tumor cells expressing estrogen
receptor of the b type such as ovarian carcinoma and others was
Colloid-chemical regression model in analysis recently reported. The isoflavone daidzein is a naturally occurring
of dynamic surface tension correlation with compound found in soybeans and other legumes. Acting as a
triglycerides and cholesterol content in the weak estrogen, daidzein recognizes a putative plasma membrane
cow blood serum estrogen receptor and a membrane-located estrogen receptor of
O. Voronina1, M. Tsarkova2, I. Milyaeva3, S. Zaitsev3 b–related protein. The conjugates between daidzein and Citrobac-
1
Federal State Budgetary Educational Institution of Higher ter freundii MGL and its mutant form C115H MGL were pre-
Education “Moscow state Academy of Veterinary Medicine and pared. Conjugates were specifically bound to ovarian carcinoma
Biotechnology - MVA by K.I. Skryabin”, Moscow, Russia, cells (SCOV-3) and the substrates (alliin, methiin and propiin)
2
Federal State Budgetary Educational Institution of Higher were added. Thiosulfinates enzymatically produced by tumor
Education “Moscow state Academy of Veterinary Medicine and anchored MGL and C115H MGL killed tumor cells. The IC50
Biotechnology - MVA by K.I. Skryabin”, Moscow, Russia, values for daidzein-enzymes conjugates with S-alk(en)yl-L-
3
Federal State Budgetary Educational Institution of Higher cysteine sulfoxides were determined. The usage of daidzein-
Education “Moscow state Academy of Veterinary Medicine and mutant form C115H MGL conjugate is more promising due to
Biotechnology - MVA by K.I. Skryabin”, Moscow, Russia its higher catalytic efficiency in the b-elimination reactions of S-
alk(en)yl-L-cysteine sulfoxides. Thus the pharmacological couples
The application of mathematical methods in biochemical sciences of the mutant form enzyme with sulfoxides might be new anti-
leads to the expansion of analytical methods for diagnostics of cancer agents. This work was supported by Russian Foundation
the physiological-biochemical status of various animals. The use for Basic Research (project # 18-04-00916-a) and by the Program
of the regression method in modeling the interrelation between of fundamental research for state academies for 2013-2020 years
some biochemical parameters and dynamic surface tension (DST) (# 01201363820).
of the animal blood serum was reported for the first time. The
254 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 255
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
256 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 257
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
258 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 259
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
260 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
Cells were seeded in 96-well plates and cultivated for 24 h before P.09-167-Tue
the onset of treatment and treated with quercetin (1–200 lM) for
Evaluation of FNDC5/irisin levels of BDNF
48 h. The cell viability and proliferation were assessed by using
Alamar Blue assay and the absorbances read at 570 nm using the heterozygous mice
spectrophotometer. Then sigmoidal graphics were drawn by S. Dogramaci1, M. Erdem1, A. Alver1, A. Mentese2, S. Aydin-
€
Abidin3, S. Ozer Yaman1, I. Ince1, A. Bodur1, S. C. Karahan1
obtained proliferation data and the data converted to IC50 val- 1
ues. Quercetin inhibited cell viability of HeLa (IC50: 27.1 lM), Department of Medical Biochemistry, Faculty of Medicine,
LNCaP (IC50: 19.4 lM), SW-620 (IC50: 13.5 lM) HEPG-2, Karadeniz Technical University, Trabzon, Turkey, 2Program of
(IC50: 79.1 lM) PC-3 (IC50: 153.9 lM) and HT-29 (IC50: Medical Laboratory Techniques, Vocational School of Health
172.4 lM) cells in a dose-dependent manner. Although quercetin Sciences, Karadeniz Technical University, Trabzon, Turkey,
3
inhibited the proliferation of HeLa, LNCaP and SW-620 cells at Department of Biophysics, Faculty of Medicine, Karadeniz
lower concentrations, it has been found less effective against pro- Technical University, Trabzon, Turkey
liferation PC-3 and HT-29 cells. We found that quercetin dis- Brain-derived neurotrophic factor (BDNF) is a factor that
plays quite different selectivity on proliferation of human cancer released by neurons and has role in the development and growth
cell lines. Our results demonstrate that quercetin may have a of neurons. BDNF heterozygous mice (BDNF (+/-)), which is
potential benefit for cancer prevention, especially colon and pros- deprived of one of the two alleles coding BDNF protein, is used
tate cancer. One of the main problems is solubility and targeting to study the physiological roles of BDNF. These mice are charac-
of quercetin towards cancerous cells, which can be overcame via terized by the reduced BDNF expression in nearly all tissues. Iri-
specialized water-soluble carrier compounds. This project was sin is formed by proteolytic cleavage of the transmembrane
supported by Selcuk University (BAP: 16401083). protein fibronectin type III domain containing 5 (FNDC5), the
expression of which is most abundant in skeletal muscle. It is
responsible for browning of white adipose tissue through enhanc-
P.09-166-Mon ing oxygen consumption and induction of expression of thermo-
Altered neuropeptide Y and ghrelin levels genin. In this study, FNDC5/Irisin parameter was aimed to be
BDNF heterozygous mice evaluated between BDNF (+/-) and wild type littermates. BDNF
M. Erdem1, S. Do gramaci1, A. Alver1, A. Mentese2, I. Abidin3, (+/-) and WT mice were identified by conventional PCR. 11 wild
S. Demir , S. C. Karahan1
4
type (WT) and 9 BDNF (+/-) mice were used. Serum FNDC5/Iri-
1 sin levels were determined by enzyme-linked immunosorbent
Department of Medical Biochemistry, Faculty of Medicine,
Karadeniz Technical University, Trabzon, Turkey, 2Program of assay method. We found that FNDC5/Irisin levels in BDNF
Medical Laboratory Techniques, Vocational School of Health (+/-) mice were higher in comparison to WT mice (718 384 pg/
Sciences, Karadeniz Technical University, Trabzon, Turkey, mL; 658 520 pg/mL, respectively). BDNF, which is the down-
3 stream mediator of FDNC5/Irisin, decreases Fndc5 gene expres-
Karadeniz Technical University, Faculty of Medicine, Department
of Biophysics, Trabzon, Turkey, 4Department of Nutrition and sion as part of potential feed-back loop. Our results were
Dietetics, Faculty of Health Sciences, Karadeniz Technical suggested that reduced BDNF may have increased FNDC5/Irisin
University, Trabzon, Turkey levels. Further studies are needed to evaluate the effects of
BDNF on metabolism.
Brain-derived neurotrophic factor (BDNF) plays a crucial role in
neuronal survival, synaptic plasticity and neural circuit develop-
ment. BDNF heterozygous mice (BDNF (+/-)), which is deprived P.09-168-Wed
of one of the two alleles coding BDNF protein, is used as an ani-
Identification of functional non-coding SNPs in
mal model to study the physiological roles of BDNF. These mice
are characterized by the reduced BDNF expression. Neuropep- the autoimmunity-associated human locus
tide Y (NPY) has a role in the regulation of various basic physio- 17q12-21 to be edited by CRISPR/Cas system
logical functions, such as food intake, metabolic functions, in order to find new therapy targets
circadian rhythm, cognition and neuronal excitability. Ghrelin is A. S. Ustiugova1,2, M. A. Afanasyeva2, K. V. Korneev1,2,
the hormone that regulate appetite and energy balance in the D. V. Kuprash1,2,3
1
hypothalamus. In this study, NPY and ghrelin parameters were Faculty of Biology, Lomonosov Moscow State University,
aimed to compare between BDNF (+/-) and wild type (WT) lit- Moscow, Russia, 2Engelhardt Institute of Molecular Biology,
termates. BDNF (+/-) and WT mice were identified by conven- Russian Academy of Sciences, Moscow, Russia, 3Moscow Institute
tional PCR. 11 WT and 9 BDNF (+/-) mice were used. Serum of Physics and technology, Dolgoprudny, Moscow Region, Russia
NPY and ghrelin levels were investigated by enzyme-linked
At the moment, protein targets for drug design are encoded by
immunosorbent assay method. We found that NPY levels of
only a small fraction of the human genome. Genome-wide associ-
BDNF (+/-) mice were lower than WT mice (2567.5 994.5 pg/
ation studies are a powerful tool for identification of loci
mL; 3821 1633.5 pg/mL, respectively). Ghrelin levels were
involved in the development of complex diseases with “missing
higher in BDNF (+/-) mice than WT mice (9 3.5 ng/mL,
heritability”, such as autoimmune disorders. However, much
8 3.5 ng/mL, respectively). BDNF and its receptor tropomyo-
effort is additionally required to reveal causative SNPs in the
sin receptor kinase B interaction lead to the induction of NPY
associated loci - mainly non-coding regulatory SNPs - as well as
gene expression. Our results were suggested that reduced BDNF
their target genes. To this end, we systematically searched for
may cause decreased NPY levels. However, reduced BDNF may
and experimentally validated potential causative SNPs in an
cause increased ghrelin levels. But, the direct mechanism between
autoimmunity-associated locus 17q12-21 for subsequent integra-
reduced BDNF and ghrelin has not been fully identified. Further
tion of the risk alleles into the genome of relevant human leuko-
studies are needed to evaluate the effects of reduced BDNF on
cyte cell lines. We focused on 6 non-coding SNPs, which are
energy metabolism.
most likely to be causal according to the PICS (Probabilistic
Identification of Causal SNPs) algorithm of fine-mapping. We
determined the coordinates of putative enhancers bearing selected
SNPs using the epigenetic data generated by the ENCODE 2
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 261
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
project for primary human immune cells. The luciferase reporter P.09-170-Tue
assay was used to test the effect of SNP alleles on the activity of
Human neuromodulator Lynx1 affects synaptic
the putative enhancers in a set of relevant cell lines. Three out of
the six non-coding polymorphisms proved to be functional in plasticity and ameliorates cognitive deficits in
model regulatory T cells and one of them was also functional in neurodegeneration
a B-lymphocytic cell line. Moreover, we compared binding of the M. Bychkov1, N. Vasilyeva2,3, A. Andreev-Andrievsky2,4,
transcription factor MEF2C to the alternative alleles of M. Shulepko1,2, A. Popova2,4, E. Lagereva2,4, I. Zueva5,
rs12946510 using modified pull-down assay. Further introduction K. Petrov5, D. Kulbatskii1,2, E. Loktyushov1,2, M. Thomsen6,
of the alternative alleles of causal SNPs into the genome of D. Dolgikh1,2, Z. Shenkarev1,2,7, P. Balaban2,3,
appropriate cell lines using CRISPR/Cas9 system will allow us to M. Kirpichnikov1,2, E. Lyukmanova1,2,7
1
determine target genes for these regulatory SNPs. Gene editing Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
efforts are supported by the Program of fundamental research Russian Academy of Sciences, Moscow, Russia, 2Lomonosov
for state academies for 2013–2020, research topic 01201363823. Moscow State University, Moscow, Russia, 3Institute of Higher
Nervous Activity and Neurophysiology, Russian Academy of
Sciences, Moscow, Russia, 4Institute of Biomedical problems,
P.09-169-Mon Russian Academy of Sciences, Moscow, Russia, 5A.E. Arbuzov
Impact of alcohol consumption on myocardial Institute of Organic and Physical Chemistry Subdivision of the
characteristics in patients with cardiovascular Federal State Budgetary Institution of Science “Kazan Scientific
Center of Russian Academy of Sciences”, Kazan, Russia,
disease 6
Department of Synaptic Transmission in Vitro, H. Lundbeck A/S,
L. Varekha1, G. Ivanov1,2,3, P. Ogurtsov1, E. Dvornikov1,
Copenhagen, Denmark, 7Moscow Institute of Physics and
G. Myandina1, E. Neborak1, S. Syatkin1
1 technology, Dolgoprudny, Moscow Region, Russia
Peoples’ Friendship University of Russia (RUDN University),
Moscow, Russia, 2Department of cardiology, First MGMU them. Many neurodegenerative diseases (e.g. Alzheimer’s disease) asso-
I.M. Sechenov, 26-2 Trofim, Moscow, Russia, 3Sechenov First ciated with cognitive impairment are accompanied by the loss or
Moscow State Medical University of the Ministry of Health of the dysfunction of nAChRs. Lynx1 is a GPI-tethered nAChR modu-
Russian Federation (Sechenov University), Moscow, Russia lator expressed in brain areas important for learning and mem-
ory. Previously, we have shown that water-soluble domain of
Alcohol consumption is related to various clinical manifestations
Lynx1 (ws-Lynx1) competes with oligomeric beta-amyloid Ab(1-
of cardiovascular diseases (CVD) ranging from hypertensive dis-
42) for binding to different nAChR subunits in the brain extracts
ease and arrhythmias to ischemic heart disease (IHD) and alco-
and abolishes the Ab(1-42) cytotoxic effect in cultured cortical
holic cardiomyopathy. Alcohol impacts on CVD via multiple
neurons. We hypothesized that ws-Lynx1 could be used for
pathways and the effects of alcohol consumption on the risk of
improvement of cognitive decline and prevent neurotoxic effects
CVD are complex as there are beneficial and detrimental effects
of Ab(1-42) in vivo. To understand the Lynx1 role in cognitive
depending on volume and patterns of drinking. The aim of this
function, we studied the effects of ws-Lynx1 preparation in wild-
study is to evaluate the damaging effect of alcohol intake on
type mice and transgenic animals modeling Alzheimer disease
myocardium characteristics in patients with CVD. The 314
(2xTg-AD). Ws-Lynx1 penetrated the blood-brain barrier upon
patients, 148 males and 166 females, the age 33 - 65 with major
intranasal administration with specific accumulation in the cor-
categories of CVD (hypertension, IHD and arrhythmia) were
tex, hippocampus, and cerebellum. Chronic ws-Lynx1 treatment
divided into three categories according to volume of alcohol con-
reduced anxiety and prevented memory impairment in wild-type
sumption: lifetime abstainers (0 doses of alcohol per day), low to
and 2xTg-AD mice. Positive modulation of synaptic plasticity
moderate drinkers (1- 4 doses per day) and heavy drinkers (5 and
associated with activation of presynaptic a7 type nicotinic acetyl-
more doses per day). The changes in electrophysiological charac-
choline receptors and neurotransmitter release facilitation was
teristics (EPC) of myocardium in patient with CVD were detected
observed in ws-Lynx1 treated mice. Ws-Lynx1 enhanced mRNA
according to analysis of echocardiography (ECG) signal by dis-
level of endogenous Lynx1, normalized AChE level in the cortex,
persion mapping method. The patients with CVD use 5 and more
and restored synaptic density in the hippocampus in 2xTg-AD
doses of alcohol per day have significantly higher sizes of left and
mice. Lynx1 could be considered a natural protector of the brain
right atria and right ventricle than patients with low alcohol con-
cholinergic system and its soluble counterpart may be a potential
sumption or abstainers. The more pronounced changes in the
candidate for the cognitive impairment treatment in neurodegen-
EPC of the myocardium, left ventricle (LV) end systolic dimen-
erative diseases. The work was supported by the Russian Science
sion (LVESD), left ventricular ejection fraction (%) and interven-
Foundation (project #16-14-00102).
tricular septum thickness were found in patients with CVD use 5
and more doses. As a conclusion, heavy drinking (≥ 5 doses per
day) provides the damaging effect on EPC of myocardium. The P.09-171-Wed
early stages of cardiovascular disease’s deterioration can be
The first experience of tomosynthesis in
identified by detection of electrophysiological abnormalities and
electric instability of the myocardium by dispersion mapping of diagnostic of nonpalpable breast cancer
ECG – signal method. The publication was supported by the M. V. Grinberg, S. M. Chibisov, I. S. Eremina,
“RUDN University Program 5-100” and the Ministry of Educa- M. L. Blagonravov, E. V. Kharlitskaya, T. V. Maksimova,
tion and Science of Russia (the Agreement No. 02.A03.21.0008). E. V. Neborak, S. P. Syatkin, G. I. Myandina
Peoples’ Friendship University of Russia (RUDN University),
Moscow, Russia
The urgency of the problem of studying the incidence of the
breast diseases is a task of modern health, as there has been a
steady increase of this problem including malignant nature. At
present time, some progress has been made in diagnosing early
forms of the diseases due to the introduction of modern
262 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
technologies of radiation diagnostics. Despite the extensive use of elucidate mutations conferring resistance. This work was sup-
mammography, a number of limitations remain. This, first of all, ported by the Russian Science Foundation grant 17-75-20060.
is associated with the complexity in interpreting images of volu-
metric formations due to superimposition of structural elements
of breast tissue, located in different planes. Tomosynthesis of the P.09-173-Tue
breast is a new technology that helps to avoid these shortcom- A polymorphism associated with wheeze
ings. The clinical data of the study were data of clinical, X-ray phenotype in asthma and regulation of IL33
and sonographic survey of 250 women in outpatient conditions. gene expression
Nonpalpable lesions in the mammary glands were found in 150
D. Kuprash1,2, N. Mitkin1, A. Muratova1,2
women. X-ray examination was carried out on the Hologic Sele- 1
Engelhardt Institute of Molecular Biology, Russian Academy of
nia Dimensions in machine «COMBO» mode. On standard mam-
Sciences, Moscow, Russia, 2Biological Faculty, Lomonosov
mograms on a background of diffuse changes nonpalpable signs
Moscow State University, Moscow, Russia
of cancer were found in 55 (93%) of women in the mode of
tomosynthesis in 58 patients (97%), and in 45 (75%) under US. Cytokine IL-33 is secreted by epithelial and endothelial cells dur-
In conclusion the use of tomosynthesis in diagnostic of non pal- ing necrosis and stimulates humoral immune response. Elevated
pable breast formations enhances X-ray method, increases the IL33 expression in pulmonary epithelium of asthmatic patients
percentage of detection of unsuspected cancer, 4% improves the correlates with exacerbation of allergen-induced inflammation
differential diagnosis of diseases associated with architectural dis- and disease progression. We hypothesized that individual varia-
tortion of the breast tissue, 5% improves the diagnosis of dis- tions in the IL33 gene expression may be explained by polymor-
eases associated with the accumulation of calcifications. This phisms of non-coding regulatory regions, in particular by SNP
publication was prepared with the support of the “RUDN rs4742170 located in an enhancer region in the il33 locus and
University Program 5-100” and the Ministry of Education and associated with asthmatic wheezing phenotype. Activities of the
Science of the Russian Federation (the Agreement No. IL33 enhancer variants containing different rs4742170 alleles
02.A03.21.0008) were assessed upon transfection of the corresponding luciferase
reporter constructs into NCIH pulmonary epithelial cell line fol-
lowed by cortisol stimulation. We observed that rs4742170 risk
P.09-172-Mon allele no longer contained a binding site for glucocorticoid recep-
Search for azolo[1,2,4,5]tetrazines biotargets in tor (GR), a transcription factor that inhibits expression of a
mycobacteria number of inflammatory mediators. Reduced binding of GR to
D. A. Maslov1, K. V. Shur1, A. A. Vatlin1, O. B. Bekker1, IL33 enhancer correlated with elevated IL-33 promoter activity
A. V. Korotina2, G. L. Rusinov2, V. N. Charushin2, in lung carcinoma cells. Our data suggest that differential binding
V. N. Danilenko1 of the rs4742170 alleles to may underlie the emergence of asth-
1
Vavilov Institute of General Genetics Russian Academy of matic wheezing phenotype in response to increased IL33 gene
Sciences, Moscow, Russia, 2Postovsky Institute of Organic expression and systemic inflammation. This study is supported by
Synthesis Ural Branch of the Russian Academy of Sciences, Program of fundamental research for state academies for 2013–
Ekaterinburg, Russia 2020 (research topic 01201363823).
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 263
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
264 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
bone implants. The aim of our study was to evaluate the effect spinal hernia and reduced activity of these enzymes in malignant
of newly synthesized amorphous calcium phosphates modifies gliomas was found. The regulatory role of these enzymes on the
with tartaric acid (ACP-TA) or (5% or 18%) citric acid (ACP- proliferative activity of lymphocytes in neurosurgical pathology,
CA-5, ACP-CA-18). Mouse (BALB/c 3T3 embryonic fibroblasts including brain gliomas with different degree of malignancy, is
and cultures from bone marrow- BMC and bone explants- BEC) suggested. The publication was supported by the “RUDN
and human (Lep-3 and BJ embryonic fibroblasts, SAOS-2 University Program 5-100” and the Ministry of Education and
osteosarcoma) cells were used as model systems in our investiga- Science of the Russian Federation (the Agreement No.
tions. Direct experiments (DE) with cells seeded on the surface of 02.A03.21.0008).
the materials were performed by MTT test and Scanning electron
microscopy (SEM) after 3–6 days (MTT test) and 7–10 days
(SEM). In indirect experiments (IDE) the cells were cultivated P.09-179-Tue
for 72 h in culture medium pre-incubated for 3, 6 and 21 days in Effects of reduced tumor burden by selective
the presence of the examined material and cell viability was esti- treatment of lung cancer on muscle mass loss
mated by MTT test. The results obtained demonstrate the ability in cachectic mice
of cells to adhere and proliferate on materials’ surface. Direct
A. Salazar-Degracia1, P. Granado-Martınez1, A. Millan-
experiments with ACP-TA reveal cell viability ≥ 100% as com-
Sanchez1, E. Barreiro1,2
pared to the control (BALB/c 3T3, BECs, Lep-3, SAOS-2). 1
Pulmonology Department-Muscle Wasting and Cachexia in
Longer pre-incubation period of culture medium with this mate-
Chronic Respiratory Diseases and Lung Cancer Research Group,
rial leads to increased cell viability (IDE). Viability of Lep-3
IMIM-Hospital del Mar, Parc de Salut Mar, Health and
human fibroblasts estimated in both DE and IDE with ACP-CA-
Experimental Sciences Department (CEXS), Universitat Pompeu
5 and ACP-CA-18 on 3rd day was ≥ 100% as compared to the
Fabra (UPF), Barcelona Biomedical Research Park (PRBB),
control and then it starts to decrease but retains >70–80%. In
Barcelona, Spain, 2Centro de Investigaci
on en Red de
contrast, the viability of human osteosarcoma SAOS-2 cells is
Enfermedades Respiratorias (CIBERES), Instituto de Salud
around 50% in the same conditions. Better viability was observed
Carlos III (ISCIII), Barcelona, Spain
with ACP-CA-5 than ACP-CA-18. In conclusion, among the
materials investigated ACP-TA exhibit the most promising cyto- Cancer-cachexia is a syndrome characterized by the pronounced
compatibility proved in direct and indirect experiments with loss of body weight and the wasting of muscle and adipose tissue,
human and murine cells. which leads to impaired quality of life and reduced survival. To
what extent tumor burden influences muscle mass independently
of specific treatments for cachexia remains to be known. To assess
P.09-178-Mon whether reduced tumor burden by selective treatment of the can-
The new approaches to understanding the cer cells may exert any effects on muscle mass loss and function in
mechanism of immunosuppression in brain mice. Body and muscle weight, grip strength, muscle morphome-
try, apoptotic nuclei, systemic levels of troponin-I, proteolysis,
glioma patients
and apoptosis markers were determined in diaphragm and gas-
R. I. Sokuev1, N. Y. Gridina2, S. P. Syatkin1, E. V. Neborak1,
trocnemius muscles of lung cancer (LP07 adenocarcinoma cells)
M. L. Blagonravov1, I. S. Eremina1, I. P. Smirnova1,
mice (BALB/c) treated with monoclonal antibodies (mAb, CD-
T. A. Lobaeva1, E. V. Velichko1
1 137, CTLA-4, PD-1, and CD-19) against immune check-point
Peoples’ Friendship University of Russia (RUDN University),
molecules (N = 10/group). Non-treated lung cancer cachectic mice
Moscow, Russia, 2The State Institution “Romodanov Neurosurgery
were the controls. Compared to non-treated cachectic mice, final
Institute” of National Academy of Medical Sciences of Ukraine,
body weight, body weight gain, and grip strength significantly
Kiev, Ukraine
increased in the mAb-treated animals. In diaphragm and gastroc-
The correlation between the peripheral blood cells aggregation nemius muscles of mAb-treated mice, the number of apoptotic
level, measured by surface plasmon resonance method (SPR), nuclei, tyrosine release, proteolysis and apoptosis markers signifi-
and chromosomal abberations of peripheral blood lymphocytes cantly decreased compared to non-treated cachectic mice. Systemic
(PBL) was investigated in patients with brain malignant gliomas. levels of troponin-I significantly decreased in treated cachectic
The results show that SPR indices of blood cells brain gliomas mice compared to non-treated animals. We conclude that reduced
patients gradually decrease with increasing of the gliomas malig- tumor burden as a result of selective treatment of the lung cancer
nancy grade and for certain ones are lower than that of healthy cells also elicits beneficial effects on muscle mass loss through
persons. The decreasing of SPR data correlate with the increase attenuation of several biological mechanisms that lead to
of chromosomal aberration frequency in PBL of glioma patients. increased protein breakdown and apoptosis.
Cytogenetic characteristics of patients with brain tumors under
treatment by Verapamil and Ketamine in vitro were investigated.
The decrease of the chromosome aberrations frequencies just like
decreasing of the aneuploid and polyploid cells quantity in
patient’s malignant tumors was shown under the Verapamil
action. The substantial reduction in aneuploid cells number
among tests of patients with malignant tumors under Ketamine
exposure was shown. Patients with benign or malignant tumors
had the very lowest magnitudes of multiaberrant cells amount
and of the extent of aberrant cell injury under the Verapamil
influence. The diamine oxidase (DAO) and polyamine oxidase
(PAO) were excreted by PBC of such patients in culture medium
during transformation of lymphocytes into blasts under the influ-
ence of phytohemagglutinin. The degree of blood cells aggrega-
tion was determined in these conditions with highly sensitive
method of SPR. Increased activity of DAO and PAO in case of
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 265
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
266 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 267
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
immunoblots. However, even after 72 h of PHA activation, the human FUS protein. It was shown that in the early stages of
number of SURF6-positive lymphocytes exceeds that of Ki-67- the course of the disease, it is possible to detect disturbances in
positive cells. Based on these results we conclude that the nucleo- the spatial memory function of transgenic mice when passing the
lar rRNA processing factor SURF6 is a novel and early marker Morris water labyrinth. Thus, 30 transgenic males were tested in
of lymphocyte activation for proliferation. The study was sup- 3 age groups - 45, 85, 105 days and the corresponding control
ported by the Russian Foundation for Basic Research (grant 18- group. The obtained results indicate the presence of statistically
34-00767). significant (P < 0.1) differences in the time of the mice in the
platform waiting sector. The difference in average time spent in
the target sector was 7.8 s or 13% of the total time of the test
P.09-186-Wed voyage. The results between the groups of 45 and 85 days were
Changes in expression of miRNAs associated not statistically different (in the 85-day group of transgenic mice
with nuclear factor kappa B in experimental a decrease in the total distance traveled during the experiment
was noted). The model under investigation seems promising for
acute respiratory distress syndrome
rapid testing of potential neuroprotective strategies and drugs for
M. Azizoglu1, L. Ayaz2, G. Bayrak3, B. Coskun Yilmaz3,
the therapy of amyotrophic lateral sclerosis and front-temporal
H. Birbicer1, N. Doruk1
1 frontal degeneration The reported study was funded by RFBR
Mersin University, Faculty of Medicine, Department of
according to the research project No. 16-04-01805 A.
Anesthesiology and Reanimation, Mersin, Turkey, 2University of
Trakya, Faculty of Pharmacy, Department of Biochemistry,
Edirne, Turkey, 3Mersin University, Faculty of Medicine, P.09-188-Tue
Department of Histology and Embryology., Mersin, Turkey
A heterozygous mutation NM_000518_c.7C>T
Acute respiratory distress syndrome is a disease that have a high (p.His3Tyr) in exon 1 in the HBB gene causing
mortality ratio. microRNAs are small, non-coding RNAs that
HbA1c interference: Hb Fukuoka
regulate gene expression by binding to specific target sites on
B. Arslan1, N. S. Yılmaz1, B. Sen1, C. Yılmaz1, G. Kayhan2,
mRNA. In recent years, it has been reported that miRNAs have
A. Pınar3, D. B. Topcu3
an important role in a number of basic physiological and patho- 1
Gazi University Medical Faculty, Department of Medical
logical processes. This study aimed to determine the expression
Biochemistry, Ankara, Turkey, 2Gazi University Medical Faculty,
response of nuclear factor kappa B-associated miRNAs in experi-
Department of Medical Genetics, Ankara, Turkey, 3Hacettepe
mental acute respiratory distress syndrome (ARDS) induced by
University Medical Faculty, Department of Medical Biochemistry,
lipopolysaccharide (LPS) in rats. Expressions of 9 miRNA were
Ankara, Turkey
determined by quantitative Real-Time PCR in each group.
Expressions of 7 miRNA were significantly changed in the study Mutations and / or deletions result in genes encoding hemoglobin
group compared to control group. While rno-miR-7a-5p, rno- variants alpha and beta chains. There are more than 700 known
miR-7b, rno-miR-9a-5p, rno-miR-21-5p, rno-miR-29a-3p and variants, but half of them are silent. Variants that do not present
rno-miR-138-5p (P < 0.05) upregulated, only rno-miR-124-3p any clinical findings usually come about as a result of interference
(P < 0.05) were also found to be down-regulated. This study sug- in the HbA1c assay and cause measurement difficulty. The HbA1c
gests that these miRNAs may have a role in the pathogenesis of result of a patient in our laboratory by ion exchange high perfor-
ARDS related to NF-jB. However, this relationship needs to be mance liquid chromatography (HPLC) method estimated as %49.
examined in new studies by evaluation of pathways and target Rerun and dilution of the sample revealed the same result. The
genes. same sample’s HbA1c estimation made by capillary electrophore-
sis (Sebia Minicap) in another laboratory, no abnormal Hb
detected, also reanalysing the sample with dilution showed the
P.09-187-Mon same result. HbA1c value of the sample via another ion Exchange
Phenotypic characteristic of a new model of HPLC analyzer (UFLC, Shimadzu Prominence) was 39.4%. This
frontotemporal degeneration caused by analyze also did not revealed the true result of the HbA1c. The
HbA1c value of the patient was studied in another cation-
aggregation of the human FUS protein in the
exchange HPLC method (Tosoh G8). HbA1c is 3.8% but does not
nervous system of transgenic mice reflect the correct result. Also a variant peak found as 41.6% in
A. Deykin1,2 Tosoh. The sample was given for further analyze at genetic depart-
1
Institute of Gene Biology, Russian Academy of Sciences, Moscow, ment. The sequence analysis of the HBB gene revealed a heterozy-
Russia, 2Institute of General Pathology and Pathophysiology, gous mutation NM_000518_c.7C>T (p.His3Tyr) in exon. In
Moscow, Russia addition, the patient was found to have a c.9T> C (rs713040)
The investigated line of transgenic mice was obtained and main- heterozygote substitution in the same codon, which did not lead to
tained based on the Center for Collective Use of the Institute of amino acid changes. Hemoglobin variants cause interference in the
Biology of the Gene of the Russian Academy of Sciences. The measurement methods of glycohemoglobin (HbA1c). This is the
genetic model was based on the functional analysis of the FUS first case in Turkey Fukuoka Hb was identified by type The
protein involved in neurodegenerative processes. The FUS pro- HbA1c and molecular analyzes of different measurement methods.
tein, like TDP-43, was associated with the pathogenesis of both
amyotrophic lateral sclerosis and front-temporal frontal degener-
ation. It has been suggested that the key role in the pathological
process associated with these proteins is played by the disorder
of RNA metabolism, since both these proteins are DNA / RNA
binding. During the work on the grant, it was possible to clarify
the time limits for the development of the pathological process
associated with neurodegeneration against the background of the
overexpression in the brains of mice of the mutant form of the
268 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 269
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
270 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 271
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
272 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
that IRF4 is essential for the transcriptional activation of ANP were male (80.70%) and 27 were female (19.30%). The mean age
during brown and beige cells differentiation, and may reveal a of the patients who were admitted to our clinic with an acute
potential therapeutic effect of ANP on obesity-related disease. chest pain complaint was 63.50 11.56 (32–86). CK-MB was
found in 2752 cases of the patients who complained of acute
chest pain, and 1256 (45.63%) of them were found. Troponin I
P.09-202-Mon (Tp I) was found in 2706 patients and 598 (22.09%) were found
Characterization of type II toxin-antitoxin to be high. The myoglobin levels were measured in thirty-five
systems in Agrobacterium tumefaciens patients and myoglobin levels were found higher in 9 patients
W. Choi, J. Park (25.7%). Troponin I was measured in 136 of AMI-diagnosed
Korea Research Institute of Bioscience and Biotechnology, patients and 133 (97.79%) were high, in 124 of 136 patient CK-
Cheongju, South Korea MB levels were high (90.51%); in 4 of 6 patients myoglobin
levels were found high (66.6%). 4.96% of patients with chest
In bacterial and archaeal genome or plasmids, there are toxin- pain complaints were diagnosed as AMI. The most specific bio-
antitoxin (TA) systems which are consisted with toxin and its chemical markers used for AMI were Tp I (97.79%) and CK-
antitoxin counterpart. TA systems were first described to func- MB (90.51%) in the second.
tion as post-segregational killing mechanisms to avoid loss of
plasmids, and then they have been reported to decrease energy
consumption by reducing cell growth as stress responses. These P.09-204-Wed
TA systems are classified into 4 to 6 categories by how their anti- The influence of selected metal ions on the
toxins inhibit toxins’ activities. In type II TA system, one of the
oligomerization of cystatin C and its variants
most studied, toxins and their cognate antitoxins are located in
J. Zygowska1, P. Czaplewska2, A. Szymanska1, Z. Pancheva1
the same operon, and antitoxins prevent their toxins from being 1
Department of Biomedical Chemistry, Faculty of Chemistry,
active. In stress conditions, however, relatively unstable antitox-
University of Gdansk, Gdansk, Poland, 2Intercollegiate Faculty of
ins are quickly degraded, toxins become active, and toxins make
Biotechnology, University of Gdansk, Gdansk, Poland
cells stop growing by various mechanisms. Until now, it has been
unknown whether Agrobacterium tumefaciens have TA systems. Neurodegenerative diseases are one of the major challenges to
In this study, we isolated 6 type II toxin-antitoxin system candi- modern medicine. The lack of effective drugs and therapy sys-
dates, Atu0939-Atu0940, Atu2018-Atu2017, Atu0674-Atu0673, tems results in the growing number of individuals suffering from
Atu0934-Atu0933, Atu8169-Atu-8168, and Atu-1004-Atu1003 in different forms of dementia and other dysfunctions arising from
the genome of Agrobacterium tumefaciens, and we found that the central nervous system degeneration. The Icelandic type of
these are homologous to mRNA interferases of E. coli, MazF, hereditary cerebral amyloid angiopathy is caused by aggregation
YeoB, YafO, and YafQ, by amino acid sequence alignment. To of a protein called cystatin C (hCC). Mutation in the hCC gene
find out if these isolated genes belong to TA system, we overex- leads to the replacement of the leucine residue in the 68th posi-
press putative toxin-antitoxin pairs in pBAD system with 0.2% tion of the chain with the glutamine residue. The result of the
arabinose on the same plates. All of 6 pairs did not inhibit cell change is a production of highly amyloidogenic variant L68Q.
growth, while toxins by themselves showed cell growth inhibition. Mutant forms deposits that accumulate in the blood vessels of
Therefore, we confirmed that these genes are TA systems. Fur- the brain. Weakened arteries burst due to blood pressure result-
thermore, we performed Northern blot assay with ompF to deter- ing in massive haemorrhages and strokes leading to the patient’s
mine if these toxins are mRNA interferases that cleave mRNAs death. Many studies on amyloidogenic proteins such as amyloid
as substrates Here, we report TA systems in Agrobacterium tume- b (Alzheimer’s disease) and a-synuclein (Parkinson’s disease)
faciens for the first time, and the understating of TA system in indicate the important role of metal ions in the oligomerization
this bacteria can help to develop the method of prevention and process. Copper and zinc, which are found as trace elements
the therapeutic agents of related diseases, such as crown gall dis- essential for proper functioning of the body, probably accelerate
eases. brain degeneration. In our project, we have studied the impact of
metal ions on the oligomerization and aggregation of human cys-
tatin C. The wild-type protein and its mutants with increased or
P.09-203-Tue lowered propensity for oligomer formation were incubated with
The percentage of acute myocardial infarction copper at different ratios. The results indicate a strong impact of
diagnosis of patients who applied to hospital copper ions on hCC oligomerization process, in most cases its
by chest pain complaint substantial acceleration connected with protein precipitation. The
effect of copper-induced protein aggregation may also apply to
A. Kahraman, A. Vurmaz, S. Celik
other transition metals and affect the cystatin C oligomerization
Department of Medical Biochemistry, Faculty of Medicine, Afyon
in vivo. A thorough examination of the deposits formation mech-
Kocatepe University, Afyonkarahisar, Turkey
anism is necessary for proper understanding of hCC and other
In this study, it was aimed to investigate the extent of acute MI amyloidogenic proteins pathogenicity. Work supported by
(AMI) and biochemical laboratory parameters of the patients National Science Centre NCN OPUS 11 grant UMO-2016/21/B/
with chest pain in the emergency department of our hospital. NZ1/02823.
Archive records of biochemistry, cardiology and emergency
departments of Afyon Kocatepe University Ahmet Necdet Sezer
Application and Research Hospital were retrospectively reviewed.
Demographic information (age, sex), biochemical markers (CK
MB, Troponin I, Myoglobin) levels were determined by the
research. Statistical analysis was performed with the information
obtained. A total of 2820 patients were admitted to our emer-
gency department with complaints of chest pain. Only 140
(4.96%) of these patients had acute myocardial infarction (AMI)
diagnosed by cardiology. Of the AMI-diagnosed patients, 113
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 273
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
274 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 275
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
P.09-212-Tue bacterial families. Recently, AI-2 was shown to affect the colo-
nization levels of a variety of bacteria in the microbiome of the
Confirmatory and orthogonal assays for
mouse gut, including members of the genus Clostridium, but no
eliminating artefactual drug bioactivities AI-2 receptor had been identified in this genus. Here, we demon-
C. C. Bataclan1, M. A. Manongdo2, A. A. Martija2, T. Ong2, strate that Clostridium saccharobutylicum possesses a functional
K. A. Roquid2, R. Garcia2 LsrB-type AI-2 receptor. The crystal structure of the C. saccha-
1
National Institute of Molecular Biology and Biotechnology, robutylicum receptor shows that it binds the same form of AI-2
University of the Philippines, Diliman, Quezon City, Philippines, as the other known LsrB-type receptors, but also reveals that it
2
Disease Molecular Biology and Epigenetics Laboratory, National has two previously unobserved variations in the amino acids of
Institute of Molecular Biology and Biotechnology, University of the AI-2 binding site. Isothermal titration calorimetry shows that
the Philippines Diliman, Quezon City, Philippines this receptor binds AI-2 with high affinity (dissociation constant
In the search for potential novel drugs, thousands of extracts in the submicromolar range). Altogether, this work represents the
derived from endemic and indigenous Philippine plants have first identification and characterization of an AI-2 receptor in the
undergone primary screening against important disease indica- Clostridium genus and shows previously unseen variations in
tions including cancer, cardiometabolic diseases and inflamma- the binding site of LsrB-type AI-2 receptors. These findings are
tion. As part of the national drug discovery pipeline, this study important for the identification of novel AI-2 receptors in thera-
has developed and performed specialized sets of orthogonal and peutically relevant microbes in the mammalian gut microbiome, a
secondary assays to validate primary hits and to rule out poten- niche where inter-species interactions are highly prevalent. More-
tial artefactual drug bioactivity. For cancer, a suite of multi-hall- over, they provide valuable structural details to aid in the design
mark assays evaluating effects on cell proliferation, apoptosis, of agonist or antagonists active against a wider range of quorum
migration, and invasion was established. Given the multiple man- sensing receptors.
ifestations of cancer, this strategy offers the advantage of provid-
ing a more comprehensive bioactivity profile against the disease.
Notably, DDHP-1508 has emerged to be a principal hit due to P.09-214-Mon
its antagonistic effects on all the hallmarks tested. For car- Up-regulation of the proapoptotic mediator
diometabolic diseases, glucose uptake assay and high-content PMAIP1/NOXA by heat shock
imaging-based assays measuring LDL-cholesterol uptake were A. Toma-Jonik1, M. Chadalski2, J. Korfanty2, P. Janus2,
performed. The orthogonal assays conducted had successfully A. Paszek2, N. Vydra2, W. Widlak2
confirmed bioactivity in 64 compounds out of the initial 918 pri- 1
Maria Sklodowskaj-Curie Institute – Oncology Center, Gliwice
mary hits for peripheral glucose uptake, and 8 compounds out of Branch, Gliwice, Poland, 2Maria Sklodowska-Curie Institute –
the initial 770 for increased LDL-cholesterol uptake. For inflam- Oncology Center, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeze
mation, a sequential workflow was developed to identify dual Armii Krajowej 15, Poland, Gliwice, Poland
COX-2/5-LOX inhibitors. Reactive Oxygen Species (ROS) activ-
PMAIP1/NOXA is a pro-apoptotic protein, a member of the
ity visualization is followed by a COX-2 activation reporter
Bcl-2 family. We found that Pmaip1 was the most induced gene
assay, then by PGE2 and 5LOX immunostaining. Validity of
in testes of transgenic mice expressing constitutively active,
top-tier hits were then confirmed by inflammation markers via
mutated HSF1 (Heat Shock Factor 1). HSF1 is the main media-
qPCR. From a pool of 263 extracts, DDHP-0175, DDHP-1975,
tor of the heat shock response typically inducing cytoprotective
and DDHP-900.10 were recognized as the top candidates for
Heat Shock Proteins (HSPs). However, cell death is induced by
dual COX-2/5-LOX inhibition. Taken together, the set of meth-
active HSF1 in mouse spermatocytes, possibly via PMAIP1.
ods used for each of the disease indications has been proven to
Based on genomic studies we stated heat-induced HSF1 binding
be effective for validating primary assay hits, narrowing down
to its consensus binding sites (HSEs), interestingly located in the
the potential novel drug candidates, and providing a prioritiza-
Pmaip1 introns, and up-regulation of the Pmaip1 transcription in
tion scheme for downstream purification, structure-ID, and clini-
spermatocytes and in certain somatic human and mouse cell lines
cal testing.
and tissues subjected to heat shock. We found the PMAIP1 pro-
tein accumulation in so-called heat-sensitive organs. TUNEL test
P.09-213-Wed demonstrated that apoptosis was rapidly induced by heat shock
in tissues exhibiting the highest Pmaip1 activation, i.e. testes, thy-
A novel receptor in Clostridium
mus, and spleen. Among cell lines, the highest Pmaip1 induction
saccharobutylicum that recognizes the was observed in HECa10 murine endothelial cells. Using
quorum sensing signal autoinducer-2 HECa10 cells with decreased HSF1 expression (shRNA) we con-
I. Torcato1,2, M. Kasal3, S. T. Miller3, K. B. Xavier1 firmed that HSF1 is important for Pmaip1 activation. Hemidele-
1
encia, Oeiras, Portugal, 2Instituto de
Instituto Gulbenkian de Ci^ tion of HSE from the second Pmaip1 intron (CRISPR/Cas9) also
Tecnologia Quımica e Biologica Antonio Xavier, Universidade led to diminished activation of Pmaip1 by heat shock, however,
Nova de Lisboa, Oeiras, Portugal, 3Department of Chemistry and we cannot exclude a clonal selection effect. Using live imaging
Biochemistry, Swarthmore College, Swarthmore, PA, United microscopy we proved that PMAIP1 overexpression led to apop-
States of America tosis. Furthermore, we deleted the Pmaip1 gene in HECa10 cells
Quorum sensing is a mechanism for cell-to-cell communication to validate its participation in the regulation of apoptosis after
that, through the exchange of small chemical molecules called heat shock. Our finding supports the idea that HSF1 may play a
autoinducers, allows bacteria to monitor their population density dual role in response to heat shock: cytoprotective, mediated by
and regulate gene expression accordingly. Autoinducer-2 (AI-2) is HSPs, or proapoptotic, mediated by PMAIP1. The final cell
a quorum sensing signal that is unique in facilitating inter-species response to stress could be determined by the balance between
communication. While it is produced and recognized by a wide antiapoptotic and proapoptotic factors differentially regulated by
variety of bacteria, to date only two classes of AI-2 receptors HSF1. This work was supported by the Polish National Science
have been identified: the LuxP-type, in the Vibrionales, and the Centre (grant no 2014/13/B/NZ3/04650)
LsrB-type, found in a number of phylogenetically distinct
276 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
P.09-215-Tue P.09-217-Mon
Targeting isocitrate lyase for the treatments of Biological activity of South African macrofungi
tuberculosis against respiratory and lung disease
R. Bhusal, K. Patel, B. Kwai, G. Bashiri, J. Reynisson, J. Sperry, J. Didloff1, G. J. Boukes1,2, T. C. Koekemoer1, M. van. de
I. Leung Venter1, S. Govender1
1
The University of Auckland, Auckland, New Zealand Department of Biochemistry and Microbiology, Nelson Mandela
University, Port Elizabeth, South Africa, 2Afrigen Biologics Pty
The enzymes isocitrate lyase (ICL) isoforms 1 and 2 are essential
Ltd, Cape Town, South Africa
for the survival of Mycobacterium tuberculosis within macro-
phages during tuberculosis infection. ICLs are not present in Macrofungi represent an untapped source of natural bioactive
humans and are therefore attractive therapeutic targets for the compounds for various diseases, which have been targeted as
treatment of tuberculosis. In this talk, we describe our work in potential therapeutic agents. Respiratory disease and lung cancer
the development and design of new ICL inhibitors. A particular places a global burden on health due to antimicrobial resistance,
focus is the application our combined high-throughput virtual non-specific drug targeting and damaging side effects. This study
screening, nuclear magnetic resonance spectroscopy and thermal investigated the antimicrobial activity and cytotoxicity of 21
shift assay strategy, which has led to the discovery of several South African macrofungi against respiratory pathogens (e.g.
novel ICL inhibitor scaffolds that are the subject of ongoing Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus
medicinal chemistry studies in our laboratories. Finally, we will pneumoniae, S. pyogenes, Staphylococcus aureus) and human lung
also describe our recent efforts in studying the structure and cat- adenocarcinoma A549 cells. Ethanol and aqueous extracts were
alytic mechanism of ICLs. We hope our work will inspire the screened for antimicrobial activity using the q-iodonitrotetrazo-
development of the next generation of ICL inhibitors that may lium chloride assay and the effect on bacterial morphology deter-
be used to treat tuberculosis. mined using transmission electron microscopy (TEM).
Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the mech-
P.09-216-Wed anism elucidated by cell cycle analysis and fluorescent staining.
Toluidine blue O reduces APLP2 and APLP2 Ethanol extracts showed higher antimicrobial activity against the
CTF levels in Hs766T cells Gram-positive bacteria than aqueous extracts. The macrofungal
K. Biberoglu, M. Y€ €
uksel, S. Onder, € Tacal
O. extracts of Fomitopsis lilacinogilva and Pisolithus tinctorius
Department of Biochemistry, School of Pharmacy, University of showed to cause cell membrane damage. Ethanol extracts of Pyc-
Hacettepe, Ankara, Turkey noporus sanguineus, F. lilacinogilva and Gymnopilus junonius and
the aqueous extract of Pseudophaeolus baudonii showed cytotoxic
Cancer and neurodegeneration are two different pathological dis- activity against A549 cells, with IC50 ranging between 7.4–
eases but they share common mechanisms in many ways. Amy- 69.2 lg/mL. Fluorescent staining confirmed cell cycle arrest and
loid precursor-like protein 2 (APLP2) and its homologous family apoptosis induced by extracts. Morphological and biochemical
member, amyloid precursor protein (APP) are overexpressed in changes included chromatin condensation, membrane blebbing,
many cancers and they are linked to abnormal growth, migra- loss of cytoskeletal structure, caspase activation and phos-
tion, and invasion. A recent study has revealed that APLP2 has phatidylserine translocation. This study demonstrated the antimi-
a role in the growth of pancreatic cancer cells. Besides, inhibitors crobial activity of South African macrofungi and their inhibition
that prevent APLP2 cleavage, leading to low APLP2 C-terminal of A549 cancer cell proliferation by means of cell cycle arrest
fragments (CTFs), decrease the viability of pancreatic cancer and induction of apoptosis.
cells. According to our recent studies, a phenothiazine-derived
compound, toluidine blue O (TBO) was found to mitigate amy-
loid pathology by reducing APP and Ab peptide levels in Chinese P.09-218-Tue
hamster ovary cells stably expressing wild type human APP and Association between serum homocysteine
PS1. In present study, we aimed to investigate whether TBO may
reduce the APLP2 and APLP2 CTFs, which have effective roles
levels and swimming stress in male and
in pancreatic cancer growth and viability. Pancreatic cancer cells female rats
(Hs766T) were treated with a dose range of TBO (0–15 lM) or L. Alic, A. Kulo, E. Kiseljakovic, S. Hasic, R. Jadric
vehicle control for 24 h. After treatment of Hs766T cells with Faculty of Medicine University of Sarajevo, Sarajevo, Bosnia and
TBO without any side effect on cell viability, the levels of APLP2 Herzegovina
and APLP2 CTFs in cell lysates were analyzed using Western Stress exposure has been associated with impaired homocysteine
blot and normalized to total protein levels. We observed a signifi- metabolism and consequently increased levels of homocysteine.
cant decrease in both intracellular APLP2 and APLP2 CTF Hyperhomocysteinemia is associated with increased risk of devel-
levels in a dose-dependent manner compared to vehicle-treated oping atherosclerosis, cardiovascular diseases, Alzheimer disease
cells. APLP2 levels were reduced by 26% (*P < 0.05) at 5 lM and other neurodegenerative diseases. Additionally, it was shown
and 42% (***P < 0.01) at 10 lM TBO. Also, APLP2 CTF levels that prolonged stress exposure is a risk factor and female gender
were decreased by 43% (*P < 0.05), 48% (**P < 0.01) and 66% shows protective role for development of stress-related diseases.
(***P < 0.001) at 5 lM, 10 lM and 15 lM TBO. In conclusion, Aim of this study was to examine gender differences in serum
these results support the idea that TBO may be used as a thera- homocysteine level (SHL) in rats exposed to swimming stress.
peutic drug in pancreas cancer. Supported by a grant from the Adult Wistar rats were distributed into three groups: control
Scientific Research Unit of Hacettepe University (HUBAB, TSA- group (n = 12; CG), repeated swimming stress (n = 12; RSS) and
2017-13929). single swimming stress (n = 12; SSS). Each of the rat groups were
further equally divided by gender (n = 6; female and male control
group – FCG and MCG, female and male repeated swimming
stress – FRSS and MRSS, female and male single swimming stress
– FSSS and MSSS). Rats exposed to repeated swimming stress
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 277
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
were forced to swim for 5 days, each day 5 min longer than pre- P.09-220-Mon
vious day, in total 25 min on 5th day. Rats exposed to single
A comparative study on essential oils
swimming stress swam only once for 25 min. RSS and SSS rats
were sacrificed on the 5th day immediately after swimming, while compositions and cytotoxic effects of wild and
CG rats were sacrificed at rest. Blood samples were obtained cultured form of Origanum acutidens (Hand.-
from abdominal aorta and serum cortisol level (SCL) and SHL Mazz.) Ietswaart. (Lamiaceae) on H1299 cells
were determined by enzyme-linked immunosorbent assay. Stress €
C. D€ulgeroglu1, A. Erdogan2, A. Ozkan 3
1
response SCLs were significantly different between all 3 groups, Akdeniz University, Science Faculty, Biology Department,
both in male and female rats (P = 0.007 and P = 0.009, respec- Antalya, Turkey, 2Genetic and Bioengineering Department, Faculty
tively). Moreover, SCLs were significantly higher in SSS rats of of Engineering, Alanya Alaaddin Keykubat University, Antalya,
both genders compared to corresponding CGs (P < 0.05). There Turkey, 3Akdeniz University, Faculty of Science, Biology
were no significant differences in SHLs between MCG, MRSS Department, Antalya, Turkey
and MSSS as well as between FCG, FRSS and FSSS. SHLs and
In this study, cytotoxic effects of essential oils from wild and cul-
SCLs showed no gender differences in tested groups (P > 0.05).
tured form of Origanum acutidens (Hand.-Mazz.) Ietswaart.
In this rat model, cortisol is a sensitive marker of acute swim-
(Lamiaceae) were determined on lung cancer cells (H1299). Also,
ming stress in both genders, whereas homocysteine is not.
the components of these two forms of essential oils were com-
pared. The air-dried aerial parts of plants were powdered and
P.09-219-Wed subjected to hydro-distillation for 3 h using the Clevenger-type
apparatus. The oils were investigated by GC and GC-MS. Cyto-
Evaluation of nuclear factor-kB with oxysterols
toxic effect of essential oil on H1299 cells were determined by
and clinical/biochemical characteristics of type Cell Titer-BlueR Cell Viability. Nineteen components in wild
1 and type 2 diabetes mellitus form of O. acutidens and twenty four components in cultured
A. Samadi1, A. G€ urlek2, S. N. Sendur2, S. Yılmaz Isıkhan3, form of O. acutidens essential oil were identified. The main com-
4
I. Lay ponents of the oils were found as carvacrol and p-cymene. The
1
Department of Medical Biochemistry, Faculty of Medicine, amount of carvacrol (72.65%) and p-cymene (15.19%) for wild
Hacettepe University, Ankara, Turkey, 2Department of Internal form were found different than cultured form carvacrol (67.98%)
Medicine, Endocrinology Unit, Faculty of Medicine, Hacettepe and p-cymene (16.01%). IC50 values (concentration that kills
University, Ankara, Turkey, 3Department of Biostatistics, Faculty 50% of cells) of wild form essential oil in H1299 cells were calcu-
of Medicine, Hacettepe University, Ankara, Turkey, 4Department lated as 149.5 lg/mL, 130.5 5 lg/mL and 109 5 lg/mL for 24, 48
of Medical Biochemistry, Faculty of Medicine, Hacettepe and 72 h respectively. On the other hand, IC50 values of cultured
University and Clinical Pathology Laboratory, Hacettepe form essential oil were found 83.3 lg/mL, 75.2 lg/mL and
University Hospitals, Ankara, Turkey 64.5 lg/mL for 24, 48 and 72 h respectively. Cytotoxic effect of
Nuclear factor-kappa B (NF-kB) is a transcription factor that is essential oil increased depend on concentrations and incubation
strategic at the crossroad between oxidative stress and inflamma- times. As a result, essential oil from cultured form of O. acuti-
tion in Diabetes Mellitus (DM). This study describes the relation- dens had more cytotoxic effect on H1299 cells than wild form
ship between NF-kB and new oxidative stress biomarkers essential oils. It can come from amount of ratio of oil composi-
oxysterols in type 1 DM (DM1) and type 2 DM (DM2). Clinical tions.
and biochemical characteristics of 26 DM1, 80 DM2 and 205
age- and gender-matched healthy controls were determined.
P.09-221-Tue
Oxysterols (7-Ketocholesterol and Cholestane-3b,5a,6b-triol)
were quantified by LC-MS/MS. NF-kB was measured by ELISA. Sphingolipidomic analysis reveals decreased
Significant higher levels of NF-kB and oxysterols that were levels of very long chain sphingomyelins and
observed in both types of DM compared to healthy controls, had ceramides in sickle cell disease patients
also strong positive correlations [NFjB and 7-Ketocholesterol €
M. Aslan1, E. Kırac1, S. Kaya1, F. Ozcan 1
, O. Salim2,
(r = 0.919, P < 0.001), NFjB and Cholestane-3b,5a,6b-triol O. A. K€ upesiz3
(r = 0.812, P < 0.001)]. Positive and strong correlations of NF- 1
Akdeniz University Faculty of Medicine, Department of Medical
kB with HbA1c (r = 0.907, P < 0.001), glucose (r = 0.885, Biochemistry, Antalya, Turkey, 2Akdeniz University Faculty of
P < 0.001), total cholesterol (r = 0.627, P < 0.001), and with the Medicine, Department of Hematology, Antalya, Turkey, 3Akdeniz
number of coronary risk factors (r = 0.915, P < 0.001) were University Faculty of Medicine Department of Pediatric
found in DM2. Significant positive correlations of NF-kB with Hematology, Antalya, Turkey
HbA1c, glucose, total cholesterol, and the number of risk factors
This study aimed to identify levels of C16-C24 sphingomyelin
were also found in DM1 (P < 0.05). Oxysterols also showed
(CerPCho) and C16-C24 ceramide (CER) in serum obtained from
strong positive correlations with HbA1c, glucose, total choles-
SCD patients and controls. Circulating levels of neutral sphin-
terol, and the number of coronary risk factors in both DM1 and
gomyelinase activity (N-SMase), ceramide-1-phosphate (C1P),
DM2 patients. NF-kB and oxysterols were increased significantly
sphingosine-1-phosphate (S1P) were also determined. Blood was
in smokers. Oral hypoglycemic drug treatment reduced the levels
collected from hemoglobin (Hb)A volunteers and homozygous
of NF-kB and oxysterols in DM2. Activated NF-jB exacerbate
HbSS patients. Serum levels of C16-C24 CerPCho and C16-C24
inflammation, oxidative stress and promote apoptosis. NFjB is
CER were determined by an optimized multiple reaction moni-
also activated by oxidative stress. It is highly likely that inhibi-
toring method using ultra fast-liquid chromatography coupled
tion of NFjB will be considered as a logical target for the treat-
with tandem mass spectrometry. Serum activity of N-SMase was
ment of DM.
assayed by standard kit methods, C1P and S1P levels were deter-
mined by enzyme-linked immunosorbent assay. A significant
decrease was observed in serum levels of C18-C24 CerPCho and
very-long-chain C22-C24 CERs in SCD patients compared to
controls. A significant positive correlation was found between
278 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 279
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
transport of carnosine, we added carnosine (20 lM – 62.5 mM) sensitive bacterium through the peptide transporter, YejABEF,
to the culture of rat cortical neurons and determined its content McC is processed by aminopeptidases, resulting in the release of
within the cells after 5, 15 and 30 min. To determine the intracel- an aspartamide-adenylate. Asp-AMP binds to aspartyl tRNA-
lular content of carnosine, the cultures were washed from external synthetase, thus causing translation inhibition and cell death.
carnosine and lysed. The content of carnosine in the lysates was Recently we have shown that genomes of various microorganisms
determined using liquid chromatography–mass spectrometry. The contain functional mcc gene clusters. Most of the mcc clusters
dependence of intracellular content of carnosine on the incuba- are comprised of only three (mccABC) genes that are sufficient
tion time was linear for all concentrations except for the maxi- for toxic peptidyl-adenylate production. Many of the newly iden-
mum one. The dependence of the rate of carnosine entry into the tified mcc clusters contain additional genes, the products of which
cells on its concentration in the medium can be decomposed into are responsible for additional post-translational modifications on
two components: the enzymatic one, described by the Michaelis- the compound, or provide immunity to the producing cell. Here
Menten equation (KM = 118 lM), and the nonenzymatic one we describe an expanded mcc cluster present in the genome of
(Kne = 0.21 9 10–4 min1). The nonenzymatic component of the the amoeba-associated bacterium, Rubidus massiliensis. The mcc
transport rate prevailed over the enzymatic one at high concentra- (Rma) cluster consists of five genes (mccABCFE2) organized into
tions of carnosine (over 12.5 mM). Thus, at physiological concen- an operon. We show that MccB(Rma) is a bona fide adenylyl-
trations of carnosine (0.1–1 mM), the rate of its active transport transferase that modifies the C-terminal asparagine residue of the
into neurons is an order of magnitude higher than that of the pas- MccA(Rma) heptapeptide with an adenosine monophosphate.
sive transport. The product of mccC(Rma) encodes an export pump belonging
to the MFS family. Two additional genes, mccF(Rma) and
mccE2(Rma), are homologous to E. coli mccF and mccE and
P.09-226-Mon provide immunity to a wide variety of McC-like compounds.
Defining how formaldehyde regulates the This work was supported by Russia Science Foundation RSF 16-
functions of biomolecules 14-10356.
R. Hopkinson
University of Leicester, Leicester, United Kingdom
P.09-228-Wed
Formaldehyde (HCHO) is a highly reactive small molecule that Association of methylenetetrahydrofolate
is toxic and carcinogenic above threshold concentrations.
reductase (MTHFR) gene variants with dental
Humans are exposed to HCHO as a consequence of its wide-
spread use in industry, including in plastics manufacturing, caries and gingivitis in Czech children
embalming, cosmetics, and as a sterilising agent. In addition, P. Borilova Linhartova1,2, Mrazkova1, D. Novak1,2,
HCHO is routinely produced during enzymatic demethylation M. Kukletova1, L. Kukla3, L. Izakovicova Holla1,2,3
1
reactions within cells, such as those catalysed by histone and Clinic of Stomatology, Institution Shared with St. Anne’s Faculty
nucleic acid demethylases. It is therefore apparent that cells are Hospital, Faculty of Medicine, Masaryk University, Brno, Czech
constantly exposed to HCHO. Despite the detrimental effects of Republic, 2Department of Pathophysiology, Faculty of Medicine,
HCHO exposure, it is unclear how HCHO induces toxicity at the Masaryk University, Brno, Czech Republic, 3Research Centre for
molecular level. Adducts between HCHO and biomolecules, e.g. Toxic Compounds in the Environment (RECETOX), Chemistry
DNA and proteins, are reported, although there is a paucity of sys- Section, Faculty of Science, Masaryk University, Brno, Czech
tematic biochemical studies. It is also unclear in many cases Republic
whether such reactions occur in cells, or whether the reactions lead Oral bacteria (cariogenic bacteria or periopathogens), host
to functional changes. I am interested in profiling HCHO biochem- immune system and genetic predisposition play a role in the
istry with a view to understanding the mechanisms underpinning etiopathogenesis of dental caries and gingivitis. Susceptibility to
HCHO-mediated disease. Here, I will discuss biochemical studies inflammation and host-pathogen interactions may be modified by
characterising the mechanisms of HCHO production and metabo- functional polymorphisms in genes linked with the folate meta-
lism, as well as work profiling the reactions of HCHO with amino bolic pathway, such as methylenetetrahydrofolate reductase
acids and nucleic acids. The combined results reveal that HCHO is (MTHFR), methionine synthase (MTR) and methionine synthase
an important endogenous metabolite with the potential to regulate reductase (MTRR). The aim of our study was to analyze four sin-
the functions of multiple cellular processes. gle nucleotide polymorphisms (SNPs) in the genes for these
enzymes in Czech children with known oral status. This case-con-
trol study included 592 children aged 13–15 years from the Euro-
P.09-227-Tue pean Longitudinal Study of Pregnancy and Childhood (ELSPAC).
Microcin C-like peptide-adenylate antibiotic Oral status was recorded using DMFT (decay-missing-filled teeth)
from Rubidus massiliensis index and gingival index, the presence of 10 selected oral patho-
A. Kulikovsky1,2, D. Tsibulskaya1,2, J. Piskunova1,2, genic bacteria was analyzed, and MTHFR C677T (rs1801133),
K. Severinov1,2,3, M. Serebryakva1,2,4, S. Dubiley1,5 MTHFR A1298C (rs1801131), MTR A2756G (rs1805087) and
1
Institute of gene biology, Moscow, Russia, 2Skolkovo Institute of MTRR A66G (rs1801394) SNPs were determined by qPCR. Chil-
Science and Technology, Moscow, Russia, 3Waksman Institute for dren with dental caries (N = 446) more often suffered by gingivitis
Microbiology, Piscataway, NJ, United States of America, than caries free children (controls, N = 145), thus gingivitis was
4
Belozersky Institute of Physico-Chemical Biology, Moscow, associated with dental caries (P < 0.001). There were no signifi-
Russia, 5Skolkovo Institute of Science and Technology (Skoltech), cant differences in the allele or genotype frequencies in both MTR
Moscow, Russia and MTRR SNPs between patients and controls. However,
MTHFR 1298 AA genotype vs. AC+CC genotypes was found as
Microcin C (McC) from E. coli is a founding member of the
protective factor for dental caries development (P < 0.01) and
growing family of ribosomally-synthetized post-translationally
MTHFR 677 CC genotype vs. CT+TT genotypes was associated
modified peptide-nucleotide antibiotics. The peptide portion of
with the non-presence of Prevotella intermedia, an important peri-
McC is bound to adenosine monophosphate though a non-
opathogen. It seems that children with common variants in
hydrolyzable phosphoramidate link. Taken into the cytoplasm of
MTHFR gene, and thus normal ability to utilize folic acid, are
280 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
protected against dental caries and gingivitis. The study was sup- In our study we investigated clonal repertoire of pediatric
ported by Ministry of Health of the Czech Republic, grant nr. 17- patients after allogenic HSCT with CD19/abT and DLI by TCR
30439A, funds provided by the Faculty of Medicine MU to junior b-chains deep sequencing. For each patient we collected two sets
researcher Petra Borilova Linhartova, grant GACR GB14- of blood samples: in 3 and 12 months after HSCT, and sample
37368G, and the project MUNI/A/1008/2017. of CD45RA--lymphocytes (DLI) from matched donor. Every set
of samples include total fraction of periphery T-cells, fraction of
CD4+ and CD8+ T lymphocytes. TCR-b repertoire profiling was
P.09-229-Mon performed using original technology with molecular barcode-
The ectonucleotidase CD73 is a target of based normalization and PCR and sequencing error correction.
profibrotic TGF-b signaling during diabetic Here we found, that 1 year after HSCT patient T-cell diversity of
nephropathy peripheral blood reached up to more than half of a healthy
repertoire, while was lower than in age matched healthy donors.
C. Cappelli, A. Tellez, C. Jara, C. Oyarzun, R. San Martin
1 We showed, that some of the recipient T cell clonotypes matched
Molecular Pathology Laboratory, Institute of Biochemistry and
with clonotypes from DLI-sample repertoire persist in patient’s
Microbiology, Science Faculty, Universidad Austral de Chile,
repertoire at least for the year after HSCT. Furthermore, some
Valdivia, Chile
of these clones expanded in a period between 3 and 12 months
Progression of diabetic nephropathy (DN) is linked to tubule-inter- after HSCT. T-cells from DLI make up from small percent of
stitial fibrosis conducted by TGF-b. The nucleoside adenosine total patient’s peripheral blood T-cells to almost half of them
increases with the progression of DN. Extracellular adenosine pro- one year after HSCT. Funding: RFBR grant 16-04-01881.
duction is dependent on the activity of ectonucleotidase CD73 in
tubule epithelial cells. Our aim was to identify CD73 induction
downstream of TGF-b signaling and demonstrate its potential as a P.09-231-Wed
molecular marker of renal tubule injury. Cells from the human Vitamin B12 status in patients with graves
renal epithelial tubule cell line HK2 were treated with TGF- thyroiditis
b 10 ng/mL, adenosine 10 mM or NECA 1 mM for 48 h. CD73 H. Vatansev1, E. Paydas Hataysal1, E. Sahin1, S. Abusoglu1,
induction was determined by RT-qPCR. The recruitment of tran- _
L. Kebapcılar2, C. O. Kırac2, S. H. Ipekci2 € u1
, A. Unl€
scription factors and changes in the histone code at the CD73 gene 1
Department of Biochemistry, Selcuk University Faculty of
promoter were determined by ChIP-qPCR. Cohorts of healthy, Medicine, Konya, Turkey, 2Department of Endocrinology, Selcuk
diabetics and diabetics with renal injury were established. CD73 University Faculty of Medicine, Konya, Turkey
content was measured in urinary exosomes and AMPase activity
mediated by CD73 was quantified in urinary sediment from Graves’ disease is an autoimmune disease that leads to over activ-
cohorts. We found Smad3 positioned at the CD73 promoter region ity of whole thyroid glands and it is the most common cause of
proximal to the transcriptional start site, in contrast to the collagen hyperthyroidism. Vitamin B12 is necessary for hematopoiesis and
1a2 promoter which exhibit heterochromatinic marks in HK2 cells. normal neuronal function. In humans, it is obtained only from
The expression of CD73 and markers of profibrotic activation of animal proteins. We aimed in this study to compare the level of
cells, fibronectin and collagen 1a2, were upregulated after exposure vitamin B12 in Graves’ Thyroiditis and healthy control groups.
of HK2 cells to TGF-b. The epigenetic environment in the CD73 Plasma samples were collected from 50 healthy control and 50
promoter was also modified by TGF-b, promoting transcriptional patients with Graves Thyroiditis who apply to the Selcuk Univer-
active chromatin. CD73 content was increased in urinary exosomes sity Hospital between in 01.04.2017–01.10.2017, prospectively.
from diabetic patients and correlated with tubular injury. CD73 The mean age for controls and patients were 38.8 11.5 and
activity was also increased in urinary sediment from DN patients. 37.4 11.2, respectively (P = 0.9). Patients with other chronic
The ectonucleotidase CD73 gene is a target of profibrotic TGF-b disease, inflammatory disorders and patients using Vitamin B12
signaling in epithelial tubule cells. Induction of CD73 is evidenced consisting drug were excluded. Vitamin B12 levels calculated in.
by increased activity in the urinary sediment of DN patients, being Roche Cobas e170 with ECLIA method. Analysis was performed
a potential marker of tubule-interstitial fibrosis. with IBM SPSS v20. Vitamin B12 levels were statistically lower in
patients with Graves Thyroiditis compared to control group
[327.8 130.3 ng/L and 398.6 185.8 ng/L] (P = 0.03). Our
P.09-230-Tue results showed that Vitamin B12 levels are lower in Graves dis-
T cell repertoire profiling after hematopoietic ease compared to control group. These findings can cause hyper-
stem cell transplantation with CD19/abT cell homocysteinemia that may lead increased cardiovascular risk in
patients with Graves Thyroiditis. Further studies are needed.
depletion and donor lymphocyte infusion
V. Fomchenkova1, E. Komech2, S. Blagov3, A. Sycheva2,
Y. Lebedev2, D. Chudakov2, M. Maschan3, I. Zvyagin2,3
1
Lomonosov Moscow State University (MSU), Moscow, Russia,
2
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, Moscow, Russia, 3Dmitry Rogachev
National Research Center of Pediatric Hematology, Oncology and
Immunology, Moscow, Russia
Depletion of abT cells substantially decrease risk of GVHD,
increasing the safety of allogeneic hematopoietic stem cell trans-
plantation. During HSCT, patient experienced a period of pro-
found immunodeficiency, which is caused by decrease of T cell
repertoire diversity and total number of T-cells. To substitute the
deficiency and to protect patient’s organism from pathogens on
early stages of immune system reconstitution infusion of
CD45RA--lymphocytes from mature donor (DLI) can be used.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 281
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
P.09-233-Tue P.09-234-Wed
Assessment of active DNA demethylation Farnesoid X receptor mRNA expression in
pathway during chemotherapy of acute non-alcoholic fatty liver disease is inversely
myeloid leukemia associated with the severity of liver damage
A. Labejszo1, M. Starczak1, L. Gackowska2, M. Modrzejewska1, and degree of insulin resistance
J. Szpotan1, M. Gawronski1, A. Koltan3, G. Charlinski4, A. Bogolyubova1, E. Mishina2, P. Belousov1, P. Bogomolov3,
J. Czyz5, J. Styczynski3, D. Gackowski1 M. Matsievich3, K. Kokina3, A. Mayorov2
1
Department of Clinical Biochemistry, Faculty of Pharmacy, 1
Engelhardt Institute of Molecular Biology, Russian Academy of
Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University Science, Moscow, Russia, 2Endocrinology Research Center,
in Torun, Bydgoszcz, Poland, 2Department of Immunology, Moscow, Russia, 3M.F. Vladimirsky Moscow Regional Research
Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus and Clinical Institute, Moscow, Russia
Copernicus University in Torun, Bydgoszcz, Poland, 3Department
of Pediatric Hematology and Oncology,, Faculty of Medicine, Non-alcoholic fatty liver disease (NAFLD) is a group of patho-
Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University logical changes which are associated with obesity and character-
in Torun, Bydgoszcz, Poland, 4Department of Hematology, ized by abnormal lipid accumulation in liver cells. NAFLD arises
Nicolaus Copernicus Specialist Municipal Hospital, Torun, Poland, as one of the most important problem of public health in devel-
Torun, Poland, 5Department of Hematology, Faculty of Medicine, oped countries. The first two stages of NAFLD are non-alcoholic
Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University fatty liver (NAFL, steatosis) and non-alcoholic steatohepatitis
in Torun, Bydgoszcz, Poland (NASH) which can progress to fibrosis and even liver cirrhosis.
Recent studies demonstrated the role of farnesoid X receptor
Patients with acute myeloid leukemias frequently harbor muta- (FXR) in the regulation of glucose and lipid metabolism. Here
tions in genes involved in the DNA hydroxymethylation path- we performed an expression analysis of FXR in core needle biop-
way. Loss-of-function mutations in TET2 have been described in sies obtained from patients with different NAFLD stages
various hematological malignancies and also TET1 has contrast- (n = 20). FXR expression in the group of NASH patients inver-
ing roles in myeloid and lymphoid transformation. To assess sely correlates with the severity of liver damage and degree of
whether active demethylation pathways are affected by standard insulin resistance as assessed by NAFLD Activity Score (NAS)
induction treatment (cytarabine and an anthracycline), we and Homeostatic Model Assessment of Insulin Resistance
compared broad spectrum of endogenously generated DNA (HOMA-IR), respectively. Our results suggest the protective role
modifications (5-methylcytosine, 5-hydroxymethylcytosine, 5-for- of hepatic FXR-mediated signaling against the progression of
mylcytosine, 5-carboxylcytosine, 5-hydroxymethyluracil and NAFLD in humans and provide a biochemical framework for
8-oxoguanine) and expression of TET dioxygenases as well as the use of FXR agonists in treatment of NAFLD and associated
TDG glycosylase in peripheral blood nuclear cells of patients metabolic disorders. This work is supported by the Russian
with acute myeloid leukemia before and after first course of ther- Science Foundation under grant No. 17-15-01475.
apy. Peripheral blood nuclear cells were isolated from hep-
arinized blood samples with Histopaque 1119 solution, according
to the manufacturer’s instruction. DNA was extracted from fro- P.09-235-Mon
zen cells by modified phenol method. Isolated material was Bacterial L-asparaginases: old friend and
hydrolysed to deoxynucleosides and analyzed using stable isotope persistent foe
dilution two-dimensional ultra-performance liquid chromatogra-
J. Lubkowski1, A. Anishkin2, W. K. Chan3, D. Fushman2,
phy with tandem mass spectrometry. The indirect multicolor flow
P. L. Lorenzi3, S. B. Rempe4, S. Sukharev2, J. M. Vanegas5,
cytometry-based method was used to assess intracellular expres-
J. N. Weinstein3, A. Wlodawer6
sion of proteins in specific subpopulations of peripheral blood 1
National Cancer Institute, Frederick, United States of America,
nuclear cells. Monocytes, lymphocytes and granulocytes after 2
University of Maryland, College Park, MD, United States of
antileukemic treatment presented with lower expression of TET1
America, 3University of Texas MD Anderson Cancer Center,
and higher expression of TET2 than cells before therapy, while
Huston, TX, United States of America, 4Sandia National
TET2 and TDG remained unaltered. The levels DNA modifica-
Laboratories, Albuquerque, NM, United States of America,
tions were not altered by therapy. This suggest, that overexpres- 5
University of Vermont, Burlington, VT, United States of America,
sion of TET3 may compensate lower expression of TET1 in the 6
National Cancer Institute, Frederick, MD, United States of
course of chemotherapy of acute myeloid leukemia. This work
America
was supported by the Polish National Science Centre (grant num-
ber 2015/19/B/NZ5/02208). Bacterial type II L-asparaginases (ASNases, EC: 3.5.1.1.) catalyze
hydrolysis of L-Asn to L-Asp and, to a lesser extent, of L-Gln to
L-Glu. Two ASNases, from E. coli and Erwinia chrysanthemi, are
used clinically in treatment of blood cancers and were also
recently shown to inhibit metastases of breast cancer (and possi-
bly other solid tumors). In all cases, therapeutic properties of
ASNases are related to the dependence of cancer cells on the cir-
culating L-Asn, with depletion of this amino acid leading to their
starvation. Despite the clinical importance of these enzymes, their
detailed catalytic mechanism and the basis of their substrate
specificity are still a matter of vigorous dispute. In contrast to
the recently reported model of catalysis proposing a single dis-
placement mechanism, our data support a double displacement
mechanism with Thr12 (EcAII, E. coli) acting as the primary
nucleophile. Based on extensive studies of EcAII utilizing X-ray
crystallography, NMR spectroscopy, kinetic measurements, and
282 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
MD/QM-MM computations, we have been able to describe all more pro-inflammatory phenotype, but experimental evidence for
major steps of the catalytic mechanism. In this project we deter- such relationship remains limited. In this study, human mono-
mined over 30 crystal structures of different mutants with differ- cytic THP-1 cells were fed with micromolar iron in three forms:
ent ligands under various conditions. Structural studies have a) transferrin, b) ferric-ammonium citrate (FAC) and c) hemin,
been complemented by kinetic measurements for over 100 differ- for 2 and 24 h. According to lactate dehydrogenase release only
ent mutants of EcAII, as well as by multiple NMR experiments hemin was toxic to the cells. All kinds of iron loading elevated
in solution, which allowed careful monitoring of the progress of the labile iron pool (spectrofluorometric assay with calcein) at
reaction. Models of the catalytic mechanism generated with these 2 h, as well as expression of ferritin (Western blot) at 24 h. As
data were evaluated by computational methods. MD simulations expected, hemin dramatically induced expression of hemoxyge-
revealed specific patterns of hydration and local electrostatics nase-1 (qPCR assay). Further qPCR analyses of selected scav-
involved in catalysis, whereas QM-MM calculations supported enger receptors (MSR-1, CD36, TLR2) and cytokines (IL-1beta,
the energetic feasibility of major stages in the proposed catalytic IL-6, TNFalpha, TGFbeta, IL-10) so far revealed only effects of
mechanism. We will present progress of our efforts with the aim hemin (decreased TNFalpha, MSR1, CD36) corresponding to the
of resolving the disagreement between the two competing models anti-inflammatory M(hem) phenotype. Only when the acid-base
of catalysis by type II L-asparaginases. indicator and antioxidant Phenol Red was omitted from the med-
ium used for iron loading, induction of MSR-1 (Western blot) in
response to iron, especially in transferrin form, could be
P.09-236-Tue observed. Degree of this induction was quite variable, ranging
Puerarin suppress on atopic dermatitis-like from zero to 5-fold. At this stage we conclude that iron loading
skin lesions through regulation of can induce both anti-and pro-inflammatory changes in the THP-
inflammatory mediators 1 monocytes, depending on the form of iron and also on other
factors, such as peroxidation of cellular lipids, whose identifica-
J. Lee1, Y. Im1, Y. Lee2, D. Kim1
1 tion seems critical for our understanding when and how iron
Department of Immunology, Medical School, Chonbuk National
within macrophages contributes to the progression of atheroscle-
University, Jeonju, South Korea, 2Department of Oriental
rotic lesions.
Pharmacy, Wonkwang University, Iksan, South Korea
Atopic dermatitis (AD) is one of the common inflammatory
immune disorders. Puerarin is the main isoflavonoid obtained P.09-238-Mon
from the root of Pueraria lobata and has been known have ame- The effects of Rooibos (Aspalathus linearis) on
liorative effects on diverse inflammatory diseases. However, the mitochondrial DNA depleted 3T3-L1
effects of puerarin on AD have not been uncovered. 2,4-dinitro-
preadipocytes
chlorobenzene (DNCB) was used to induce atopic dermatitis
A. Hattingh, T. Koekemoer, M. van. de Venter
(AD)-like skin lesions on BALB/c mice for 17 days. Further, the
Department of Biochemistry and Microbiology, Nelson Mandela
BALB/c mice were orally administered puerarin. Puerarin amelio-
University, Port Elizabeth, South Africa
rated DNCB-induced AD-like symptoms in the mice by regulat-
ing skin thickness, degranulation of mast cells, and serum Rooibos (Aspalathus linearis), a well-known South African fyn-
immunoglobulin E (IgE). Human keratinocytes (HaCaT cells) bos plant, has become increasingly popular as a cosmetic ingredi-
were also used to clarify the effects of puerarin on the secretion ent, however little scientific information exists to support such
of pro-inflammatory cytokines. Puerarin inhibited the secretion use. Human aging is characterised by a loss of subcutaneous adi-
of inflammatory cytokines and chemokines. The aim of this study pose tissue resulting in a redistribution of fat to visceral depots, a
was to investigate the protective and alleviative effect of puerarin feature strongly associated with many age related diseases. Adi-
on AD in vitro and in vivo. The results in this study indicated pocyte progenitor cells, preadipocytes, appear to be the most vul-
that puerarin ameliorates AD-like skin lesion and skin inflamma- nerable and account for the age related decline in dermal adipose
tion via regulation of various atopic and inflammatory mediators. tissue mass. Mitochondrial dysfunction is recognised as a contrib-
Therefore, puerarin might be useful in treating AD and other utory factor in aging process and is identified as a potential ther-
skin diseases. apeutic target to preserve tissue function. In the present study the
anti-aging potential of rooibos was investigated using 3T3-L1
preadipocytes depleted of mitochondrial DNA (mtDNA) through
P.09-237-Wed long-term exposure to sub-lethal concentrations of ethidium bro-
Iron loading can induce both pro- and anti- mide. MtDNA depleted 3T3-L1 preadipocytes showed a signifi-
inflammatory changes in cultured human THP- cantly reduced rate of proliferation, delayed cell cycle
1 monocytes progression (G0/G1 arrest), decreased mitochondrial membrane
potential (MMP), as well as an increase in glucose utilization and
J. Platenık1, P. Risko2, R. Buchal1, P. J. Kraml2,
lactate production, confirming mitochondrial dysfunction. Treat-
A. Rybnık arov
a1, J. Potockova2
1 ment with rooibos stimulated cell growth which was reflected in
Institute of Medical Biochemistry and Laboratory Medicine, First
an attenuation of the G0/G1 arrest as well as an improved
Faculty of Medicine, Charles University, Praha, Czech Republic,
2 MMP, indicating that rooibos improves mitochondrial function.
Second Department of Internal Medicine, University Hospital
However, glucose utilization and lactate production were not
Kr alovske Vinohrady, and Third Faculty of Medicine, Charles
diminished as would be expected for improved mitochondrial
University, Praha, Czech Republic
function raising the possibility that rooibos increases glycolytic
Cardiovascular diseases due to atherosclerosis remain a dominant ATP production to sustain favourable cell function and resis-
medical problem worldwide. Macrophages play a crucial role tance to mitochondrial dysfunction. Cellular ATP levels and
both in the atherosclerosis progression and recycling of body AMPK activation provide support for this hypothesis. Taken
iron. Epidemiological data point to elevation of the body iron together the data indicates that rooibos treatment alters preadi-
stores as one of the non-classical cardiovascular risk factors, and pocyte cellular energy metabolism providing some degree of resis-
we know the iron must be within macrophage to be atherogenic. tance to mitochondrial dysfunction and may thus have potential
Presumably the iron within macrophages turns the cells to a to recover adipose tissue health.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 283
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
284 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 285
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
286 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 287
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
P.09-251-Tue Comparing the mean values of GPx obtained before dry period
with the values obtained in the following periods, it was deter-
The impact of highly active antiretroviral
mined that a significant increase in the D1 (P < 0.05) started and
therapy on adverse pregnancy outcomes these values reached the highest on PP-d3 and PP-d8
K. Pinda1, V. Reid1, M. Matjila2, A. Katz1 (P < 0.0001). It was determined that the ratio of the C classified
1
Institute of Infectious Disease and Molecular Medicine, University in all the obtained GPx values was very low at D4-5W and D
of Cape Town, Cape Town, South Africa, 2Department of periods, respectively 6.7% and 13.3%; on the other hand, this
Obstetrics and Gynecology, University of Cape Town, Cape Town, rate increased significantly at D1 (P < 0.05) and was highest on
South Africa PP-d3 and PP-d8, respectively 80% and 70% (P < 0.0001). How-
Combination antiretroviral drugs such as Highly Active ever, it was determined that the ratios in category A dropped sig-
Antiretroviral Therapy (HAART) are now being widely used by nificantly in PP-d3 and PP-d8 periods (P < 0.01). SOD values in
HIV-infected pregnant women for reducing mother-to-infant the period of PP3-4w show a significant decrease compared to
HIV transmission and improving maternal health. Recent studies the other periods (P < 0.05; P < 0.01). The mean values of SOD
associate in utero HAART exposure with adverse pregnancy out- significantly decreased in PP3-4w compared to D4-5W period. In
comes such as pre-eclampsia, premature delivery, low birth conclusion, the results obtained show that GPx levels increased
weight and small for gestational age births. However, there is significantly in early PP period. Cows especially undergoes oxida-
limited research on the molecular mechanism for these HAART tive stress in this period compared to other periods.
induced pregnancy toxicities. In order to investigate whether
these toxicities are due to placenta insufficiency secondary to the
impact of HAART on trophoblast cell proliferation and migra- P.09-253-Mon
tion, the first trimester extravillous trophoblast cell line, Antioxidant effects of Ribes nigrum tincture
HTR8SVneo, was used as model trophoblast cell line. The cells against oxidative stress induced in human
were exposed to Tenofovir, Emtricitabine and Efavirenz the indi- colon cells
vidual drugs of first-line HAART Atripla and Zidovudine, S. N. Voicu1,2, M. S. Stan1, A. Hermenean3, A. Dinischiotu1
Lamivudine, Lopinavir and Ritonavir the individual drugs of the 1
University of Bucharest, Faculty of Biology, Department of
second-line HAART drug Aluvia. The effects of HAART on Biochemistry and Molecular Biology, Bucharest, Romania,
trophoblast proliferation were investigated by counting viable 2
Faculty of Pharmacy, Titu Maiorescu University, Bucharest,
cells and by MTT assays. Cell migration was assessed by wound- Romania, 3Department of Experimental and Applied Biology,
healing assay. Our results show that first and second line Institute of Life Sciences and Department of Histology, Faculty of
HAART drugs, individually and combined inhibited trophoblast Medicine Pharmacy and Dentistry, Vasile Goldis” Western
proliferation in a dose and time dependent manner, in addition, University of Arad, Arad, Romania
the drugs reduced trophoblast migration. The inhibition of cell
proliferation and migration was observed in the presence of phys- Natural antioxidants found in the various parts of plants have
iological concentrations of the HAART drugs. These results sug- attracted an increased interest from consumers and scientific
gest that the adverse pregnancy outcomes of HAART may be community. This study aimed to evaluate the antioxidant effects
due to placenta insufficiency secondary to the inhibition of tro- of Ribes nigrum tincture on human colon cells (C2BBe1 clone of
phoblast cell proliferation and migration by HAART. Caco-2). Cells were grown in standard culture conditions and
treated firstly with an oxidant agent (H2O2) in order to induce
oxidative stress and then with different concentrations of tincture
P.09-252-Wed (5, 25, 50 and 100 lg/mL) for 24 and 48 h. The cell viability was
Monitoring of changes in serum antioxidative tested by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazo-
lium bromide) assay. The reactive oxygen species (ROS) were
system parameters in different gestation
measured fluorimetrically using 5, 6-carboxy-2, 7-dichlorodihy-
periods of cows drofluorescein-diacetate (carboxy-DCFDA) as reagent. Malondi-
S. Sayiner1, O. Ergene2, I. Darbaz2, D. Ceylanli1, S. Gencosman1, aldehyde (MDA) and reduced glutathione (GSH) were assayed
S. Aslan2 by a fluorimetric respectively colorimetric method. MTT tests
1
Department of Biochemistry, Faculty of Veterinary Medicine, showed values similar with control cells after the treatment with
Near East University, Nicosia, North Cyprus, Turkey, Ribes nigrum tincture for both time intervals. After 24 h, a slight
2
Department of Obstetrics and Gynaecology, Faculty of Veterinary increase of GSH level was noticed for all tincture concentrations
Medicine, Near East University, Nicosia, North Cyprus, Turkey (in the absence and presence of oxidant), while at 48 h this
The purpose of this study was to provide a unique perspective remained at the level of control. Moreover, the levels of MDA
into the oxidative status of cows in different gestation periods. and ROS production were unchanged in human colon cells, for
Thirty Holstein cows were assigned to this study. Blood samples all conditions studied. In conclusion, our results suggest that
were collected during different periods; 4–6 weeks before dry per- Ribes nigrum tincture may be used as a natural therapeutic agent
iod (D4-5W), at the beginning of dry period (D), first month of and functional food ingredient to support treatment of ROS –
dry period (D1), antepartum day (AP- 8), postpartum (PP) day 3 mediated colon maladies, such as inflammatory bowel disease.
(PP-d3), PP day 8 (PP-d8), PP between 3–4 weeks (PP3-4w) and Acknowledgements to Project Bridge Grant 12BG/2016.
PP between 80–90 days (PP-d80-90). Glutathione peroxidase
(GPx) and Superoxide dismutase (SOD) activities were measured
in serum by using commercial assay kits (Randox, UK).
“Repeated Measures Define Factors” test and X2 test was per-
formed. The values were classified as low (A; GPx = 300.67–
594.68 U/L; SOD = 0.009–0.099 U/mL), medium (B; GPx:
600.22–798.19 U/L; SOD = 0.101–0.150 U/mL) and high (C;
GPx:831.44–2666.83 U/L; SOD = 0.133–0.998 U/mL) groups
and the percentage changes of the group were compared.
288 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 289
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
290 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
disruption of enzyme-inhibitor complex by changing of the con- treatments, making development of new compounds a priority.
tact network in the inhibitor binding site. This work was sup- Our group has synthesized steroidal compounds with antiprolifer-
ported by Russian Science Foundation grant # 14-50-00131. ative activities against human tumor cells. In addition, local
medicinal plants traditionally used for reproductive or hormonal
problems have been collected. To screen for compounds or plant
P.09-260-Tue extracts with potential for treatment of hormone-dependent can-
Effect of the ubiquitin-proteasome system cers, we developed yeast-based hormone-sensitive fluorescent
inhibitors on the energy metabolism of nerve biosensors and in vitro enzymatic assays. Yeast vectors encoding
steroid receptor ligand binding domains for estrogen receptor iso-
cells
forms, androgen receptor or glucocorticoid receptor, were created
V. Pershin1, N. Maksimova1, I. Mukhina1, M. Gainullin2,
fused to a yellow fluorescent protein biosensor. Yeast treated
A. Yudintsev3, T. Sergeeva1
1 with cognate ligand display a dose-dependent increase in fluores-
PRMU, Privolzhsky Research Medical University, Nizhny
cence. Compounds/extracts with specific affinity for estrogen
Novgorod, Russia, 2Oslo University Hospital, Oslo, Norway,
3 receptor beta, estrogen receptor alpha or glucocorticoid receptor
Lobachevsky State University of Nizhny Novgorod, Nizhny
have been identified. Other drug targets involved in steroidogene-
Novgorod, Russia
sis (human aromatase, human aldoketo reductase 1C3/AKR1C3)
Different nerve cells have specific metabolic status to maintain have been expressed in E. coli for purification, screening and X-
brain energy metabolism. Neurons were shown mainly generate ray analysis. Steroidal inhibitors of AKR1C3 have been identi-
energy through oxidative phosphorylation, while astrocytes also fied. Human AKR1C3 has been crystallized in complex with a
produce significant levels of ATP through glycolysis. The protea- steroidal inhibitor and the X-ray structure is undergoing refine-
some system plays a key role in modulating metabolism via regu- ment. Molecular docking simulations and molecular dynamics
lation of protein degradation and cell signaling processes. support our in vitro screening results. This combined approach is
However, the role of the ubiquitin-proteasome system (UPS) in low-cost, reusable, and has identified compounds and plant
the regulation of nerve cell metabolism is little studied. The aim of extracts with affinity for anticancer targets.
this investigation was to analyze the metabolic changes in nerve
cells under the UPS inhibitors treatment using confocal fluores-
cence microscopy and fluorescence lifetime imaging (FLIM). Neu- P.09-262-Mon
rons and astrocytes were isolated from freshly dissected embryonic Identification of CC2D1A homozygous
mouse hippocampus (E18). For metabolism study, primary hip- mutation as a cause of Joubert Syndrome
pocampal culture was treated with the UPS inhibitors (MG132
with obsessive compulsive disorder
and PR619) before fluorescence imaging. Detection of fluorescence
M. C. Ergoren1,2, Y. Engindereli3, B. Kaymakamzade Culhaoglu4
intensity and fluorescence lifetime of the metabolic cofactors 1
Near East University, Medical Faculty, Department of Medical
(NAD(P)H) and FAD) was performed using the FLIM system
Biology, Nicosia, Cyprus, 2Experimental Health Research Center
(Becker&Hickl GmbH., Germany) on LSM 710 microscope (Carl
of Health Sciences, Near East University, Nicosia, Cyprus, 3Near
Zeiss, Germany). NAD(P)H fluorescence lifetime imaging revealed
East University, Faculty of Medicine, Department of Child And
an increase in bound NAD(P)H amplitude in hippocampal neu-
Adolescent Psychiatry, Nicosia, Cyprus, 4Near East University,
rons upon PR619 treatment. It can be associated with a shift
Faculty of Medicine, Department of Neurology, Nicosia, Cyprus
towards more oxidative metabolism. At the same time, the ampli-
tude of bound NAD(P)H was slightly elevated under MG132 Joubert syndrome (JS) is a rare autosomal recessive disorder with
action. Astrocytes did not show significant changes in energy partial or complete agenesis of cerebellar vermis. This syndrome
metabolism in comparison with intact cells after the UPS inhibi- is identified mainly by the presence of molar tooth sign in mag-
tors treatment. These results demonstrate the different mecha- netic resonance imaging of the brain since it has a varied pheno-
nisms of action of MG132 and PR619 on cellular metabolism. typic presentation. Obsessive-compulsive disorder (OCD) is a
Modulation of the UPS activity may be a useful therapeutic strat- neuropsychiatric disorder, commonly having an early age of
egy for regulation of the energy metabolism of nerve cells and onset, and is characterized by the presence of obsessions and
treatment of neurodegenerative disease. This work was supported compulsions that can become incapacitating. Here in, we report
by the Russian Science Foundation (grant number 17-75-10202). a 17 year-old male patient with JS who shows the symptoms of
OCD. He was admitted to the neurology department with the
chief complaint of imbalance and abnormal eye movements since
P.09-261-Wed his birth. He had also delayed motor and cognitive milestones.
Use of hormone-sensitive fluorescent His mother described neonatal episodic hyperpnea and hypotonia
biosensors and in vitro assays for for the early childhood period. On neurological examination he
had oculomotor apraxia and truncal ataxia. The brain MRI
characterization of steroidal compounds with
revealed typical molar-tooth sign. The patient presented to child
antiproliferative activity against breast and and adolescent psychiatry department, 2 years duration of illness
prostate cancer cell lines characterized by repetitive questions, extreme anxiety and anger
c, E. Petri, S. Bekic, M. Marinovic, J. Plavsa, M. Sakac
A. Celi when non-answered by his mother and worrying about getting
University of Novi Sad, Faculty of Sciences, Novi Sad, Serbia sick. Neuropsychological testing performed after clinical inter-
Hormone-dependent cancers, including breast and prostate can- view using the Wechsler Intelligence Scale for Children, revised
cers, are leading causes of mortality. Because hormonal steroids edition, he demonstrated moderate intellectual disability overall.
influence proliferation of hormone-sensitive cancers, reduction of There is increasing understanding that the interconnectivity
circulating estrogen and androgen levels is an important treat- between association neocortex, limbic cortex and the cerebellar
ment strategy. Some anticancer drugs reduce steroid hormone hemispheres may play a key role in the processing of multimodal
levels by inhibition of enzymes involved in steroidogenesis, or by information. A missense homozygous mutation (c.1739C>T;
targeting steroid receptors, such as estrogen receptor. However, p.Thr580Ile) within the CC2D1A gene has been identified by
breast and prostate cancers often become resistant to these sequencing. This mutations (rs202057391) has been associated
with mental retardation before. To the best of our knowledge,
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 291
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemistry and medicine POSTERS
this is the first case of Joubert syndrome with OCD associated CA-mediated cell protection against oxidative stress. More inter-
with CC2D1A. Studies focusing on psychiatric symptoms in JS estingly, CA largely inhibited the adhesion of U937 cells to
are necessary for clarifying their relation. HUVECs by decreasing the expression level of VCAM-1. These
results suggest that CA protects HUVECs dysfunction under
oxidative stress conditions, and this effect is mediated by Nrf2
P.09-263-Tue activation and the up-regulation of HO-1. Overall, these observa-
Partially oxidized glyceraldehyde-3-phosphate tions suggest that CA has the potential for use as an anti-athero-
dehydrogenase is inactivated by alpha- sclerotic agent.
synuclein
V. Muronetz1,2, K. Barinova1, P. Semenyuk1, E. Schmalhausen1 P.09-265-Mon
1
Belozersky Institute Lomonosov Moscow State University,
Moscow, Russia, 2Faculty of Bioengineering and Bioinformatics,
Localization of LEA proteins in the cells of
Lomonosov Moscow State University, Moscow, Russia anhydrobiotic insect
A. Nesmelov, T. Voronina, S. Kondratyeva, E. Shagimardanova
Colocalization of glycolytic enzyme glyceraldehyde-3-phosphate Institute of Fundamental Biology and Medicine, Kazan Federal
dehydrogenase (GAPDH) with alpha-synuclein in Lewy bodies University, Kazan, Russia
was demonstrated by immunofluorescent analysis in brain of
patients with Parkinson’s disease. Co-expression of GAPDH and The larvae of Polypedilum vanderplanki and its relative P. pembai
alpha-synuclein in COS-7 cells results in the formation of Lewy are the only known insects and the most complex animals with
body-like inclusions, suggesting possible GAPDH involvement in an ability to survive complete body desiccation, termed anhydro-
Lewy body formation in synucleinopathies in vivo. However, biosis. Desiccation of P. vanderplanki larva is associated with
there is still no direct evidence of the interaction between these induction of some protective proteins, including LEA (Late
proteins. Our molecular modeling data predicted the binding of Embryogenesis Abundant) proteins. LEA proteins stabilize vitri-
alpha-synuclein to the positively charged groove in the tetrameric fied sugar glasses which preserve cell components in dried state.
GAPDH molecule that comprises NAD+-binding pocket. Direct High redundancy of LEA genes in P. vanderplanki genome (27
interaction between alpha-synuclein and GAPDH was confirmed members) suggests their functional difference. In this study, we
by different experimental approaches. It was shown that the aimed to examine the subcellular targeting of LEA proteins in P.
binding of alpha-synuclein to partially oxidized GAPDH results vanderplanki cells. Genes encoding 27 LEA proteins were cloned
in the inactivation of the enzyme, but does not lead to dissocia- in fusion with GFP in pP121K vector under control of P. vander-
tion of the tetrameric GAPDH into dimers or monomers. At the planki-specific promoter. P. vanderplanki-derived cultured cells
same time, the addition of the partially oxidized GAPDH to Pv11 were transfected with LEA-containing vectors using NEPA
alpha-synuclein prevents its amyloid transformation. Thus, the 21 electroporator. After incubation for 24 h., cell membranes
interaction between GAPDH and alpha-synuclein could play a and nuclei of Pv11 cells were stained with CellVue Claret Far
role in the onset and development of synucleinopathies. Further Red and Hoechst stain, respectively. Cells were also stained with
investigation of specific interaction between these two proteins Cytopainter kit containing a mixture of Golgi-selective, ER-selec-
could lead to better understanding pathogenetic mechanisms of tive and nucleus-selective dyes. Then cells were investigated with
synucleinopathies and help in searching for drug targets for pre- a confocal laser-scanning microscope LSM 780 (Carl Zeiss). We
vention and curing of these disorders. Acknowledgments: This found that three LEA proteins have very specific localization in
work was supported by the Russian Science Foundation (project Pv11 cells. These are targeted to cell membrane LEA1 protein,
No. 16-14-10027). localized in endoplasmic reticulum LEA3 protein and localized in
vacuole-like compartment LEA22 protein. Other LEA proteins
are uniformly distributed across the whole cell or localized in
P.09-264-Wed cytosol with exclusion from nucleus or both from nucleus and
Cinnamaldehyde protects oxidative stress and vacuole-like compartment. Thus, we revealed different localiza-
tion of LEA proteins in P. vanderplanki cells, suggesting their dif-
inhibits TNF-a-induced inflammatory response
ferent spatial roles and different functions in anhydrobiosis. This
by heme oxygenase-1 induction in human study was supported by Russian Science Foundation grant No
umbilical vein endothelial cells 17-44-07002.
N. Kim, S. Kim
College of Korean Medicine Dongguk University, Gyeongju, South
Korea P.09-266-Tue
Oxidative stress and inflammation play a critical role in the Withdrawn
development of cardiovascular diseases. Cinnamaldehyde (CA) is
a natural compound from Cinnamomum cassia which is widely
studied regard to its anti-cancer, anti-microbial, anti-inflamma-
tory, and anti-oxidant activities. In this study, we investigated
the cytoprotective effects and anti-inflammatory properties of CA
in hydrogen peroxide- and TNF-a-treated human umbilical vein
endothelial cells (HUVECs). The results showed that CA did not
have any cytotoxicity or cause morphological changes at concen-
trations up to 50 lM. CA treatment strongly up-regulated the
cellular protein level of heme oxygenase-1 (HO-1) and promoted
Nrf2 translocation to the nucleus. CA-mediated Nrf2/HO-1 acti-
vation reduced the level of ROS and protected the HUVECs
from hydrogen peroxide-induced oxidative stress, which induces
apoptosis. Moreover, HO-1 depletion by siRNA attenuated the
292 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemistry and medicine
P.09-267-Wed P.09-268-Mon
Three-spined stickleback of the White Sea is a Influence of 5-substituted 3-pyridylisoxazoles
new source of long-chain omega-3 oils on human platelet membrane receptors and
S. Murzina, Z. Nefedova, S. Pekkoeva, V. Voronin, N. Nemova platelet activation
Institute of Biology of the Karelian Research Centre of the Russian O. Demina1, N. Podoplelova2, I. Melnikova3, N. Belikov3,
Academy of Sciences, Petrozavodsk, Russia M. Panteleev2, F. Ataullakhanov2, S. Varfolomeev3,
Eicosapentaenoic fatty acid (EPA), and docosahexaenoic fatty A. Khodonov3
1
acid (DHA), are the most well- known essential polyunsaturated N.M. Emanuel Institute of Biochemical Physics of Russian
fatty acids (PUFA) for humans. Their function as beneficial Academy of Sciences, Moscow 119334, Russia, 2Dmitry Rogachev
health constituents of lipids from aquatic organisms, especially National Research Center of Pediatric Hematology, Immunology
fish, has been reported and proofed in much research: brain and and Oncology, Moscow, Russia, 3N.M. Emanuel Institute of
eyes systems development, preventing atherosclerosis, dementia Biochemical Physics of Russian Academy of Sciences, Moscow,
and more. Throughout the last decades, the third fatty acid – Russia
docosapentaenoic acid (DPA), has been included in the group of Design and synthesis of the series of 5-substituted 3-pyridylisoxa-
essential fatty acids, which is because of its biological role in zoles with the substituent variation in positions 3 (2-, 3- and 4-
wound-healing processes and combined effect together with EPA. pyridyl) and 5 (various groups, including alkyl C3-C8, Ph, C(=O)
The White Sea is considered as an ecologically clean marine NH2, etc.) of isoxazole ring as potential anti-platelet agents were
ecosystem with almost no human impact, in addition the done by us earlier [1, 2]. The main goal of this work is the study
resources of the sea are still unstudied and its bioresources are of relative selectivities of 5-substituted 3-pyridylisoxazoles with
misjudged from the position of potential sustainable sources of respect to the platelet membrane receptors and the influence of
products for biotechnology such as high quality long-chain this compound class on the platelets activation induced by
omega-3 oils. Three-spined stickleback, Gastrosteus aculeatus, is thrombin and collagen-relative peptide (CRP) with the generation
the most abundant non-commercial fish species in the White Sea. of two platelets subpopulations. The influence of a series of the
In our study, we accessed the lipids and their fatty acids con- most active compounds, 5-alkyl-3-(3-pyridyl)isoxazoles, on the
stituents profiles with focus on the amount of EPA, DPA and platelet membrane receptors was studied using the samples of
DHA in juveniles and adults of the three-spined stickleback from platelet-rich plasma and washed human platelets suspensions.
the White Sea in summer. Shared Equipment Facilities of KarRC The experimental data indicate that 5-substituted 3-pyridylisoxa-
RAS was used to analyze lipid classes and fatty acids, and the zoles are not the selective inhibitors of ADP, epinephrine, PAF
technological platform for the research was HPTLC and GC. In and thrombin receptors. These investigations allowed to identify
flesh, juveniles had higher amounts of PUFA (up to 45% sum the role of tested 5-substituted 3-pyridylisoxazoles in the platelet
FAs), due to omega-3 acids, than adults in muscle (28% sum signal transduction as the thromboxane A2 receptor antagonists.
FAs), in liver (35% sum FAs), and in female gonads (up to 45% All tested compounds were shown to partly inhibit the formation
sum FAs). Among omega-3 PUFA, EPA and DHA were domi- of serine-positive platelets subpopulation induced by thrombin
nating, the levels were high and accounted for 8% and 24% in and CRP. The possibility of tested compounds to influence on
juveniles and less in adults – 6% and 13%. The amount of DPA the pro-coagulating serine-positive activated platelets subpopula-
was 3.5% and 4.3% in juveniles and adults respectively, which is tion demonstrates their availability as possible new drugs. All
higher than commonly reported for other fish – from 1% to 3%. results allow to understand platelet responses formation and to
The research was carried out in the frame of the budgetary theme predict the possible cell response controlling. This work was
№ 0221-2017-0050. partly supported by Russian Foundation of Basic Research
(grant No 17-04-01326a).
References
1. Demina O.V., Khodonov A.A., Sinauridze E.I., Shvets V.I.,
Varfolomeev S.D., Rus. Chem. Bull., 2014, 2092–2113. 2. Demina
O.V., Belikov N.E., Varfolomeev S.D., Shvets V.I., Khodonov
A.A./ Russian Pat. No 2565754.
P.09-269-Tue
Determination of toxicity of ammonia on
narrow clawed crayfish (Astacus leptodactylus
Esch. 1823)
R. Tural1, F. Dalogulları2, A. Ç. G€
unal3, A. Sepici Dincel4
1
Vocational School of Health Services, University of Sinop, Sinop,
Turkey, 2Gazi University, Ankara, Turkey, 3Division of Biology
Education, Department of Mathematics and Science Education,
Faculty of Education, University of Gazi, Ankara, Turkey,
4
Department of Medical Biochemistry, Faculty of Medicine, Gazi
University, Ankara, Turkey
In this study, the toxic effects of ammonia on fresh water cray-
fish were investigated. Four different measuring times (24, 48, 96
and 168 hour) and two different sub-lethal doses of ammonia
(12.3 mg/L and 60.25 mg/L) exposed to fresh water crayfish
(Astacus leptodactylus). Changes in biochemical parameters
hemolymph of fresh water crayfish, total hemocyte counts and
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 293
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Cancer biology POSTERS
294 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
of 160 pM, 50 pM, and 1.1 nM for inhibitors VR16-09, VD11-4- P.10-003-Wed
2, and VD12-09, respectively. VR16-09 was the most selective for
TSG101, a tumor susceptibility gene,
CA IX. Crystal structures of CA IX with VD11-4-2 and VD12-
09 showed a conventional coordination bonds. Western blot bidirectionally modulates cell invasion in an
analysis demonstrated increased CA IX expression in response to MMP-9-dependent manner
hypoxia in HeLa, H460, MDA-MB-231, and A549 cells. Mass H. Shirataki1, S. Higashi1, T. Makiyama2, H. Sakane3
1
spectrometric gas-analysis revealed nanomolar IC50 for tested Graduate School of Medicine, Dokkyo Medical University, Mibu,
inhibitors to inhibit extracellular CA activity in MDA-MB-231 Japan, 2Kyorin University School of Medicine, Mitaka, Japan,
3
cells. Compounds significantly reduced hypoxia-induced extracel- Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama
lular acidification in a dose-dependent manner, whereas the effect University, Fukuyama, Japan
on extracellular pH in normoxia was negligible. Functional effect TSG101 was initially identified in fibroblasts as a tumor suppres-
was the most pronounced for VR16-09. Viability EC50 values sor gene. TSG101 has diverse biological functions in endosomal
were lower in normoxia than hypoxia in 2D cells. In contrast, trafficking, transcriptional regulation, and cell proliferation.
hypoxia-dependent effects on clonogenic survival of VR16-09 Recently, TSG101 has received attention in the field of exosome
were observed in H460 spheroids. In conclusion, novel inhibitors research because of its involvement in multivesicular body forma-
exhibited high affinity and strong selectivity toward recombinant tion as a component of the endosomal sorting complex required
CA IX and reached nanomolar functional effects in hypoxic can- for transport protein machinery. Since accumulating evidence
cer cells. Interestingly, hypoxia-dependent reduction of clono- demonstrates that TSG101 also functions as a tumor-enhancing
genicity was observed only in spheroids, highlighting the gene in some epithelial cells, it is assumed that TSG101 functions
importance of testing CA IX-targeting compounds in 3D cell as a tumor-declining gene or a tumor-enhancing gene in different
models resembling naturally occurring hypoxic microenvironment cell types. However, the precise mechanism underlying the bidi-
with clonogenic survival as endpoint. Overall, our newly designed rectional and multifaceted functions of TSG101 in carcinogenesis
compounds appear to be promising agents for CA IX-specific and tumor progression remains unclear. In this study, we found
therapy. that TSG101 bidirectionally modulates cell invasion through reg-
ulating matrix metalloproteinase-9 (MMP-9) expression in differ-
ent cell types. In HT1080 fibrosarcoma cells, TSG101 depletion
P.10-002-Tue
promoted cell invasion through enhancing MMP-9 expression. In
Lactate-mediated mitoribosomal defects contrast, in HeLa S3 cervical carcinoma cells, TSG101 depletion
impair OXPHOS and promote hepatoma cell repressed cell invasion through reducing MMP-9 expression. To
invasiveness reveal a mechanistic context for the role of TSG101 in cell inva-
Y. Lee, J. J. Lim, U. Jeoun, S. Min, E. Lee, G. Yoon sion as a multifaceted gene, we further investigated the molecular
Ajou University School of Medicine, Suwon, South Korea mechanism underlying the regulation of MMP-9 expression by
TSG101 and found that TSG101 regulated MMP-9 expression at
Impaired mitochondrial oxidative phosphorylation (OXPHOS)
the transcriptional levels.
capacity, accompanied by enhanced glycolysis, is a key metabolic
feature of cancer cells, but its underlying mechanism remains
unclear. Previously, we reported that human hepatoma cells that P.10-004-Mon
harbor OXPHOS defects exhibit high tumor cell invasiveness via
Altered miR-17-5p expression pattern in
elevated claudin-1 (CLN1). In the present study, we show that
OXPHOS-defective hepatoma cells (SNU354 and SNU423 cell response to chemotherapeutic drugs for
lines) exhibit reduced expression of mitochondrial ribosomal pro- metastatic colorectal cancer
tein L13 (MRPL13), a mitochondrial ribosome (mitoribosome) J. Despotovic1, J. Urosevic2, R. R. Gomis2, A. Nikolic1
1
subunit, suggesting a ribosomal defect. Specific mitoribosomal Institute of Molecular Genetics and Genetic Engineering,
translation inhibition with doxycycline and chloramphenicol or University of Belgrade, Belgrade, Serbia, 2Institute for Research in
siRNA-mediated MRPL13 knockdown decreased mitochondrial Biomedicine (IRB Barcelona), Barcelona, Spain
protein expression, reduced the oxygen consumption rate (OCR), The conventional first-line chemotherapy for metastatic colorectal
and increased CLN1-mediated tumor cell invasiveness in cancer (mCRC) consists of fluorouracil (5-FU) in combination
SNU387 cells, which have active mitochondria. Interestingly, we with either oxaliplatin (FOLFOX) or irinotecan (FOLFIRI).
also found that exogenous lactate treatment suppressed MRPL13 With an overall response rate of approximately 50% for either
expression and OCR and induced CLN1 expression. A bioinfor- treatment combination, a major unsolved problem is that there
matics analysis of the open RNA-Seq database from The Cancer are currently no available predictive markers for these
Genome Atlas Liver Hepatocellular carcinoma (TCGA-LIHC) chemotherapeutics. Due to the high tissue specificity and stability
cohort disclosed a significant negative correlation between of microRNAs (miRNAs) and their altered expression in drug
MRPL13 and CLN1 expression. Moreover, in patients with low resistance, miRNAs have been suggested as predictive biomarkers
MRPL13 expression, two oxidative metabolic indicators, pyru- for therapeutic response. miR-17-5p belongs to the miR-17/92
vate dehydrogenase B expression and the ratio of lactate dehy- cluster which is important in cell cycle, proliferation, apoptosis
drogenase (LDH) type B to LDH type A, significantly and and other pivotal processes. Furthermore, expression of miR-17-
negatively correlated with CLN1 expression; this implied that the 5p is frequently altered in colon tumors. The aim of this study
combination of elevated glycolysis and deficient MRPL13 activity was to determine whether 5-FU, oxaliplatin, irinotecan and their
was closely linked to CLN1-mediated tumor activity in LIHC. combinations modify the expression of miR-17-5p in colorectal
These results suggest that OXPHOS defects may be initiated and cancer cell lines SW620 and HCT116. Total RNA was isolated
propagated by lactate-mediated mitoribosomal deficiencies and after three-day treatment with these drugs and expression level of
that these deficiencies are critically involved in LIHC develop- miR-17-5p was measured by real-time RT-PCR using specific
ment. TaqMan miRNA assay. The apoptotic effects of the anti-cancer
drugs were studied by flow cytometry using an Annexin V and
DAPI staining. It was confirmed that all tested chemotherapeutic
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ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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POSTERS Cancer biology
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298 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 299
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
cytometry in some cases are not applicable for MRD detection, the function of C3G in human glioblastoma (GBM), U87 cell
NGS-based detection of rearrangements of Ig and TCR loci is line, as well as in different patient-derived cell lines as in vitro
likely to become routine procedure for MRD diagnostics. The models. In U87 cells C3G knock-down enhances invasion by
study was funded by RFBR grant No. 17-29-06052, Russian Sci- inducing actin-cytoskeleton reorganization. C3G silencing also
entific Foundation grant No. 17-75-10113 and Russian Presi- increases the expression of transcription factors and proteins
dent’s Fellowship SP-671.2018.4 involved in the epithelial-mesenchymal transition. Moreover,
C3G down-regulation increases the number of foci in anchorage-
dependent and –independent assays, although with a reduced
P.10-016-Mon number of cells within each focus. C3G silencing also modifies
Simultaneous administration of probiotic the activation of different signalling pathways in U87 cells, such
lactobacilli and antibiotics displays anti-tumor as p38MAPK or ERKs cascades, preventing c-Met and EGFR
effect in in vivo colorectal carcinoma model activation upon stimulation with their ligands. In addition, we
have recently characterized the role of C3G in patient-derived
R. Link, L. Ambro, L. Strojny, A. Bomba
arik cells (named 12Ф12D and HCO1D). U87, 12Ф12D and HCO1D
Institute of Experimental Medicine, Pavol Jozef Saf
cells differ in their genetic alterations (mutations, amplifications,
University, Faculty of Medicine, Kosice, Slovakia
etc), which are involved in the onset and development of glioblas-
Probiotic lactobacilli exhibit various positive effects on human toma. Therefore, it is important to compare the impact of C3G
health including immunostimulatory and immunomodulatory in all of them. According to the results obtained in U87, C3G
effect. In the pilot experiment we aimed to study possible gut silencing enhances cell motility and appears to regulate in vitro
microbiome modulating and anti-tumor effect of the Lactobacil- tumour growth in 12Ф12D and HCO1D cells. C3G also regulates
lus plantarum LS/07, a novel isolate from our previous research, cell signalling in these patient-derived cells. We are currently
using in rat vivo model of the colon carcinoma. Experimental comparing them with U87 in order to further characterize how
animals (male rats) were divides in three groups (n = 5): control C3G regulates GBM initiation, progression and generation of
group (CTRL+PRO), colorectal carcinoma group (CRC+PRO) metastasis, to confirm its promising role in clinic.
and colorectal carcinoma group treated with antibiotics
(CRC+PRO+ATB). All experimental groups received probiotic
culture 5 days a week during the 25-week long experiment. We P.10-018-Wed
observed no significant differences in feed and water consump- Sensitive analysis of somatic mutations in
tion and animal weight changes between three experimental circulating tumor DNA in melanoma patients
groups. Gut microbiome of the experimental animals was
using biochip-based assay
assessed from the feces samples collected at the end of the experi-
M. Emelyanova1, E. Telysheva2, K. Orlova3, I. Abramov1,
ment. Gut microbiome analysis (total bacteria, Bacteroidetes, Fir-
G. Snigiryova2, O. Ryabaya3, V. Shershov1, T. Nasedkina1
micutes and Lactobacillus) using PCR-DGGE experiments 1
Engelhardt Institute of Molecular Biology, Russian Academy of
showed no significant changes between CTRL+PRO and
Sciences, Moscow, Russia, 2FBSI “Russian Scientific Center of
CRC+PRO groups, but we observed reduced diversity of the
Roentgenology & Radiology” Ministry of Health, Moscow, Russia,
microbiome between CRC+PRO+ATB and two other groups. 3
N.N. Blokhin Russian Cancer Research Center, Ministry of
Due to the application of antibiotics (amoxicillin and metronida-
Health of the Russian Federation, Moscow, Russia
zole), we could not detect any Lactobacillus plantarum LS/07
presence using PCR-DGGE in CRC+PRO+ATB group. Surpris- Melanoma is a highly aggressive cancer. Analysis of BRAF status
ingly, we have observed significant reduction of the macroscopic is mandatory prior to targeted therapy. Circulating tumor DNA
tumors in both CRC+PRO and CRC+PRO+ATB groups when (ctDNA) includes the same genetic alterations as an initial tumor.
comparing with our data reported in our previous in vivo experi- The analysis of ctDNA presents great potential in the process of
ments on the same model. Based on our findings we conclude cancer treatment, including diagnostic and prognostic informa-
that alive but also dead lactobacilli could exhibit preventive anti- tion before and during therapy, as well as at tumor progression.
tumor effect. Although mechanisms of the preventive effect need For detection somatic mutations in ctDNA next generation
to be studied, these data support idea of preventive use of probi- sequencing and droplet digital PCR most often are used. These
otics in humans. methods are very powerful and extremely sensitive but for simple
clinical task, they are, probably, too expensive and time-consum-
ing. We developed a biochip for detection somatic BRAF muta-
P.10-017-Tue tions (V600E, V600M, V600K, V600R, V600D) in ctDNA. For
Dissecting the function of C3G in preferable amplification of mutated DNA we used nested LNA
glioblastoma: from the U87 cell line to patient- clamp PCR. The PCR product size was 74 bp. PCR fragments
were labeled via incorporation of fluorescence-labeled nucleotides
derived cells
during the second round of PCR and hybridized with specific
S. Manzano1, C. Sequera1, C. Baquero1, N. Palao1,
oligonucleotides immobilized on a biochip. Method allows detect-
M. Arechederra1, C. Guerrero2, A. Porras1
1 ing 0.05% mutated DNA in a background of WT DNA. The
Universidad Complutense de Madrid, Madrid, Spain, 2Centro de
thirty control DNA samples with known genotype were tested.
Investigaci
on del C
ancer IBMCC (USAL-CSIC), Departamento
All genotypes were determined correctly using biochip. More-
de Medicina, IBSAL, Salamanca, Spain
over, we tested 36 plasma samples from melanoma patients with
C3G is a guanine-nucleotide exchange factor (GEF) for Rap-1, known BRAF status: 9 tumors were WT and 27 ones were
although it can act through GEF-independent mechanisms. The mutated. The ctDNA was isolated from 2–5 ml of plasma col-
role of C3G in human cancer is controversial, acting as either a lected prior to surgical intervention. In 19/27 BRAF-positive
tumour suppressor or mediator depending on the context. Simi- patients the consistency of results between ctDNA and tumor
larly, C3G also regulates migration. In colon carcinoma, we DNA was observed. Most of patients (8/9) with discordant geno-
found that C3G represses migration and invasion through down- typing results had no metastases in contrast to patients with coin-
regulation of p38aMAPK activity and promotes tumour growth. cidental results. We detect no BRAF mutations in all ctDNA
Using permanent gene silencing approaches, we have analysed samples from nine BRAF WT patients. The biochip assay is a
300 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
sensitive method for BRAF mutation detection in ctDNA. This Valproic acid (VPA, histone deacetylase inhibitor) on breast
method can be useful in diagnosis and monitoring of melanoma CSCs. The effect of niclosamide (1 lM, 24 h pre-treatment) and
treatment. This work was supported by Grant of the President of VPA (0.63–5 mM) combination on the viability of MCF-7s cells
the Russian Federation (#MK-2519.2017.4). (CSCs-enriched population) were demonstrated by the ATP
assay. Acetylated histone H3 levels at selected doses for the com-
bination were assessed by ELISA. Protein levels associated with
P.10-019-Mon the Wnt/b-catenin signaling pathway, EMT and histone modifica-
Subcellular distribution and anticancer activity tions were shown by western blotting. Cell death mode was inves-
of 3,6,9-trisubstituted acridine derivatives on tigated via Hoechst 33342/PI double staining, M30 ELISA, real-
selected cancer cell line time PCR (gene levels associated with apoptosis and autophagy)
and western blotting (protein levels associated with apoptosis,
E. Konkol’ov a1, P. Nunhart1, L. Janovec2, J. Vargova3,
3, P. Fedorocko3, M. Kozurkova1 autophagy and ER stress). As a result, combination therapy
R. Jendzelovsky3, J. Sevc
1 arik, Institute of chemistry, exhibited a marked decrease in cell viability by inducing apopto-
University of Pavol Jozef Saf
sis along with the stronger Wnt inhibition and increased histone
Department of biochemistry, Kosice, Slovakia, 2University of Pavol
arik, Institute of Chemistry, Department of Organic H3 acetylation in MCF-7s cells. Furthermore, it was found that
Jozef Saf
the epithelial markers were re-expressed in which H3K9ac and
Chemistry, Kosice, Slovakia, 3University of Pavol Jozef Safarik,
H3K4me3 were also increased. In addition, ER stress and block-
Institute of Biology and Ecology, Department of Cellular Biology,
ade of autophagic flux have also been shown to be involved in
Kosice, Slovakia
this process. In conclusion, the future success of this combination
The many of acridines are known cytotoxic or cytostatic agents approach in targeting CSCs and converted CSCs to non-CSCs
used in cancer treatment. Unfortunately, selectivity of these com- may hold significant promise for successful treatment of breast
pounds is still at a low level. For these reasons a many of teams cancer.
manipulate with structure of acridine with the aim of increasing
the selectivity and at the same time decrease cytotoxic effect on
organism. In this study, a series of novel 3,6,9-trisubstituted acri- P.10-021-Wed
dine derivatives (3,6,9-AKR) was prepared with structural modi- Prognostic impact of immune cytolytic activity
fications in position 9 and then, these derivatives were and its association with checkpoint molecules
biologically evaluated for their ability to intercalate into molecule
and TIL/TAN load in human malignancies
of DNA and for their inhibitory effect on topoisomerase I/II
A. Zaravinos, K. Roufas, D. Chasiotis, A. Makris,
enzymes. The derivatives at different concentrations were anal-
C. Efstathiades, C. Dimopoulos
ysed against human lung adenocarcinoma A549 cell line, with the
European University Cyprus, Nicosia, Cyprus
aim of detecting the effect of changing the substituent in position
9 on cancer cells. The anticancer activity was assessed using vari- Immune cytolytic activity (CYT) is determined by the expression
ous techniques, such as MMT assay, viability and total cell num- of GZMA and PRF1, both secreted by effector CTL and NK
ber measurements, cell cycle distribution analyses and cells, and exhibiting high levels upon CD8+ T-cell activation and
clonogenicity assay, after 24 h and 48 h incubation. The ability during a productive clinical response against immune checkpoint
of 3,6,9-AKR derivatives to penetrate cell membranes and subse- blockade therapies. Still, how different tumors induce and adapt
quently enter the cell was monitored using flow cytometry and to immune responses is not completely understood. Here, we cal-
confocal microscopy. On the basis of these experiments, we can culated CYT across different cancer types and focused on differ-
conclude that the changing of shape or length substituent in posi- ences between primary and metastatic tumors. We screened the
tion 9 lead to significant change in efficiency and the way of variation of CYT across 32 different cancer types and 28 differ-
antiproliferative effect. This study was supported by Internal ent normal tissue types and correlated it with patient overall sur-
Grant Programme of University of P. J. Saf arik in Kosice vival, the expression of several immune checkpoint molecules,
VVGS-PF-2018-754 and VEGA 1/0016/18. and the load of TILs and/or TANs in different tumors. We
found diverse levels of CYT across different cancer types, with
highest levels in kidney, lung, and cervical cancers, and lowest
P.10-020-Tue levels in glioma, adrenocortical carcinoma, and uveal melanoma.
Dual inhibition of Wnt/b-catenin signaling and CYT was significantly higher in metastatic skin melanoma and
histone deacetylation as a new strategy to correlated significantly to the TIL load. In adrenocortical carci-
noma, skin melanoma and bladder cancer, cytolytic activity asso-
eliminate breast cancer stem cells by
ciated with an improved patient outcome and high levels of both
augmentation of apoptosis GZMA and PRF1 synergistically affected patient survival in
N. Aztopal1, M. Erkisa1, F. Ari2, E. Dere2, E. Ulukaya1 these cancers. In bladder, breast, colon, esophageal, kidney, ovar-
1
Istinye University, Faculty of Medicine, Department of Clinical ian, pancreatic, testicular and thyroid cancers, high cytolytic
Biochemistry, Istanbul, Turkey, 2Uludag University, Science and activity was accompanied by upregulation of at least one
Art Faculty, Department of Biology, Bursa, Turkey immune-checkpoint inhibitor, indicating that similar to mela-
Epigenetic changes play a critical role in the regulation of cancer noma and prostate cancer, immune response in cytolytic-high
stem cell (CSC) properties and the development of drug resis- tumors elicit immune suppression in the tumor microenviron-
tance. Modulation of histone acetylation program is closely ment. Overall, our data highlight the existence of diverse cytolytic
related to differentiation and apoptosis process. CSCs, a subset levels across different cancer types and suggest that along with
of tumor cells, are responsible for disease relapse because of an the existence of complicated associations among various tumor-
acquired resistance to apoptosis and the Wnt signaling which is infiltrated immune cells, it is capable to promote or inhibit the
associated with cell survival/self-renewal and differentiation, is establishment of a permissive tumor microenvironment, depend-
re-activated in these cells. Therefore, in the present study, we ing on the type of cancer.
focused on a possible cytotoxic/apoptotic effect of the combina-
tion of niclosamide (Wnt/b-catenin pathway inhibitor) and
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6
Engelhardt Institute of Molecular Biology, Russian Academy of P.10-033-Wed
Sciences, Moscow, Russian Federation, Moscow, Russia
The investigation of the effects of
MiRNAs and methylation of miRNA genes play a key role in Cinnamomum cassia extracts on Type 1
epigenetic deregulation in malignant tumors. The aim of this diabetes in vitro model
work was to define the role of methylation of novel tumor sup- G. Çanaklı1, B. Agrap2, F. Lermioglu Erciyas3
pressor miRNA genes in clear cell renal cell carcinoma (ccRCC) 1
Izmir Biomedicine & Genome Centre (IBG), Izmir, Turkey, 2Ege
pathogenesis and identify markers for diagnostics and metastasis University, Izmir, Turkey, 3Ege University Faculty of Pharmacy
prediction. The set of 70 paired (tumor/normal) ccRCC samples, Department of Pharmaceutical Toxicology, Izmir, Turkey
19 post-mortal renal tissues from individuals without cancer his-
tory, methylation-specific PCR, and RT-qPCR were applied. The There are a large number of studies suggesting that increased
14 miRNA genes (MIR-124-1/-2/-3, -125b-1, -129-2, -132, -137, - oxidative stress plays a pivotal role in the development of compli-
193a, -34b/c, -9-1, -9-3, -107, -130b, -1258) among 19 analyzed cations of diabetes in patients with Type 1 diabetes mellitus
were significantly hypermethylated and 2 miRNA genes (MIR- (T1DM). It is alleged that increased oxidative stress induces
191, -212) were hypomethylated. Using the subset of 14 ccRCC oxidative DNA damage and that it may contribute the develop-
samples, 11 miRNA genes were found to be downregulated, and ment of cancer due to insufficient DNA repair capacity. There-
significant correlation was revealed between miRNA level alter- fore, studies focused on the investigation of the protective drugs
ations and methylation aberrations for 7 genes: MIR-124-3, - and herbal products against to oxidative stress and DNA damage
125b-1, -129-2, -137, -34b/c, -375, -9-3. Moreover, hypermethyla- in diabetic patients. Cinnamon is a herbal product that its diverse
tion of most miRNA genes studied was associated with ccRCC biological activities including antidiabetic and anti-tumor proper-
progression (advanced clinical stage, tumor size, differentiation ties has been proved by scientific studies. Cinnamomum cassia
grade). In particular, hypermethylation of 6 miRNA genes (C. cassia) is a cinnamon species commonly presents in commer-
(MIR-125b-1, -129-2, -203a, -375, -107, -1258) significantly corre- cial cinnamon preparations. The purposes of our study were; 1-
lated with metastasis presence. Of interest, MIR-203a and MIR- To investigate the effects of C. cassia bark extracts on cell viabil-
375 were hypermethylated only in primary tumors from patients ity and the apoptotic activities of the extracts on type 1 diabetes
with metastases. Novel marker systems were suggested for in vitro model; 2- To investigate of the effects of the extract(s)
ccRCC diagnostics (MIR-125b-1, -375, -137, -193a) and metasta- with no apoptotic activity on endogenous and H2O2-induced
sis prediction (MIR-125b-1, -375, -107, -1258, -203). Both sys- oxidative DNA damage; 3- To investigate the implication of
tems were characterized by high enough clinical sensitivity (86%) reactive oxygen species (ROS) generation on these effects. In the
and specificity (95%) on examined sample set (AUC 0.93). In present study, IC50 values of the extracts were determined by
conclusion, hypermethylation of 14 miRNA genes and associa- WST-1 test. Annexin V/PI and TUNEL tests were performed for
tion of 6 miRNAs with metastasis were revealed in ccRCC, as determination of apoptotic activities, by using flow cytometry.
well as novel marker systems for diagnostics and metastasis pre- The effects of butanol and water extracts with non-apoptotic
diction were suggested. This work was supported by the Program activities on endogenous and H2O2-induced oxidative DNA dam-
of fundamental research for state academies for 2013-2020 years age were determined by alkali comet assay and confirmed by
(№ 0520-2014-0030). TUNEL test. The effects on the production of ROS were investi-
gated by DCF-DA test using flow cytometry. Our results showed
that the C. cassia water extract has protective effect against to
P.10-031-Mon H2O2-induced oxidative DNA damage and that this effect might
Novel inhibitors of lysine (K)-specific be related to its reducing effect on ROS production in T1DM
demethylase 4A with anticancer activity in vitro cell model.
H. J. Lee, S. Han
Gyeongsang National University, Jinju, South Korea
P.10-034-Mon
Lysine (K)-specific demethylase 4A (KDM4A) is a histone Arctigenin induces cell death through
demethylase that removes methyl residues from trimethylated or oxidative mitochondrial damage against
dimethylated histone 3 at lysines 9 and 36. Overexpression of
KDM4A is found in various cancer types. To identify KDM4A
acidity-tolerant prostate carcinoma PC-3 cells
Y. Lee, J. Oh, K. Park, S. Lee
inhibitors with anti-tumor functions, screening with an in vitro
Department of Biochemistry, College of Medicine, Soonchunhyang
KDM4A enzyme activity assay was carried out. The benzylidene-
University, Cheonan, South Korea
hydrazine analogue LDD2269 was selected, with an IC50 of
6.56 lM of KDM4A enzyme inhibition, and the binding mode Extracellular acidity in the tumor microenvironment contributes
was investigated using in silico molecular docking. Demethylation to the chemoresistance of malignant tumors. Herein, the
inhibition by LDD2269 was confirmed with a cell-based assay chemosensitizing effects of arctigenin, a novel anti-inflammatory
using antibodies against methylated histone at lysines 9 and 36. lignan from the seeds of Arctium lappa, on the acid-tolerant pros-
HCT-116 colon cancer cell line proliferation was suppressed by tate cancer PC-3AcT cells and their parental acid-sensitive PC-3
LDD2269, which also interfered with soft-agar growth and cells were studied. The PC-3AcT cells manifested increased toler-
migration of HCT-116 cells. Annexin V staining and PARP ance to low-pH media together with enhanced percent cell viabil-
cleavage experiments showed apoptosis induction by LDD2269. ity, and an increase in the resistance to docetaxel, as compared
Derivatives of LDD2269 were synthesized and the structure–ac- with their parental PC-3 cells. A preferential antiproliferative
tivity relationship was explored. LDD2269 is reported here as a activity of arctigenin against PC-3AcT cells was associated with a
strong inhibitor of KDM4A in in vitro and cell-based systems, non-necroptotic, necrotic type of cell death, which was accompa-
with anti-tumor functions. nied by an increase in the cell fraction with a sub-G0/G1 peak,
an increase in the levels of ROS production and depolarization
of mitochondrial membrane potential, and an increase in viable
cells exhibiting intense LysoTracker Red staining. A series of
changes caused by arctigenin were significantly recovered by
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 305
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
306 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
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POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 307
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
308 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
branched chain amino acid pathway to show the contribution of main limit of native SST for anticancer therapy is the very short
the mentioned metabolic reactions to proliferation and viability half-life, relatively low receptor subtype selectivity and low aque-
of breast cancer cell lines. For this purpose we used isotopic ous solubility. For these purposes, a novel synthetic analog of
labelling, where citric acid pathway metabolites have been veri- SST with higher stability to enzymatic degradation in means of
fied for fully labelled C-13 atoms when lipid and branched chain including D-amino acids with higher selectivity for specific SST
amino acids degradation pathways were blocked by appropriate subtypes was synthesized and characterized. A peptide drug con-
siRNAs. We demonstrated that lipid synthesis and oxidation can jugate represents analog of the somatostatin hormone receptor
be simultaneous in breast cancer cells. platform, linked to two known hydrophobic anticancer drugs,
camptothecin and chlorambucil, acting through different onco-
genic mechanisms. To solve the solubility problem and increase
P.10-044-Tue potential activity of the drug, a novel approach for encapsulation
Trace elements deficiency and integrin beta3a was applied and the anticancer activity against HeLa cells has
Leu33Pro polymorphism and cancer been checked. The approach to Triton X-114 micellar coordina-
L. Muhana, G. Myandina, A. Syroeshkin, T. Maksimova, tion clusters (MCCs) formation is based on partial entrapment
L. Varekha, E. Neborak, S. Syatkin, I. Smirnova and stabilization by hydrophobic forces of chelate ligands found
Peoples’ Friendship University of Russia (RUDN University), on the non-ionic surfactant micelles interface of the Triton X-114
Moscow, Russia with further “cross-linking” of the formed structures by various
metal ions. An incubation of the HeLa cells culture with drug-
The genetic variations alone do not explain the observed differ- loaded MCCs showed that during the the process, the MCCs are
ences in incidence of prostate cancer (PC) and bladder cancer captured by the immobilized cells without intrusion into them
(BC) in the populations, indicating that environmental and diet- and stayed on their surface. After 16 h of incubation, the total
ary factors are of importance. The glycoprotein alpha (IIb) amount of living cells didn’t exceed 5–10%. MCCs are unable to
beta3a (GPIIb/IIIa) is the main fibrinogen receptor on platelets. penetrate into the cells, but could attach to the cells surface and
Using participants (n = 150) from the Moscow population, we release the drug. The incubation of the drug with the culture has
assessed the risk of PC and BC in individuals with the beta3a only a slight cytostatic effect. MCCs themselves without a drug
Leu33Pro polymorphism (allelic variants PLA1 and PLA2) and do not influence on the cells. The work was supported by the
zinc (Zn) and iron (Fe) deficiency. A study group of 90 patients Grant of the President of the Russian Federation No. MK-
with PC, 30 patients with BC and 30 age-matched controls 2124.2017.3 (2017–2018).
healthy volunteers were measured for Leu33Pro polymorphism
with the PCR-RFLP assay. Detection of trace elements in the
hairs of the patients with PC and BC and healthy adults was P.10-046-Mon
done by using atomic absorption spectrophotometer. Relative Combined epigenetic therapy with HMT and
risks of cancer and 95% confidence intervals were calculated.
HDAC inhibitors caused acute promyelocytic
The frequency of PLA2 carriers (heterozygous and homozygous)
was significantly higher in patients with PC and BC, comparing leukemia cell differentiation and apoptosis
to population frequency (42.2% vs 24.0%). For the patients with in vitro and ex vivo
PLA2 the tumor progression has a greater rate of local invasion A. Vitkeviciene1, G. Skiauteryte1, M. Stoskus2, E. Gineikiene2,
and metastases. Concerning the metastases group for BC the fre- A. Zucenka2, L. Griskevicius2, R. Navakauskiene1
1
quency of Leu33Pro polymorphism carriers was higher than in Vilnius University, Life Sciences Center, Institute of
non-invasive group (41.7% vs 23.3%). For the carriers of Leu33- Biochemistry, Vilnius, Lithuania, 2Vilnius University Hospital
Pro polymorphism (PLA1/PLA2) the frequency of local PC and Santaros klinikos, Hematology, Oncology and Transfusion
of metastases PC is 2.8 and 2.4 times higher than that for non- Medicine Centre, Vilnius, Lithuania
carriers patients respectively. The concentration of Zn for the Acute promyelocytic leukemia (APL) is a subgroup of acute mye-
patients with invasive forms of PC and BC were significantly loid leukemia, possessing a PML-RARA translocation. APL
lower than for patients with non-invasive cancers. The tumor
patients are conventionally treated with combinations of Retinoic
progression, local invasion and metastases have a greater rate for acid and chemotherapeutics. Although, prognosis after such
the patients with Zn deficiency and for the carriers of Leu33Pro treatment is quite satisfactory, relapses and patients resistant to
polymorphism (PLA1/PLA2) than that for non-carriers. The retinoic acid occur. Thus, we investigated the effect of epigenetic
publication was supported by “RUDN University Program 5-
agents’ 3-Deazaneplanocin A (histone methyl transferase inhibi-
100” and the Ministry of Education and Science of Russia (the tor, HMTi) and Belinostat (histone deacetylase inhibitor,
Agreement No. 02.A03.21.0008) HDACi) in combination with Retinoic acid on APL cells in vitro
and ex vivo. NB4 cell line was used for in vitro experiments.
P.10-045-Wed Samples of APL patient bone marrow cells were collected at
diagnosis stage and used for ex vivo experiments. Both NB4 cells
Triton-X based micellar coordination clusters and patient bone marrow cells possess PML-RARA transloca-
loaded by a novel peptidyl platform drug tion. Both cell types were treated with 3-Deazaneplanocin A,
conjugates and their activity against HeLa cells Belinostat, and Retinoic acid combination. We showed that such
A. Solomonov1, E. Rumyantsev1, E. Ragozin2, T. Shekhter- treatment induced granulocytic cell differentiation both in vitro
Zahavi3, G. Gellerman2 and ex vivo. Also, we observed increase of differentiation
1
Ivanovo State University of Chemistry and Technology, Ivanovo, (CEBPE, PPARG), DNA damage (ATM), and cell cycle arrest
Russia, 2Ariel University, Ariel, Israel, 3Tel Aviv University, Tel (p53, p21) associated gene expression, meanwhile, oncogene
Aviv, Israel (MYC, WT1) expression significantly decreased. After the treat-
ment, pro-apoptotic protein BAX level increased, while, anti-
Among natural receptor-binding peptides, the neuroendocrine
apoptotic protein Bcl-2 level decreased showing triggered apopto-
hormone somatostatin (SST) and its analogs received much
sis process both in vitro and ex vivo. Elevated phospho-H2AX
attention because of their high affinity to five human receptors
histone modification level suggests that apoptosis might be
(SSTR1–SSTR5) overexpressing in cancer cells. However, the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 309
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
triggered by DNA damage response. Analysis of H3K4me3, is secreted in the surrounding medium. Interestingly S100A4 is
H3K14Ac, and Hyper-Ac-H4 histone modifications showed that also present on the surface of CD4+ CD25+ PGRPs+ S100A4+
the levels of these active chromatin markers increased after treat- lymphocytes as a component of complex involved in their cyto-
ment. In conclusion, treatment with 3-Deazaneplanocin A, Beli- toxicity. Here we investigated the relation between S100A4
nostat and Retinoic acid combination induced acute expression in cancer cells and CD4+ CD25+ PGRPs+ S100A4+
promyelocytic leukemia cell differentiation, apoptosis, and chro- lymphocytes and the cytotoxicity of lymphocytes to cancer cells.
matin remodeling in vitro and ex vivo. Therefore, we suggest that We show that the decrease of extracellular S100A4 led to increase
this combination might be a promising acute promyelocytic leu- of lymphocyte cytotoxicity against CSML100 and M3 melanoma
kemia treatment approach. cells. The decrease in S100A4 level in CD4+ CD25+ PGRPs+
S100A4+ lymphocytes inhibits their cytotoxic activity. Thus, the
resistance to CD4+ CD25+ PGRPs+ S100A4+ lymphocyte cyto-
P.10-047-Tue toxicity depends on S100A4 expression level in target cells and in
Autocrine cytokine stimulation of MPM SCs lymphocytes. The effect of several anti-cancer drugs on cancer
might be responsible for chemoresistance in cells survival on the background of decreased S100A4 level was
MPM also studied. The decreased intracellular S100A4 level declines
the resistance of malignant lymphoid Namalwa cells to dexam-
V. Milosevic1, J. Kopecka1, I. C. Salaroglio1,
ethasone. In Namalwa cells, dexamethasone decreased the effect
P. Ananthanarayanan1, S. Novello2, L. Righi3, G. Scagliotti4,
of camptothecin but the decrease of intracellular S100A4 level
C. Riganti1
1 retained their antitumor effect to the level of combined effect of
Department of Oncology, University of Torino, Turin, Italy,
2 dexamethasone and camptothecin. Studying the sensitivity of
Thoracic Unit, San Luigi Gonzaga Hospital and Department of
MDA MB 231 breast cancer cells having the decreased level of
Oncology, University of Torino, Turin, Italy, 3Pathology Unit, San
S100A4 to different combinations of anti-cancer drugs revealed
Luigi Gonzaga Hospital and Department of Oncology, University
that combination of Dexamethasone and Docetaxel highly
of Torino, Turin, Italy, 4Medical Oncology Unit, San Luigi
increased their cumulative effect. Dexamethasone and Doxoru-
Gonzaga Hospital and Department of Oncology, University of
bicin had a lower joint effect which was negated by the Doc-
Torino, Turin, Italy
etaxel. Studying the mechanisms of these pharmaceutical
Standard therapy of malignant pleural mesothelioma (MPM) is combinations has determined the potential role of S100A4 in
based on cisplatin and pemetrexed which is so far considered as MDA MD 231 cell anticancer sensitivity. This work was sup-
“golden standard”. Only partial response is obtained with exist- ported by Russian Science Foundation grant 14–15–01032 and
ing therapy due to the intrinsic chemoresistance of MPM. the Program of fundamental research for state academies for
Tumor-derived stem cells (SCs) are responsible for MPM dissem- 2013–2020 years (№ 01201363822).
ination and progression, but it is not known if they can con-
tribute to its chemoresistant phenotype. The aim of this work
was to investigate the mechanisms by which MPM SCs could P.10-049-Mon
exert chemoresistant properties of this tumor. From biopsies and MiR-106b mediates sorafenib refractory
pleural effusions of MPM patients we collected and stabilized hepatocellular carcinoma progression
MPM cell lines. The SC component was isolated by sorting the W. Wang, Z. Su, W. Hsieh, C. Yen
SOX2+ Oct4+ Nanog+ ALDHbr1ght cells and then confirming National Cheng Kung University Hospital, Tainan, Taiwan
their clonogenicity and self-renewal potential. High-throughput
PCR array was used to examine interleukins genes expression. Hepatocellular carcinoma (HCC) is the leading cause of cancer-
Interleukins production were measured using Elisa. By using related death in Taiwan. Curative options for HCC are limited
CRISPR/CAS system we created KO SCs subclones which we and exclusively available for 15% patients carrying an early stage
tested for cytotoxicity and cell viability. MPM SCs showed HCC. Systemic target therapy sorafenib which could inhibit
higher resistance to cisplatin and pemetrexed compared to non angiogenesis and Raf-1 protein is the only target therapy drug
SCs. High-throughput PCR array showed higher expression of a proved in advanced HCC patient’s treatment. However, the sora-
vast number of cytokines in MPM SCs, particularly of IL1b and fenib had only marginal effects and prolonged 3–4 months sur-
IL8. Higher production of these two cytokines were confirmed by vival times in advanced HCC patients. The resistance to
ELISA. KO IL1b and IL8 MPM SCs sub-clones showed restored sorafenib caused the rapid disease progression and poor clinical
chemosensitivity to cisplatin and pemetrexed. Our study showed outcome. Thus, it is emergent to understand the sorafenib resis-
that MPM SC are resistant to a broad spectrum of first-line tant mechanisms and develop a new strategy to improve the
drugs such as cisplatin and pemetrexed. Knocking out experi- effective treatment. In this study, we want to explore the roles of
ments suggested that IL1-b and IL-8 autocrine stimulation might miR-106b in sorafenib refractory HCC progression. Sorafenib
play a significant role in MPM SC resistance to cytotoxic effects treatment inhibited RAS signaling and proliferation in HCC
induced by chemotherapeutic drugs. cells. Meanwhile, reduced expression of E2F1, MCM7 mRNA,
and miR-106b was significantly observed in sorafenib-treated
HCC cells. Our previous studies were showed that high expres-
P.10-048-Wed sion of miR-106b with poor prognosis in HCC. Moreover, we
The role of S100A4 in response to anti- found that overexpression of miR-106b resulted in downregula-
neoplastic drugs and lymphocyte anticancer tion of PTEN and upregulation of PD-L1. HCC cells which were
long-term exposed with sorafenib led to a greatly decreased level
cytotoxicity in PTEN. Furthermore, PD-L1 levels were elevated in these cells.
T. Portseva1,2, E. Dukhanina1, A. Dukhanin2, S. Georgieva1 These findings suggested that miR-106b mediated sorafenib resis-
1
Engelhardt Institute of Molecular Biology, Russian Academy of tance by regulation of PD-L1 in HCC. The mechanisms of miR-
Sciences, Moscow, Russia, 2Pirogov Russian National Research 106b and its regulatory axis in sorafenib-refractory HCC patients
Medical University (RNRMU), Moscow, Russia might apply to develop a new drug therapy in advanced HCC
Mts1/S100A4 is a calcium binding protein involved in metastasis. patients.
S100A4 is present in cytoplasm of many types of cancer cells and
310 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
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ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
312 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 313
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
314 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 315
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
(MK2P) are expected to be located near the local adhesion mole- P.10-067-Mon
cules associated with the cell’s stress fibers, cytoskeleton and
Role of PDLIM4 in triple-negative breast cancer
adhesion molecules. Thus, epithelial mesenchymal transition
A. Ivanova, D. Kravchenko, S. Chumakov, E. Frolova
(EMT) can affect gating of channel proteins that can interfere
Shemyakin–Ovchinnikov Institute of bioorganic chemistry of the
with cell homeostasis by altering cell membrane potential. The
Russian Academy of Sciences, Moscow, Russia
phenomenon of EMT in cancer cells is involved in invasion,
metastasis, recurrence, chemotherapy and drug resistance, but it PDLIM4 (PDZ and LIM domain protein 4) is an adaptor pro-
is not known that MK2P is involved in EMT. In this study, we tein belonging to ALP subfamily of PDZ and LIM domain pro-
used MK2P small interfering RNA (MK2Psi) and rat MK2P teins. PDLIM4 is located on human chromosome region 5q31.1
plasmid DNA (rMK2Pp) to determine whether MK2P contribute which is frequently deleted in tumor cells of patients with lym-
to EMT regulation. Among the EMT markers, alterations in phoproliferative disorders. According to several reports,
vimentin, E-cadherin, and N-cadherin were determined using PDLIM4 gene methylation is frequently observed in some can-
MK2Psi or rMK2Pp alone and MK2P siRNA and MK2P plas- cers; and subsequent loss of expression may serve as an adverse
mid DNA simultaneously (MK2Psi plus rMK2Pp) in prostate prognostic factor of disease. As a result, PDLIM4 is thought to
cancer cell line. The mRNA levels of cells treated with MK2Psi act as a tumor suppressor protein in some cancers, particularly in
or rMK2Pp alone and cells treated with negative control siRNA colon cancer and prostate cancer. We conducted a systematic
(NCsi) were observed using real-time RT-PCR. Cells treated with analysis of PDLIM4 status and function in the panel of triple-
MK2Psi twice for 6 days showed an increase in vimentin and N- negative breast cancer cell lines (TNBC) and found an ambigu-
cadherin compared to NCsi, whereas rMK2Pp-overexpressing ous role of the protein. TNBC cell lines were characterised by
cells showed a decrease in mesenchymal marker vimentin com- wide range of PDLIM4 expression levels varying from higher-
pared to non-transfected control cells. The epithelial marker E- than-normal to complete loss. PDLIM4 knock-in/knock-out in
cadherin was slightly decreased in MK2Psi treated cells. Cells lines with respective low and high PDLIM4 expression levels had
transfected with MK2Psi and rMK2Pp restored increased vimen- a distinct impact on cell migration activity and morphology.
tin and decreased E-cadherin compared to cells transfected with Assessment of functional interaction between PDLIM4 expres-
MK2Psi alone. Immunocytochemistry or real-time RT-PCR anal- sion levels and c-Src activity – a previously described possible
ysis showed that MK2Psi increased the binding homeobox tran- mechanism of PDLIM4-mediated malignization, revealed no evi-
scription factor 1 (Zeb1) and Slug compared to NCsi, and the dent correlation. Instead, the alternative theory of PDLIM4 func-
factors were restored by rMK2Pp. These results suggest that the tion via CD74/Scribble/E-cadherin axis was proposed and
MK2P is involved in the EMT process of cancer cells. confirmed for TNBC cell lines. Moreover, for the first time
PDLIM4 was detected in nuclei of several breast cancer cell lines.
To establish the role of nuclear PDLIM4, the protein was
P.10-066-Wed immunoprecipitated and the possible new binding partners were
An adhesion molecule, ARHGEF7, regulates studied by LC-MS/MS. The results suggest a new mechanism of
the cell cycle progression in colon cancer PDLIM4 function in breast cancer.
E. Shin1, K. Kim2, E. Kim3
1
Chungbuk National University, Cheongju, South Korea,
2
Biomedical Engineering, College of Medicine, Chungbuk National
P.10-068-Tue
University, Cheongju, South Korea, 3Department of Biochemistry, NNMT knockdown enhances autophagy for
College of Medicine, Chungbuk National University, Cheongju, resistance to nutrient starvation in liver cancer
South Korea S. M. Hong1, G. Yoon1,2
1
Department of Biochemistry & Molecular Biology, Ajou
ARHGEF7 is a guanine nucleotide exchange factor for Rac/
University School of Medicine, Suwon, South Korea, 2Department
Cdc42 that regulates the important cellular processes such as cell
of Biomedical Science, The Graduate School, Ajou University,
migration, differentiation and proliferation. However, little is
Suwon, South Korea
known about how plays a role in colon cancer. In this study, we
investigated whether the expression level of ARHGEF7 was Nicotinamide N-methyl transferase (NNMT) transfers a methyl
increased, and the cell cycle by ARHGEF7 was regulated on group from S-adenosyl-L-methionine (SAM) to nicotinamide
colon cancer. First, we analyzed the ARHGEF7 expression in (NAM), producing 1-methylnicotinamide (1MNA). NNMT levels
colon cancer tissues. ARHGEF7 were overexpressed in cancer are variably dependent on tumor types and the underlying roles
tissues compared to paired normal tissues. Knockdown of ARH- of NNMT in liver cancer have not been fully investigated. In this
GEF7 using specific siRNAs inhibited the kinase activity of p21- study, we found that NNMT expression levels were downregu-
activated kinase 1 (PAK1) in HCT116, colon cancer cells. Also, lated in liver cancer cell lines and NNMT knockdown promoted
overexpression of GFP ARHGEF7-wild type increased the activ- tumor cell survival under nutrient deprivation condition by
ity of endogenous PAK1 in dose dependent manner. Overexpres- enhancing autophagy. Consistently, NNMT knockdown
sion of GEF-negative ARHGEF7 couldn’t induce the PAK1 enhanced tumor growth by decreasing the regions of cell death in
activation as well as Rac1. These results implicate that overex- a xenograft model. Increased autophagy flux in liver cancer cells
pression of ARHGEF7 regulates PAK1 activity through Rac1 with NNMT knockdown was dependent on ULK1 activation.
activation in colon cancer. Furthermore, we observed that the NNMT knockdown increased both methylation and phosphatase
proliferation of colon cancer cells and cell cycle progression were activity of PP2A, resulting in down-regulation of p-ULK1
attenuated compared with control. These results suggest that (S638). Accordingly, NNMT downregulation rescued tumor cells
overexpressed ARHGEF7 in colon cancer regulates cell prolifera- under nutrient deficiency in vivo, which was alleviated by ULK1
tion by promoting the cell cycle progression. inhibitor treatment. Taken together, our findings suggest a novel
function of NNMT in liver cancer and provide insight into
ULK1 inhibition as its effective therapeutic strategy.
316 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
P.10-069-Wed showed that PLK1 degradation contribute to avoid the cell pro-
liferation after DNA damage in the S phase of the cell cycle. In
New synergic biomaterials for anti-cancer
the present study, we continue analyzing the role of FBXW7 on
therapy cell proliferation after UV radiation. We compared proliferation
B. Swanepoel1, M. van de Venter1, L. Venables1, O. T. Olaru2, of wild type and FBXW7/ HCT116 cells after UV treatment,
G. M. Nitulescu2 and found that FBXW7 enabled a faster recovery of cell prolifer-
1
Department of Biochemistry and Microbiology, Nelson Mandela ation. The analysis of the amount of TP53 in both cell lines
University, Port Elizabeth, South Africa, 2Carol Davila University determined a faster decrease of TP53 protein level in the wild
of Medicine & Pharmacy, Bucharest, Romania type compared with knock out cells. These results led us to study
Recent anti-cancer research has focused on finding novel com- TP53 as a new potential substrate of SCF(FBXW7). The results
pound/product combinations with non-overlapping mechanisms obtained will be shown. In summary, we suggest that FBXW7
of action, due to limited toxicity in normal tissues, harmful side has a double function in regulation of cell proliferation. On the
effects and the development of drug resistance. The first objective one hand, it regulates the cell cycle arrest when DNA damage
of this study was to determine the mechanism of action of Ane- occurs and, on the other, it allows the cell proliferation when the
mone nemorosa, Artemisia afra, N-[[3-(4-bromophenyl)-1H-pyra- DNA damage has been repaired.
zol-5-yl]-carbamothioyl]-4-chloro-benzamide (BC-7) and cisplatin
through cell cycle arrest, phosphatidylserine translocation, cas-
pase activation and mitochondrial membrane depolarization. The P.10-071-Tue
second objective was to investigate mixtures of these extracts and Molecular mechanism of action of classical
compounds for their synergistic cytotoxic activity and thus for- and next-generation taxanes for their potential
mulating the mixtures as potential anti-cancer agents. Thirty effects in resistant ovarian tumors
combination mixtures were prepared using the IC50 values of R. Vaclavikova1,2, K. Koucka1, M. Ehrlichova1,2,
each extract or compound at ratios of 1:3, 1:2, 1:1, 2:1 and 3:1, V. Brynychova1,2, L. Rob3, B. Mohelnikova-Duchonova4,
respectively. The cytotoxic/anti-proliferative activity of each mix- M. Hruda3, I. Ojima5, V. N. Kristensen6, P. Soucek1,2
ture was determined by the bis-Benzamide H 33342 trihydrochlo- 1
NIPH, Prague, Czech Republic, 2Biomedical Center, Faculty of
ride/propidium iodide (Hoechst/PI) dual staining method on Medicine in Pilsen, Charles University, Pilsen, Czech Republic,
HeLa cervical cancer cells. The combination index (CI) values, at 3
Department of Gynecology and Obstetrics, Third Faculty of
inhibition of 50% for each mixture was determined by means of Medicine and Vinohrady University Hospital, Charles University,
the Chou and Talalay method. The combined effect can then be Prague, Czech Republic, 4Department of Oncology, Palacky
indicated as CI < 1, synergism; CI = 1, additive effect or CI > 1, University Medical School and Teaching Hospital, Olomouc,
antagonism, respectively. Most combination treatments showed Czech Republic, 5Institute of Chemical Biology & Drug Discovery,
to have an antagonistic effect except for cisplatin:BC-7 and cis- State University of New York at Stony Brook, Stony Brook, NY,
platin: A. afra combinations that showed synergism. The 1:2 United States of America, 6Institute of Clinical Epidemiology and
ratio of cisplatin:BC-7 and 1:1 and 3:1 ratios of cisplatin:A. afra Molecular Biology (EpiGen), Akershus University Hospital, Oslo,
were additive. The current finding is that BC-7 and A. afra could Norway
lower the dose of cisplatin in combination to achieve a similar
anti-cancer efficacy compared to the higher cisplatin dose when Taxanes are widely used anticancer drugs. However, drug resis-
used alone. The lower dosage in combination could result in tance of tumor cells to classical taxanes presents one of the major
reduced drug resistance as well as limit the toxicity on normal obstacles in successful therapy. At present, new anticancer drugs
cells associated with cisplatin treatment. on the basis of taxanes are developed as potential drugs effective
in resistant type of tumors. We explored efficacy of classical (pa-
clitaxel) and next-generation Stony Brook taxanes (SB-taxanes)
P.10-070-Mon in vitro in sensitive and resistant models of cancer cells. Twelve
Role of SCF(FBXW7) on cell proliferation examined SB-taxanes (SB-T-1214 and SB-T-1216 derivatives and
fluoro-derivatives) demonstrated significantly higher cytotoxicity
recovery after UV treatment
and accumulation than paclitaxel in resistant tumor cells. SB-T-
M. Galindo-Moreno1, S. Giraldez1, M. C. Lim on-Mortes1,
1214, SB-T-1216 and SB-T-12854 were also efficient in vivo in rat
E. Crespo-Martın1, A. Belmonte-Fernandez1, C. Saez2,
Jap lymphomas and mouse xenograft models of pancreatic carci-
M. A. on2, M. Tortolero1, F. Romero1
1 noma. Accordingly, SB-taxanes seem to be potential candidates
Departamento de Microbiologıa, Facultad de Biologıa,
for treatment of classical taxane-resistant tumors. In the frame of
Universidad de Sevilla, 41012-Sevilla, Spain, 2Instituto de
molecular mechanism of action, we have measured mRNA and
Biomedicina de Sevilla (IBIS) and Departamento de Anatomıa
miRNA profile changes using Affymetrix arrays in resistant ovar-
Patol
ogica del Hospital Universitario Virgen del Rocıo de Sevilla,
ian carcinoma cells (NCI/ADR-RES) after the treatment with
41013-Sevilla, Spain
paclitaxel and compared data with one of the most efficient tax-
SKP1-CUL1-F-box protein (SCF) complex is probably the best- anes, SB-T-1216. We have found significant differences in mRNA
characterized Cullin-RING ubiquitin ligase. The E3 ligases are and miRNA profiles linked to the action of taxanes and identi-
one of the three key enzymes involved in protein ubiquitination fied key pathways and genes for molecular mechanism of taxane
and degradation. The F-box protein is the variable substrate- action (e.g., SLC transporters, NOTCH and AhR signaling path-
recognition subunit, being FBXW7 one of the most well studied ways, Hedgehog pathway). Subsequently, NOTCH signaling
F-box proteins. FBXW7 is a tumor suppressor that targets multi- pathway genes were selected for estimation of their genetic profile
ple oncoproteins for ubiquitination-mediated proteolysis, includ- in ovarian carcinoma patients. High inter-individual variability of
ing cyclin E, c-MYC, c-JUN or MCL1. For this reason, gene expression from NOTCH pathway was found and particular
approximately six percent of all human cancers harbor FBXW7 associations of expression with prognosis of ovarian carcinoma
mutations. In addition to his role in cancer, FBXW7 also plays a were also identified. NOTCH signaling pathway was suggested as
key role in metabolism and other processes. We have previously potential therapeutic target in ovarian carcinoma therapy suitable
demonstrated that SCF(FBXW7) modulates the intra-S-phase for further research. Supported by the research grants CSF no.
DNA-damage checkpoint by regulating PLK1 stability. We P303/12/G163, AZV no. 15-25618A and NIH/CA103314.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 317
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
318 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
mitosis. Timely release of separase is essential for the proper sis- gene expression analysis of EGFR and ERBB2, was performed
ter chromatids separation. Securin also acts as a transcriptional using real-time reverse transcription– polymerase chain reaction.
regulator for more than a thousand genes mainly involved in As a result, we observed that A. calamus and A. graveolens
proliferation, cell cycle progression and DNA repair. It is chiefly extracts affected EGFR and ERBB2 levels in LNCaP cells.
expressed in the G2 phase of cell cycle. We discovered a novel
minor isoform of securin, which lacks exons 3 and 4. These exons
contain the main APC recognition site (D-box), which is neces- P.10-077-Tue
sary for the swiftness of degradation. The presence of the new Apium graveolens extract induces apoptosis
short isoform mRNA is confirmed in multiple cancer cell lines via Bax and p-53 proteins in the LNCaP human
and primary cells by qRT-PCR at levels 0.4–2% of the major prostate cancer cell line
isoform. Preliminary results obtained on synchronized cells sug-
T. K€oken1, B. Koca1, T. Koca2, K. Altunbas3
gest that the short isoform has higher mRNA level in some 1
Afyon Kocatepe University, School of Medicine, Department of
phases of the cell cycle as compared to the full-length isoform.
Clinical Biochemistry, Afyonkarahisar, Turkey, 2Afyon Kocatepe
We have shown that overexpression of the short isoform acti-
University, Ataturk Vocational School of Health Services,
vates transcription of several growth factors, for instance FGF2
Department of Medical Laboratory, Afyonkarahisar, Turkey,
and VEGFA and cyclins such as CCND3. However, the absence 3
Afyon Kocatepe University, Faculty of Veterinary Medicine,
of the DNA-binding domain in the short isoform of securin leads
Department of Histology and Embryology, Afyonkarahisar,
to its inability to affect expression of some critical cell cycle regu-
Turkey
lators, for example MYC and TP53. Our preliminary data sug-
gest that short isoform has transcriptional effect on a distinct set Apium graveolens (A. graveolens) have shown significant antipro-
of genes. We propose that this functional dissimilarity from the liferative and apoptotic properties in various types of cancer cells,
major isoform mainly stems from the difference in their expres- including prostate cancer cells. However, the mechanism by
sion throughout the cell cycle. This work is supported by the which A. graveolens induces apoptosis is not completely under-
Program of fundamental research for state academies for 2013– stood. In this study, LNCaP cells were treated with increasing
2020, research topic 01201363823. concentrations of an ethanolic extract of A. graveolens (1500 lg/
ml, 2000 lg/ml and 2500 lg/ml) and viability was determined
after 24 and 48 hr using the XTT cell proliferation assay. The
P.10-076-Mon levels of cleaved poly (ADP-ribose) polymerase (PARP), one of
Effect of Acorus calamus and Apium the best biomarkers of apoptosis, were analyzed. Finally, protein
graveolens extracts on Egfr and Erbb2 in content of Bcl-2, Bax and p53 were measured. We found that
A. graveolens extract inhibited the proliferation and viability of
LNCaP cells
human prostate cancer LNCaP cells via induction of apoptosis.
H. B. Koca1, G. Sadi2, T. Koken1, T. Koca3
1 LNCaP cells showed clear apoptosis within 48 h after A. grave-
Department of Medical Biochemistry, Faculty of Medicine, Afyon
€ Science olens (2000 and 2500 lg/ml) treatment. We observed no changes
Kocatepe University, Afyonkarahisar, Turkey, 2K.O.
in Bcl-2 protein levels when increased Bax and p53 protein levels
Faculty, Department of Biology, Karamanoglu Mehmetbey
48 h after A. graveolens (2000 and 2500 lg/ml) treatment. These
University, Karaman, Turkey, 3Department of Medical
results indicate that the induction of apoptosis by A. graveolens
Laboratory, Ataturk Vocational School of Health Services, Afyon
may be mediated by induction of proapoptotic mechanism rather
Kocatepe University, Afyonkarahisar, Turkey
than suppression of antiapoptotic mechanisms.
Prostate cancer is one of the most commonly diagnosed cancers
in men of developed Western countries. Several treatments are
available for treatment of prostate cancer. These include surgery, P.10-078-Wed
radiation, radioactive implants and hormonal therapy. However, Characterization of the ubiquitin ligase for
the treatment often impacts the quality of life due to side-effects S6K2 may overcome apoptotic barrier for
or complications. Thus, numerous investigators have focused on breast and lung cancer cells
discovering novel drugs or treatments. Among all the agents
N. Sever, S. Cengiz Sahin
tested, natural products derived from medicinal plants are among
Pamukkale University, Denizli, Turkey
the most favorable. Migration and invasion of cancer cells is reg-
ulated by multiple pathways that employ various growth factor The younger member of S6 Kinase family, S6 Kinase 2 (S6K2)
and their receptors, integrins and cytoskeletal elements. A key was displayed to have anti-apoptotic roles in breast and lung can-
role is played by the EGF receptor (EGFR), which, following cer cells. S6K2 was shown to complex with anti-apoptotic pro-
interaction with the integrin a6b4, promotes cell migration teins BRAF and protein kinase C-epsilon (PKCe) in Fibroblast
through activation of PI3K and other downstream pathways. Growth Factor (FGF-2)-stimulated non-small cell lung cancer
The androgen receptor can be activated indirectly by growth fac- cells (NSCLC). This complex helps NSCLC cells to evade apopto-
tor receptors, mainly the transmembrane tyrosine kinases EGFR sis [1]. In another study, S6K2 was displayed to use an indirect
and ERBB2, members of the epidermal growth factor receptor way to escape from apoptosis in HEK 293T cells. S6K2 phospho-
(EGFR) family, which are activated inappropriately in many rylates and therefore degrades PDCD4, a tumor suppressor pro-
human cancer types. It has been reported that the EGF receptors tein. The disappearance of PDCD4 yields to the translation of
EGFR (HER-1) and ERBB2 (HER-2/neu) are overexpressed in anti-apoptotic proteins Bcl-XL and XIAP [2]. Two studies by
metastatic prostate tumors. In this study, we investigated the Basu and Sridharan uncovered the role of S6K2 in breast cancer
effect of A. Calamus and A. Graveolens extracts on Egfr and cell survival. S6K2 downregulation inactivates Akt in MCF-7 cells
Erbb2 on the human prostatic carcinoma cell line LNCaP. and decreases the level of Mcl-1 in T47D cells. Therefore, apopto-
LNCaP cells were treated with increasing concentrations of an sis of MCF-7 and T47D cells induced by TNF and TRAIL,
ethanolic extract of A. graveolens ranging from 1000 to 3000 lg/ respectively, is stimulated. In the former study, S6K2 was dis-
ml, A. Calamus 250 to 750 lg/ml and viability was determined played to inactivate Bid, a proapoptotic protein as well [3,4]. All
after 24 and 48 h using the XTT cell proliferation assay. The the aforementioned studies point out the importance of regulation
levels of EGFR and ERBB2 were analyzed. Finally, quantitative of steady-state level of S6K2 in a cell. The acetylation of S6K2 by
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 319
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
the acetyltransferase p300 contributed to the stability of S6K2 pro- migration and apoptosis. They play biological roles that are
tein [5]. Although ROC1 is known to be the ubiquitin ligase for related both to tumor suppression and progression. Bioinformatic
S6K1 [6], the ubiquitin ligase for S6K2 still remains to be discov- analysis of high-throughput sequencing data generated by The
ered. Therefore, characterization of the ubiquitin ligase responsible Cancer Genome Atlas project (TCGA) using original CrossHub
for S6K2 degradation promises a very important potential in software revealed that expression of CTDSP1/2/L genes is often
induction of apoptosis in breast and lung cancer cells. A large-scale reduced in tumors as compared to normal tissues. This data was
interactome analysis of S6K2 by Pavan et al. [7] reveals that confirmed in the study of clinical specimens of clear cell renal cell
TRIM25 might be a candidate for the ubiquitin ligase of S6K2. We carcinoma (N = 32, paired samples) using qPCR, which showed
aim to search and find out the ubiquitin ligase of S6K2 to overcome frequent and significant down-regulation of CTDSP1/2/L genes.
the apoptotic barrier for breast and lung cancer cells. For further analysis, protein-coding DNA sequences of the genes
CTDSP1/2/L were cloned into the pT2/HB vector and trans-
fected into Caki cells by electroporation together with the plas-
P.10-079-Mon mid pSB100 encoding the Sleeping Beauty transposase. Caki is a
Specific effectors of topoisomerase II poisons human clear cell renal cell carcinoma line that displays epithelial
contribute to cytotoxicity in AML cell lines morphology and grows as an adherent culture. These cells are a
P. More, U. G€odtel-Armbrust, L. Wojnowski useful preclinical model to study renal cancer. Exogenous expres-
Department of Pharmacology, University Medical Center, sion of CTDSP1/2/L inhibited (P ≤ 0.01) growth of renal cancer
Johannes Gutenberg University Mainz, Mainz, Germany cells (Caki) in vitro. Proportion of cells in the G1 phase of cell
cycle was increased. The size of the transfected cell colonies was
The use and efficacy of topoisomerase II (TOP2) poisons, such decreased in most cases, the cells became enlarged and less
as anthracyclines and epipodophyllotoxins, is constrained by mobile. A partial mesenchymal-epithelial transition was observed.
indiscriminate toxicity towards normal cells and by the resistance These findings demonstrate that CTDSP1/2/L genes can play the
of cancer cells. There is a need for drugs with similar efficacy but role of tumor-suppressors in renal cancer. Study of cell growth
higher specificity. To identify targets of such drugs, we first inves- suppression was supported by the Program of fundamental
tigated the relevance of pre-treatment basal gene expression levels research for state academies for 2013–2020 years (№
(GEL) in response to the epipodophyllotoxin etoposide in 11 01201363823, № 01201363819) and gene expression analysis was
AML (Acute Myeloid Leukemia) cell lines. We identified both supported by grant 14-50-00060 of the Russian Science Founda-
genes positively correlating (assisting) and negatively correlating tion.
(impeding) with the etoposide IC50. The impeding genes BIRC7,
ESRRB and PARP9 exerted effects synergistic with etoposide, as
demonstrated using inhibitors. Furthermore, gene induction pre- P.10-081-Wed
dominated among etoposide-evoked gene expression changes Salivary squamous cell carcinoma antigen 1
(GEC) in these AML cell lines. By integrating public resources
and 2 in oral cancer patients: a pilot study
Project Achilles and Connectivity Map, we predicted 111 GEC
I. Karmelic1, I. Salaric2, K. Bazdaric3, J. Lovric1, D. Macan2
essential for AML cell survival. Out of the five essential GEC 1
University of Zagreb, School of Medicine, Department of Medical
investigated in more depth, BCL2A1, IGF1R and PLK1 exerted
Chemistry, Biochemistry and Clinical Chemistry, Zagreb, Croatia,
cytotoxic effects when targeted with inhibitors. We also identified 2
University of Zagreb, School of Dental Medicine, University
genes, modulation of which results in GEC either similar (TOP2-
Hospital Dubrava, Department of Oral & Maxillofacial Surgery,
like targets) or opposite (TOP2-contrary targets) to those evoked
Zagreb, Croatia, 3University of Rijeka, School of Medicine,
by etoposide. Functional validation using inhibitors revealed
Department of Medical informatics, Rijeka, Croatia
effects synergistic with etoposide for TOP2-contrary targets
ROCK1 and HDAC9. Altogether, these results demonstrate that According to the World Health Organization, oral cancer (OC) is
pre-treatment GEL and drug-evoked GEC of some genes con- the eighth most common cancer in the world, with a five-year
tribute to the cytotoxic effects of etoposide. These genes may survival rate of 50%. Squamous cell carcinoma antigens (SCCA)
serve as targets for etoposide dose reduction, or for overcoming are part of the family of inhibitory serine protease inhibitors (ser-
resistance to this drug. This approach is applicable to other anti- pins), involved in inflammation, apoptosis, cell migration and
cancer drugs interfering with DNA integrity and gene expression. invasiveness and expressed in the squamous cell epithelium.
SCCA2 protein is 92% homologous to SCCA1, however these
antigens are not expressed concurrently and play different roles
P.10-080-Tue in the human body. Apart from inactivation of certain enzymes,
Serine phosphatases of the CTDSP/SCP family their role in the healthy and malignant tissues is not clearly
show tumor-suppressing activity for kidney understood. The origin of SCCA antigens in serum and body flu-
ids remains unclear. Unstimulated whole saliva (UWS) and stim-
tumors: bioinformatic approach, study of
ulated whole saliva (SWS) was sampled from 10 patients with
clinical samples and experiments in vitro OC (2 female, 8 male) and 15 control subjects (6 female, 9 male).
G. A. Puzanov1, E. B. Dashinimaev2, G. S. Krasnov1, Only patients with histologically diagnosed oral squamous cell
A. D. Beniaminov1, K. S. Vishnyakova1, M. A. Afanasyeva1, carcinoma were included in the study. Sandwich human SCCA1
T. T. Kondratieva3, V. N. Senchenko1, Y. E. Yegorov1 and SCCA2 ELISA Kits, My BioSource, San Diego, USA, were
1
Engelhardt Institute of Molecular Biology, Russian Academy of used. Respondents’ alcohol consumption, papilla bleeding index
Sciences, Moscow, Russia, 2Koltzov Institute of Developmental (PBI), smoking, drug consumption and medical condition was
Biology of Russian Academy of Sciences, Moscow, Russia, registered. This research was funded by the Croatian Science
3
Blokhin National Medical Research Center of Oncology of the Foundation (IP-2014-3796). Salivary SCCA1 was significantly
Russian Ministry of Health, Moscow, Russia higher in OC patients, both in UWS and SWS (Mann Whitney U
Proteins of the CTDSP/SCP (Small C-terminal domain Phos- test, U = 14, P = 0.0004). Although no statistically significant dif-
phatase) family, which include CTDSP1, CTDSP2, and CTDSPL ference in SCCA2 levels in UWS and SWS between the OC and
(CTDSP1/2/L), are involved in a variety of cellular and biologi- control group was found (U = 43, P = 0.081), the average value
cal processes including cell proliferation, differentiation, in the control group was 895.92 pg/ml, while in OC patients
320 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
319,45 pg/ml. Due to the small sample size, smoking, PBI, medi- P.10-084-Wed
cal conditions, drug and alcohol consumption had no statistical
A link between alternative splicing regulation
significance. Salivary SCCA1 could serve as a satisfactory bio-
marker for OC. However, a greater sample is needed to establish and the efficacy of cancer treatment
the diagnostic value of the investigated biomarkers. To our K. Anufrieva1, V. Shender1, G. Arapidi1, M. Pavlyukov2,
knowledge, SCCA1 and SCCA2 have not yet been measured in P. Shnaider1, M. Lagarkova1, V. Govorun1
1
the saliva of OC patients. Federal Research and Clinical Center of Physical-Chemical
Medicine of Federal Medical Biological Agency, Moscow, Russia,
2
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the
P.10-082-Mon Russian Academy of Sciences, Moscow, Russia
Oncogenic potential of Wip1 and its role in Dysregulation of pre-mRNA splicing often occurs in cancer cells
modulation of DNA damage response in and many studies report about an interplay between alternative
human non-hodgkin lymphoma (hNHL) splicing and cancer resistance to chemotherapy. However, most
H. Pilevneli1, M. Kılıc Eren1, N. B. Kartal2, D. Kozacı3, of these studies were focused on the functions of specific isoforms
F. D€oger4 rather than global changes in the transcriptome. In our study, we
1
Adnan Menderes University, Faculty of Medicine, Department of revealed that different types of chemotherapy lead to similar
Medical Biology, Aydin, Turkey, 2Adnan Menderes University, changes in alternative splicing. Moreover, the most pronounced
Faculty of Medicine, Department of Clinical Biochemistry, Aydin, alternative splicing differences after exposure to various types of
Turkey, 3Yildirim Beyazit University, Department of Biochemistry, chemotherapy were observed for intron retention of splicing-
Ankara, Turkey, 4Adnan Menderes University, Faculty of related genes. To reveal the mechanism underlying this effect, we
Medicine, Department of Medical Pathology, Aydin, Turkey analyzed changes in gene expression, abundance of proteins,
phosphorylation level after exposure of ɣ-irradiation, hypoxia
Wild-type p53-induced phosphatase 1 (Wip1/PPM1D), is a serin/ and 10 different types of chemotherapeutic drugs. After treat-
threonine phosphatase induced upon DNA damage in a p53- ment of cancer cells with the various type of chemotherapies, we
dependent manner. Upon induction, Wip1 dampens the cellular demonstrated a decrease the number of spliceosomal proteins by
stress responses by dephosphorylating multiple proteins including multiple mechanisms, such as intron retention, reduced gene
p53, Chk1, CHK2, H2AX, ATM/ATR. Wip1/PPM1D is overex- expression, phosphorylation, and extracellular export. We con-
pressed, amplified and mutated in human solid tumors hence dis- clude that a decrease of active spliceosomal protein levels in
playing typical oncogenic properties. However, in hematological response to stress is aimed at cell cycle arrest. We demonstrated
cancers, so far no data has shown whether Wip1 has an onco- a strong interplay between splicing- and cell cycle-related genes
genic potential. Thus, we evaluated the role of Wip1 in modula- in stressed cancer cells at the expression and processing levels.
tion of cellular stress responses as well as its oncogenic potential Disturbance of the splicing components by these processes can
in human NHL cell lines and specimens, respectively. Therefore, benefit the survival of cancer cells after chemotherapy. We
human FFPE NHL and non-cancer lymphoid tissue as well as showed that this mechanism can be inhibited by Pladienolide B,
cell lines were assessed for mRNA and protein levels of Wip1 by which significantly increased the sensitivity of cancer cells to cis-
qRT-PCR, WB and immunohistochemistry analysis. PPM1D platin treatment. We suggest that Pladienolide B is a potential
locus was analysed by Taqman gene copy number assay. PPM1D therapeutic drug improving the efficiency of cancer therapy. Also,
exons were amplified by PCR and subjected to sequencing analy- we found that dysregulation of pre-mRNA splicing in splicing-
sis. Apoptosis was measured by Annexin V and Caspase3/7 related genes is evolutionary conserved and could be observed in
assays. RNA interference was used to target Wip1. Here we show other organisms. This work was supported by the Russian
for the first time Wip1 overexpressing NHL cell lines maintain Science Foundation 17-75-20205.
resistance to cell cycle arrest and apoptosis through decreased
phosphorylation of ATM, ATR, Chk1, Chk2 and p53 in
response to etoposide or doxorubicin treatment. Specific knock P.10-085-Mon
down of Wip1 significantly induced phosphorylation of Chk1, Hsp70-GAPDH complex is a potential target for
Chk2, p53, ATM and increased apoptosis in cell lines expressing
rat glioma therapy
wt p53. Moreover, Wip1 is overexpressed and amplified in
V. Lazarev, A. Nikotina, E. Mikhaylova, I. Guzhova,
human NHL specimens. Overexpression of Wip1 was indepen-
B. Margulis
dent of p53 status of tumors. Further, heterozygous mutations
Institute of Cytology RAS, St. Petersburg, Russia
were detected within exon 6 of Wip1 gene in particular in diffuse
large B cell lymphoma. Hence, Wip1 may have an oncogenic Hsp70 protein carries a protective function in the cell. It
potential in hematological cancers in particular in NHL which increases the resistance of cells to stress of different types, includ-
may play an important role in the initiation, progression or ther- ing suppressing toxic effects of anticancer drugs. In cancer cells,
apeutic outcome of the disease. (This work is supported by Hsp70 is able to bind damaged proteins and peptides and take
TUBITAK Gr. No. 214S200 to MKE). part in ubiquitination processes. Its other function – binding pro-
teins involved in apoptotic signaling. One of the proteins sensitive
to oxidative stress in the cell is the enzyme glyceraldehyde 3-
phosphate dehydrogenase (GAPDH); this protein is capable of
forming insoluble intracellular aggregates and induce apoptosis.
According to our earlier data Hsp70 is able to bind GAPDH.
We hypothesized that in oxidative stress conditions Hsp70 can
increase cell survival by binding to oxidized GAPDH. Work was
carried out on the rat glioma cell line C6. For the analysis of
GAPDH aggregates we used confocal microscopy and filter trap
assay. The interaction of Hsp70 and GAPDH was monitored by
enzyme linked immune sorbent assay and immunoprecipitation.
Toxic effects of oxidative stress was evaluated using the CytoTox
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 321
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
96 kit (Promega). Using rat glioma cell line C6, we have found P.10-087-Wed
that in response to oxidative stress intracellular GAPDH dena-
The dose-dependent effects of brassinin on
tures and cells die. Using tests developed in our laboratory, we
found that Hsp70 can bind to oxidized GAPDH in ATP-depen- apoptosis in SW480 colorectal cancer cells
dent manner. Varying the level of hsp70 expression by triptolide through MMP-9 related pathway
(expression inhibitor) or U133 (a derivative of echinochrome, €
E. Ozkan, F. Bakar-Ates
expression inducer) demonstrated that the number of GAPDH Ankara University Faculty of Pharmacy Department of
aggregates and cellular resistance to oxidative stress depends on Biochemistry, Ankara, Turkey
intracellular Hsp70 level: the greater the Hsp70, the smaller Brassinin (BSN) is an essential indole phytoalexin first identified
aggregates of GAPDH and less mortality. These results suggest as a constituent of Chinese cabbage. Studies have reported signif-
that to reduce the resistance of cancer cells to oxidative stress it icant roles of brassinin including chemopreventive, antiprolifera-
is advisable to use chemical preparations – inhibitors of Hsp70 tive, antifungal and anticarcinogenic activities against lung
synthesis. This work was supported by grant of Russian Science carcinoma. Furthermore, brassinin showed strong antiprolifera-
Foundation № 14-50-00068. tive effects on several human cancer cell lines. The present study
aimed to evaluate the cytotoxic and apoptotic effects of brassinin
in SW480 human colorectal cancer cells through MMP-9 related
P.10-086-Tue
pathway. Cytotoxicity of the compound was determined by MTT
Construction of an apoferritin nanoparticle test and apoptotic effects were evaluated by annexin V binding
bearing ellipticine and a study of release of and fluorescence imaging methods. The alteration in mRNA
this drug from the nanocarrier expression of MMP-9 in brassinin treated cells was measured by
R. Indra1, M. Wilhelm1, Z. Heger2, S. Dostalova2, V. Adam2, real-time PCR method. Results showed that brassinin signifi-
M. Stiborov a1 cantly induces cell death at concentrations of 200 lM and 50 lM
1
Department of Biochemistry, Faculty of Science, Charles following 24 and 48 h of treatment, respectively. According to
University, Prague 2, Czech Republic, 2Faculty of Science and cell cycle analysis, brassinin induced a cell cycle arrest at G0/G1
CEITEC – Central European Institute of Technology, Masaryk level. Apoptosis detection assays also showed that brassinin dis-
University, Brno, Czech Republic plays a potential apoptotic effect on SW480 cells and it correlates
with the decrease of enzyme activity and mRNA expression of
Ellipticine (Elli) is an anticancer agent exhibiting high efficiencies
MMP-9 protein, which has an important role on metastasis of
in antineoplastic action. There are, however, several phenomena
cancer cells. Taken together, these results confirm that brassinin
that can cause a limited usage of ellipticine and/or its limited
has a significant apoptotic and anti-metastatic activity against
anticancer efficiencies. Because of its adverse effects, novel meth-
SW480 colorectal cancer cells and could be considered as a
ods of targeting the drug to cancer cells are being investigated.
potential therapeutic candidate.
Nanocarriers are a suitable platform for this specific delivery.
Apoferritins (APO), which are responsible for the storage and
transfer of iron, can provide these much needed properties. APO P.10-088-Mon
protein subunits assemble to form a hollow cage into which
Cobalt(II) complexes with various ligands
diverse substances, such as drugs, can be placed. It was shown
that while disassembled, APO can be mixed with drug molecules express different cytotoxic activity
and they are encapsulated within APO cavity once reassembled. A. Abudalleh1, D. Dinev1, T. Zhivkova1, M. Georgieva2,
In this study, the simple-to-use encapsulation protocol to con- G. Miloshev2, I. Pantcheva3, M. Miteva3, G. Marinescu4,
struct naturally found protein nanocarrier apoferritin encapsulat- D. Culita4, L. Patron4, R. Tudose5, O. Costisor5,
ing ellipticine (creating a nanocarrier construct ApoElli) was R. Alexandrova1
1
developed and the prepared nanocarrier was characterized. The Institute of Experimental Morphology, Pathology and
visualization of nanocarrier prior to removal of released ellip- Anthropology with Museum, Bulgarian Academy of Sciences,
ticine was performed using transmission electron microscopy Sofia, Bulgaria, 2Institute of Molecular Biology, Bulgarian
(TEM). The nanocarrier exhibits narrow size distribution suggest- Academy of Sciences, Sofia, Bulgaria, 3Faculty of Chemistry and
ing to be suitable for entrapping of the hydrophobic molecule of Pharmacy, Sofia University “St; Kliment Ohridski”, Sofia,
ellipticine. Ellipticine is gradually released from its ApoElli form Bulgaria, 4Institute of Physical Chemistry “Ilie Murgulescu”,
into the water environment under acidic pH; more than 80% Romanian Academy, Bucharest, Romania, 5Institute of Chemistry,
ellipticine was released after 48 hrs incubation at pH 6.5 at 37°C. Romanian Academy, Timisoara, Romania
In contrast, ApoElli is stable after its storage at physiological pH Demand for effective new anti-tumor agents is one of the major
(7.4) up to 1 month at 4°C; less than 5% ellipticine was released challenges facing modern biomedicine and medical chemistry. Par-
after this storage. Furthermore, the prepared ApoElli nanocarrier ticulary promising in this respect are the compounds of essential
is capable of the interaction with microsomal subcellular particles metals that act as biological response modifiers, such as cobalt.
resulting in its release and subsequent oxidation by microsomal The aim of our study was to evaluate the cytotoxic activity of ten
cytochromes P450 to its hydroxylated metabolites. This nanopar- Co(II) complexes with various ligands: i) Mannich bases N,N’-bis
ticle form of ellipticine is considered to be promising tool to be (4-antipyrylmethyl)-piperazine (BAMP) and N,N’-tetra-(antipyryl-
used in cancer treatment. Supported by GACR 17-12816S. l-methyl)-1,2-diaminoethane (TAMEN) – Group A; ii) Schiff
bases derived from condensation reactions between o-vanillin and
S-tyrosine, L-threonine, DL-tryptophan or L-serine – Group B;
monensic acid – Group C. The following cell lines served as model
systems in our investigations: human MCF-7 (luminal A type
breast cancer), HepG2 (hepatocellular carcinoma), 8MGBA
(glioblastoma multiforme); rat – LSR-SF-SR (sarcoma induced by
SR-RSV) and chicken – LSCC-SF-Mc29 (hepatoma induced by
the myelocytomatosis virus Mc29). The compounds were applied
322 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 323
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
P.10-094-Mon
POU2F1(Oct-1) isoforms contribute to
hematopoiesis in human and the variation in
the structure of their N-terminal parts results
in different patterns of genes regulated by
them
E. Pankratova1, A. Stepchenko2, I. Krylova3, T. Portseva4,
S. Georgieva2
1
Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences, Moscow, Russia, 2Engelhardt Institute of Molecular
Biology, Russian Academy of Sciences, Moscow, Russia, Moscow,
Russia, 3Moscow State Pedagogical University, Moscow, Russia,
Moscow, Russia, 4Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences, Moscow, Russia, Pirogov Russian
National Research Medical University (RNRMU), Moscow,
Russia, Moscow, Russia
The multiplicity of functions fulfilled by the transcription factor
POU2F1(Oct-1) may be accounted for by the existence of iso-
forms which result from the transcription from alternative pro-
moters and alternative splicing. We have studied the expression
of the Oct-1 isoforms differing by their N-terminal parts: Oct-1A
324 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
(GenBank: NM_002697), Oct-1L (AY113189), Oct-1X was also able to arrest the cell cycle in G2/M phase in HeLa
(KT438684.1), Oct-1Z (AK091438.1) during the hematopoiesis cells. The high phototoxicity and low dark toxicity make the
using human hematopoietic progenitor cells (HPCs) (CD34+), B- newly prepared colchicine-iodo-BODIPY conjugate a suitable
cells (CD19+) and T-cells (CD3+), and monocytes (CD14+). candidate for use in PDT and an effective tool for multimodal
Using RT-PCR, we revealed high levels of Oct-1A,L mRNAs, therapy.
and low level of Oct-1X mRNAs in the HPCs. The differentia-
tion of HPCs to B and T cells and monocytes led to an increase
in Oct-1X and Z levels. The dramatic decrease in the Oct-1L P.10-096-Wed
mRNA levels was observed in T cells and monocytes. Oct-1 iso- The investigation of antimetastatic effects of
form expression levels also changed during the in vitro differenti- colchicine through MMP-9 downregulation in
ation of the HL-60 cells to granulocytes induced by DMSO. The MCF-7 breast cancer cells
differentiation of HL-60 caused a decrease in the mRNA levels
E. Kaya-Sezginer, N. Ozmen, F. Bakar-Ates
for the Oct-1A,X,Z, as well as for the surface antigen CD14+,
Ankara University, Faculty of Pharmacy Department of
while it led to a considerable increase in the Oct-1L expression
Biochemistry, Ankara, Turkey
level. In such a way, both in vivo and in vitro differentiation of
cells is associated with the dynamic changes in the Oct-1 isoform Colchicine is an alkaloid agent and it’s been clinically used for a
composition and relative ratio. We have shown that the variation long time because of its microtubule destabilizer effect. Beside its
in the structure of N-terminal parts of the Oct-1 isoforms results strong binding capacity to tubulin to perturb the assembly
in the difference in the patterns of genes regulated by them. Our dynamics of microtubules, colchicine may also increase cellular
findings indicate that during hematopoiesis, Oct-1 isoforms show free tubulin and limit mitochondrial metabolism in cancer cells.
cell type-specific expression, and the regulatory interplay between Since the use of colchicine is limited because of its toxicity, the
the Oct-1 isoforms contributes to hematopoietic cell differentia- studies targeting to demonstrate the significant effects of the drug
tion. The work was supported by the Fundamental Research at lower doses gain importance in cancer investigations. This
Program for State Academies for the years 2013–2020 (№ study is planned to evaluate the effects of colchicine on prolifera-
01201363822), the Russian Science Foundation (№ 14-15-01032- tion, apoptosis and metastasis of MCF-7 human breast carci-
P) (Oct-1 expression in hematopoiesis and microarrays). noma cells. In this study, proliferation of MCF-7 cells was
determined by MTT assay. Annexin V binding, fluorescence
imaging studies and multi-caspase assay were performed to evalu-
P.10-095-Tue ate apoptosis. The expression levels of MMP-9 as a metastatic
Novel photosensitive conjugates as a tool for enzyme were measured by real-time PCR. We found colchicine
multimodal therapy significantly inhibited cell proliferation at 10 lg/ml and higher
V. Pavlıckova1, S. Rimpelova1, M. Jurasek2, I. Krızova1, concentrations when compared to nontreated control (P < 0.001)
P. Drasar , T. Ruml1
2 and it has induced cell cycle arrest at G2/M phase. Colchicine
1
Department of Biochemistry and Microbiology, University of has also induced apoptosis of MCF-7 cells by increasing Annexin
Chemistry and Technology Prague, Technick a 3, 166 28, Prague 6, V binding and caspase levels in a dose dependent manner. There
Prague, Czech Republic, 2Department of Chemistry of Natural was a significant decrease in IjBa levels in colchicine treated
Compounds, University of Chemistry and Technology Prague, group, whereas we didn’t find any difference in the expression of
Technick a 3, 166 28, Prague 6, Prague, Czech Republic NF-jB protein among treated and nontreated groups. Colchicine
induced the downregulation of MMP-9 protein in a dose depen-
Currently, there are many methods to treat cancer, the most uti- dent manner. This study indicates that colchicine has promising
lized of which is chirurgical dissection of a tumor tissue, effects on MCF-7 cancer cells at lower concentrations and may
chemotherapy, and radiotherapy. It has been shown that the be a candidate compound for the treatment of metastatic breast
most effective treatment method is a combination of several cancer.
approaches, so-called multimodal therapy. In this study, we have
employed this principle of dual/multimodal effect in the develop-
ment of a cytotoxic agent combined with photosensitizer (PS) for P.10-097-Mon
photodynamic therapy (PDT) of cancer. PDT belongs among Requirement of the IRE1-XBP1 axis for long-
noninvasive methods of cancer treatment. Its principle lies in term hypoglycemia-induced reversible
photoactivation of PS in the presence of molecular oxygen. After
that, reactive oxygen species are generated, which leads to the cell resistance to anticancer therapies
death. in order to reach this goal, we prepared a series of conju- M. Jeong1,2, M. Lee1,2, S. Jo1,2, S. Park1,2, J. Sohn1,2,
gates of iodo-BODIPY will colchicine, which is the inhibitor of Y. G. Park1,2
1
microtubule polymerization leading to cell cycle arrest in the G2/ Department of Biochemistry & Molecular Biology, Korea
M phase followed by apoptosis. Using live-cell fluorescent micro- University College of Medicine, Seoul, South Korea, 2Korea
scopy, we found that the newly synthesized conjugates localized Institute of Molecular Medicine and Nutrition, Korea University
predominantly in endoplasmic reticulum od MCF-7 and PC-3 College of Medicine, Seoul, South Korea
cells. Further, we determined cyto- and phototoxicity of the con- Cancer cells survive in harsh microenvironments that are influ-
jugates as well as of pure colchicine and pure BODIPY/iodo- enced by various factors including pH, hypoxia and nutrient
BODIPY in a panel of human cell lines. Any of the conjugates deprivation. Emerging evidence suggests that hypoxia is a cause
did not exhibit significant toxicity (without illumination) in can- of resistance of cancer cells to various anticancer therapies.
cer cell lines of LNCaP, PC-3, U-2 OS, HeLa, MiaPaCa-2, and Despite the accumulation of new knowledge, it is still unclear
MCF-7 up to 1 lM concentration. An iodo-BODIPY conjugate whether and how nutrient deficiency makes resistance to anti-
was very potent in terms of phototoxicity upon illumination with cancer therapies. In this study, human lung adenocarcinoma cells
the light of 450–800 nm (the light dose of 4Jcm2). After pho- (A549 and PC-9) were cultured in the medium containing differ-
toactivation, the iodo-BODIPY conjugate resulted in a 50% ent low concentrations of glucose. The cells which were cultured
decrease in cell proliferation already at 5 nM concentration for under hypoglycemic condition for more than 7 weeks became
HeLa cells and 10 nM for U-2 OS and PC-3 cells. This derivative resistant to cisplatin and taxol. Interestingly, it was also observed
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 325
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
that the resistant cells were resistant to immunotherapy, and their P.10-099-Wed
migration capacity was remarkably enhanced compared with
European HPV DNA test external quality
those of control group maintained under normal condition. Cis-
platin-induced apoptosis was diminished in hypoglycemic condi- assurance scheme (EHEQAS)
tion, but the reversion to normal glucose condition only for short P. Neophytou1, J. Konya2, C. Kroupis3, R. Tachezy4,5
1
term periods made the cells susceptible to cisplatin-induced apop- Mendel Center for Biomedical Sciences, Egkomi, Nicosia, Cyprus,
2
totic cell death. Among ER stress responses, BiP-XBP1 axis was Department of Medical Microbiology, University of Debrecen
increased by long-term hypoglycemia, whereas PERK axis was Medical Faculty, Debrecen, Hungary, 3Attikon University General
decreased. Inhibition of XBP1 splicing rendered the cells suscepti- Hospital, University of Athens Medical School, Athens, Greece,
4
ble to cisplatin and immunotherapy. This study demonstrated Department of Genetics and Microbiology, Faculty of Science,
that hypoglycemia-induced cisplatin resistance is reversible and Charles University, Prague, Czech Republic, 5Institute of
dependent upon IRE1-XBP1 pathway. This research was sup- Hematology and Blood Transfusion, National Reference
ported by the National Research Foundation of Korea (2016R1 Laboratory for Papillomaviruses and Polyomaviruses, Prague,
D1A1A0293, 2017R1D1A1B03036008 and 2013M3A9D3045881). Czech Republic
EHEQAS is a program of external quality control used to assess
and improve the quality of laboratories in HPV detection and
P.10-098-Tue typing. Batches of 5–7 samples are sent from the coordinator to
Effect of cisplatin treatment on protein participants 1–2 times per year. Samples are either real patient
expression of vitamin D metabolizing CYP24A1 samples or prepared from international standards. Samples that
in PC3 cell line are not international standards are pre-tested by reference labora-
€ Durukan2, M. Akkulak1, O. Adalı1
E. Evin1, O. tories. To test for reproducibility, samples are used in duplicate
1
Department of Biological Sciences, Middle East Technical in the same and in different rounds. Linearity is evaluated by dif-
University, Ankara, Turkey, 2Department of Biological Sciences, ferent dilutions of the same sample in the same and in different
Graduate Program of Biochemistry, Middle East Technical rounds. Results are evaluated and consensus results are issued
University, Ankara, Turkey and announced to participants in a confidential way. Marks are
awarded to participants based on defined rules that reflect the
Vitamin D is a sterol derivative, composed of four rings and clinical value of the result (e.g. higher penalty for errors regard-
found in two common forms; ergocalciferol (D2) and cholecalcif- ing types 16 and 18). Certificates of competence that reflect the
erol (D3). Vitamin D3 is produced in the skin and metabolized performance of a laboratory during the past 4 years are issued.
in liver and kidney by cytochrome P450 enzymes to its active During the period 2006–2017 a total of 233 samples have been
form (1,25-dihydroxycholecalciferol). This active metabolite is tested in 24 rounds: 65 negative, 67 single infections, and 101 co-
then inactivated to calcitroic acid by hydroxylation with infections. 32 different types (6, 11, 16, 18, 31, 33, 34, 35, 39, 40,
CYP24A1 enzyme. 1,25-dihydroxycholecalciferol binds to its 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 68, 70,
receptor and controls expression of several genes that have roles 73, 81, 82, 83, and 84) were detected. Surprisingly even laborato-
in differentiation, proliferation, and apoptosis of cancer cells. ries using IVD tests made significant errors in HPV detection
Because of this, vitamin D deficiency is considered as risk factor and typing, depending on the skills of laboratory personnel and
for cancer development and high rates of mortality. Cisplatin is a on whether they correctly followed manufacturer’s instructions.
chemotherapeutic drug used to treat wide variety of cancer types. EHEQAS improves quality with the coordinating team providing
The aim of this study is to examine the effect of cisplatin on pro- feedback to participants on how to improve their methodology.
tein expression of vitamin D metabolizing CYP24A1 on prostate EHEQAS assesses the quality of laboratories in: (a) detecting a
cancer cell line PC3. Cytotoxicity of cisplatin was determined shift in sensitivity and specificity over time. (b) HPV typing
with Alamar Blue Assay and IC50 value was calculated. The (high- or moderate- or low-resolution). Successful participation
effect of cisplatin on CYP24A1 protein expression was deter- in EHEQAS is extremely helpful to high-quality HPV labs that
mined by western immunoblotting technique. Results were nor- also have to verify or validate their methods: success in an EQA
malized with b-actin as an internal standard. The intensity of is an absolute prerequisite for granting ISO15189 accreditation.
each band was analyzed by Image J software. IC50 value of cis-
platin on PC3 cell line was found as 30 lM. CYP24A1 protein
expression was significantly decreased (0.66 fold) in cisplatin trea- P.10-100-Mon
ted PC3 cells with respect to control groups. The decreased pro- STING-related innate immunity pathways,
tein expression of CYP24A1 in cisplatin treated PC3 cells may
inflammation and mediators of invasive
result in increased active vitamin D metabolite. In conclusion,
due to downregulation of CYP24A1, cisplatin may have potential growth: transcriptome-wide profiling of HPV-
to prevent rapid inactivation of active vitamin D and this may positive microcarcinoma of the cervix
improve beneficial effects of vitamin D for cancer patients. O. Kurmyshkina, A. Bogdanova, P. Kovchur, T. Volkova
Institute of High-Tech Biomedicine, Petrozavodsk State University,
Petrozavodsk, Russia
Antitumor innate immunity pathways are inherently related with
angiogenesis and epithelial-mesenchymal transition (EMT), the
key determinants of epithelial tumor invasiveness, via common
mechanisms of inflammation and functioning of immunosuppres-
sive cells. We aimed to study which of these mechanisms may
undergo the most pronounced changes upon induction of inva-
sive growth in virus-related cervical cancer. By means of RNA-
seq, transcriptome-wide profiles of gene expression were obtained
for preinvasive and early-stage invasive cervical cancer; selected
markers of angiogenesis and EMT and tumor-infiltrating
326 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 327
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
proliferating nonmotile and noninvasive; JUN-3, rapidly prolifer- CASPASE-8, 9, 10, BID, P53, NOXA, TIMP-1 and TIMP2. It
ating, motile and invasive and JUN-2fos-3, with slow prolifera- was also found that FA has effect in MDAH 2774 cells on sup-
tion, but pronounced motility and invasiveness. This made us pressed invasion, migration and colony formation. In conclusion,
possible to identify two separate groups of genes tentatively we demonstrate that FA significantly affect cell cycle, apoptosis,
involved in sarcoma progression in a single transcriptomic invasion, migration and colony formation of MDAH 2774 cells.
analysis. Proliferation-related genes could be identified by their FA may be a potential candidate as chemotherapeutic agent for
differential expression in JUN-3 compared to both JUN-2 and the treatment of ovarian cancer. More studies have to be per-
JUN-2fos3. Motility and invasiveness-related genes showed com- formed to profile the mechanisms and genome wide effects of FA
mon expression pattern in JUN-2fos3 and JUN-3 cells compared to understand its therapeutic potential.
to JUN-2. The high-throughput gene expression analysis has
been performed using the GeneChip Mouse Genome 430 2.0
Array (ThermoFisher Scientific). We identified 277 up- and 212 P.10-105-Wed
down-regulated unique transcripts in JUN-2 and JUN-2fos3 Determination of metabolites and DNA
compared to the JUN3 cells, and 29 up- and 112 down-regulated adducts formed by nitro-reduction of a
unique transcripts in JUN3 and JUN2fos3 compared to JUN2 carcinogen aristolochic acid I in vitro
cells (adjusted P < 104). The chemokine Ccl-8 was identified as
M. Stiborova1, A. Hudecova1, K. Kotalik1, P. Hodek1,
a possible druggable target responsible for sarcoma motility, as it
V. M. Arlt2, H. H. Schmeiser3
is overexpressed in both motile cell lines and its pharmacological 1
Department of Biochemistry, Faculty of Science, Charles
inhibition significantly downregulated JUN-3 cell motility. Inter-
University, Prague 2, Czech Republic, 2Department of Analytical,
estingly, the scrutiny of differentially expressed genes in the
Environmental & Forensic Sciences, MRC-PHE Centre for
motile cell lines JUN-2fos3 and JUN-3 revealed a significant
Environment & Health, King’s College London, London, United
downregulation of several stemness genes like Abcg2 (21x reduc-
Kingdom, 3German Cancer Research Center, Heidelberg, Germany
tion) or CTGF (10x reduction). This is reminiscent of the phe-
nomenon “go or grow”, known for brain tumours. Our sarcoma Balkan endemic nephropathy (BEN) is a chronic tubulointersti-
cell lines could thus provide a valuable model to identify key tial nephropathy characterized by a gradual progression to end-
genes responsible for sarcoma progression as well as to decipher stage renal disease that is similar to another renal fibrosis, aris-
a relationship between stemness and cancer invasion. Supported tolochic acid nephropathy (AAN). A characteristic feature of
by the Czech Grant Agency project No 17-17636S and by the AAN and BEN is their close association with upper urothelial
project of Faculty of Medicine in Pilsen (grant no. SVV-2017- carcinomas (UUC). Aristolochic acid (AA), a plant alkaloid, has
260393). been shown to be the major cause of BEN/AAN/UUC develop-
ment. Nitro-reduction of AAI, the major component of AA, is
required to exert its genotoxicity (AA-DNA adduct formation)
P.10-104-Tue and carcinogenicity. Initial reduction of AAI to the correspond-
Determining the effects of ferulic acid on ing N-hydroxyaristolactam is the activation pathway responsible
apoptosis, cell cycle, invasion, migration and for this genotoxic effect. During this reaction AAI is enzymati-
cally reduced to the cyclic acylnitrenium ion that is capable of
colony formation in MDAH 2774 ovarian
binding to the exocyclic amino groups of adenine and guanine in
cancer cell line DNA. The most abundant DNA adduct detected in AAN and
M. Secme, L. Elmas, Y. Dodurga, N. Sirin, E. Demir BEN patients is 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-
Pamukkale University, School of Medicine, Department of AAI) which causes characteristic AT?TA transversion mutations
Medical Biology, Denizli, Turkey (e.g. in TP53 gene). NAD(P)H:quinone oxidoreductase and cyto-
Increasing understanding of the molecular biology of ovarian chromes P450 1A1 and 1A2 are the major enzymes reducing AAI
cancer has led to the development of numerous targeted therapies to N-hydroxyaristolactam I, the acylnitrenium ion, the final
such as cellular signalling pathway inhibitors, immunotherapies, reductive metabolite aristolactam I and forming also dA-AAI
antiangiogenic molecules and new effective novel anticancer adducts. Here, we investigated formation of the reductive inter-
agents. Ferulic acid is a common dietary plant phenolic com- mediates of AAI, the end reduction product aristolactam I and
pound abundant in fruits and vegetables and have powerful AAI-DNA adducts. Human NQO1 and hepatic cytosols rich in
antioxidant capacity and anti-cancer effects. The aim of this this enzyme were used. Whereas AA-DNA adducts measured by
study is to investigate the potential therapeutic effects of FA on 32P-postlabeling were formed by both used enzymatic systems,
cell proliferation, apoptosis, invasion, migration, colony forma- no reductive intermediates of AAI and aristolactam I measured
tion and wound-healing in MDAH 2774 human ovarian by HPLC have been detected. Two reasons can be responsible
endometrioid adenocarcinoma cell line. The cytotoxic effects of for the results; (i) a lower sensitivity of HPLC detecting AAI
FA were determined by XTT method. The mRNA expression metabolites than 32P-postlabeling determining AA-DNA adducts
analysis of BAX, BCL-2, BCL-XL, CASPASE-3, 8, 9, 10, BID, and (ii) reactive reductive intermediates of AAI can be trapped
CYLIND1, CDK6, P53, P21, FADD, PUMA, NOXA, MMP-2, by proteins in incubation mixtures, thereby forming AAI-protein
MMP-9, TIMP-1, TIMP2 was performed on Real-time PCR adducts. Supported by GACR 17-12816S
(RT-PCR) according to the SYBR Green qPCR Master Mix.
Effects of FA on cell invasion, migration and colony formation
were detected by Matrigel-chamber assay, wound-healing and
colony formation assay, respectively. The statistical analysis of
the findings has been made with the DDCT method with RT2
Profiler PCR Array Data Analysis. IC50 value of FA in MDAH
2774 cells was detected as 150 lM at 24th hours. It was deter-
mined that FA caused a decrease in the expression of BCL-2,
BCL-XL, CYLIND1, CDK6, PUMA. It is also observed that
FA caused a significant increase in the expression of BAX,
328 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
P.10-106-Mon enriched with ovarian cancer cells and a small amount of tumor-
associated immune cells (up to 5%) which create a unique
Investigation of the effect of flavonoid
microenvironment actively contributing to progression of the dis-
nobiletin on TLR4 signaling pathway in ease. However, it is remain unclear how cancer cells communicate
prostate cancer cell lines to its local environment under the influence of chemotherapy. To
€
A. Deveci Ozkan 1 €
, S. Kaleli1, H. I. Onen2 €
, O. Aksoy3, address this issue, we performed LC-MS/MS analyses of ovarian
H. N. Kaleli4, E. M. G€ uler5, A. Kalayci Yigin6, M. Akdogan7 cancer ascites from the same patients before and after chemother-
1
Sakarya University, Medicine Faculty, Department of Medical apy. Moreover, we investigated how ascites before and after
Biology, Sakarya, Turkey, 2Gazi University, Faculty of Medicine, chemotherapy modulates the behavior of cancer cells. In general,
Department of Medical Biology and Genetics, Besevler, Ankara, we identified 1775 proteins in tumor ascites. Intriguingly, a large
Turkey, 3Kocaeli University, Faculty of Arts and Sciences, number of known intercellular signaling mediators such as cytokines,
Department of Biology, Kocaeli, Turkey, 4Sabanci University chemokines and growth factors were decreased in abundance in
Faculty of Engineering and Natural Sciences Molecular Biology ascites samples after chemotherapy. These data were independently
Genetics and Bioengineering Program, Istanbul, Turkey, confirmed by cytokine profiling assay. Functional annotation of
5
Bezmialem Vakif University School of Medicine Department of upregulated proteins with the use of KEGG and GO databases
Medical Biochemistry, Istanbul, Turkey, 6Istanbul University, revealed that malignant ascites after chemotherapy were enriched
Cerrahpasa Medical Faculty, Department of Medical Genetics, with the cluster of spliceosomal proteins. These splicing factors were
Istanbul, Turkey, 7Sakarya university, Medicine Faculty, linked to induction of epithelial-to-mesenchymal transition leading
Department of Medical Biochemistry, Sakarya, Turkey to more aggressive phenotype of cancer cells. We showed that ascites
Toll-like receptors (TLRs) are a well-known family of pattern after chemotherapy increases cell mobility, induces the change of sur-
recognition receptors that expressed in epithelial, immune and face markers from epithelial (E-cadherin) to mesenchymal (N-cad-
cancer cells. Nobiletin is an O-methylated flavonoid that pos- herin) and finely promoting cancer chemoresistance. In summary,
sesses anti-cancer properties and has been reported to reduce the our study uncovers previously unrecognized signaling networks in
risk of prostate cancer. In this study, we investigated the effects the ovarian cancer microenvironment that are of potential clinical
of Nobiletin on TLR4 in LNCaP, PC-3 and HUVEC cell lines. relevance. This work was supported by the Russian Science Founda-
Lipopolysaccharide (LPS) was used for TLR4 stimulation. Cell tion 17-75-20205.
viability was analyzed with the WST-1 assay. Inhibitory concen-
trations (IC50) of Nobiletin were found 20 lM for LNCaP and
P.10-108-Wed
40 lM for PC-3 and HUVEC cell lines (P < 0.05). Gelatinase
activity and protein expression were examined by zymography Kaempferol sensitizes human hepatoma cells
and western blotting, respectively. Gelatinase activity of MMP-9 to TRAIL-mediated apoptosis through
and MMP-2 was found high in PC-3 and LNCaP, although there inhibition of Akt activity
was high MMP-2 activity in HUVEC, MMP-9 activity was not S. S. Park, S. Jeong, E. K. Choi
observed (P < 0.05). Also, it was found that Nobiletin reduced Asan Medical Center, University of Ulsan College of Medicine,
TLR4 and IRF-3 protein levels in LNCaP and PC-3 and there Seoul, South Korea
was no significant change in the amount of these proteins in
Kaempferol is polyphenolic compounds that are widely distributed
HUVEC (P < 0.05). TLR4 gene expression was examined by
in fruits and vegetables. Tumor necrosis factor (TNF)-related apop-
Real-Time Quantitative Reverse Transcription Polymerase Chain
tosis-inducing ligand (TRAIL) is a promising cancer therapy that
Reaction (qRT-PCR). It was observed that when Nobiletin and
preferentially induces apoptosis in cancer cells. However, many hep-
LPS were co-treated, TLR4 gene expression decreased in LNCaP
atoma cells are resistant to TRAIL by mechanisms that are poorly
(P < 0.05). Cytokines (INF-a and INF-b) were analyzed with
understood. Here, we show that treatment with tumor necrosis fac-
Enzyme-Linked Immunosorbent Assay (ELISA). It was found
tor (TNF)-related apoptosis-inducing ligand (TRAIL) in combina-
that Nobiletin reduced the amount of INF-a and INF-b in
tion with subtoxic doses of kaempferol significantly induces
LNCaP (P < 0.05). It is observed that TLR4 signaling pathway
apoptosis in TRAIL-resistant hepatoma cells. Western blot analysis
is suppressed by Nobiletin only LNCaP cells. When all the
showed that combined treatment with TRAIL and Kaempferol did
results are evaluated together; the effect of Nobiletin is AR-
not change the levels of DR5 or anti-apoptotic proteins (inhibitor of
dependent and shows a reducing effect on TLR4 signaling path-
apoptosis, Bcl-2 and Bcl-xL). However, Kaempferol induced potent
way. Conclusion, Nobiletin may be effective on prostate cancer
inhibition of Akt phosphorylation. Taken together, the present stud-
via TLRs. And also TLR4-dependent signaling pathway with
ies suggest that kaempferol enhances TRAIL-induced cytotoxicity
great potential may be important for new therapeutic approaches
by activating caspases and inhibiting phosphorylation of Akt.
in prostate cancers.
Kaempferol may be a safe strategy for treating resistant hepatomas.
P.10-107-Tue
P.10-109-Mon
Chemotherapy-induced microenvironment of
Mitochondrial 2HG production as a function of
ovarian cancer cells promotes tumor resistance
V. Shender1,2, P. Shnaider1,2, K. Anufrieva1,2,3, M. Pavlyukov1,
IDH2 and HOT in breast cancer cells
ckova1, L. Vıtek2, P. Jezek1
K. Smolkova1, A. Dvorak2, J. Spa
G. Arapidi1,2,3, M. Lagarkova2, V. Govorun1,2 1
1 Institute of Physiology, Department of Mitochondrial Physiology,
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS,
Czech Academy of Sciences, Czech Republic, Prague, Czech
Moscow, Russia, 2Federal Research and Clinical Center of
Republic, 2Institute of Medical Biochemistry and Laboratory
Physical-Chemical Medicine, Moscow, Russia, 3Moscow Institute
Diagnostics, 1st Faculty of Medicine, Charles University, Prague,
of Physics and Technology (State University), Dolgoprudny,
Czech Republic
Moscow Region, Russia
Cancer metabolic alterations result from complex genetic and epi-
Ovarian cancer is characterized by transcoelomic metastasis into
genetic adjustments which include mitochondrial pathways glu-
the peritoneal cavity. The peritoneal malignant ascites are
taminolysis, reductive carboxylation (RC), 2-hydroxyglutarate
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 329
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
330 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 331
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
332 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
deubiquitinase that was described to play a role in DNA damage polypeptides. The reports related to role of O-GlcNAcylation in
as well as in response to loss of centrosome, and SPINT2, a mitochondria are very limited. At the same time, growing body
tumor suppressor frequently inactivated in cancer. Depletion of of evidence shows that mitochondrial dysfunction may be caused
these two candidates improves proliferation of tetraploid cells by by hyperglycemia, although mechanism of this regulation is still
independent mechanisms. Taken together, we describe novel play- not fully understood. Therefore, the aim of our study was to
ers in regulation of cell cycle arrest upon tetraploidization, whose determine whether glucose and glucose dependent mitochondrial
dysfunction may lead to tolerance of genome duplication. isoform of O-GlcNAc transferase (mOGT) impacts on core
mega-channel proteins in breast cancer cell lines (MCF-7, MDA-
MB-231, Hs578t). To find out how mOGT affects on mitochon-
P.10-119-Tue drial mega-channels, cells growing in hypo-, normo- and hyper-
Apoptotic effect of essential oils from cultured glycemia was treated with plasmid coding mOGT and its variant
and wild form of Origanum acutidens (Hand.- without catalytic domain as well as plasmid bearing no insert.
We have investigated that the mitochondrial expression of
Mazz.) Ietswaart. (Lamiaceae) on lung cancer
mOGT and some of core mega-channel proteins such as porins
cells (A549) VDAC and adenine nucleotide translocases (ANT) are glucose
€
A. Ozkan 1
, A. Erdogan2, C. D€ulgeroglu1
1
dependent. In addition, upregulation of mOGT impacts on
Akdeniz University, Faculty of Science, Biology Department, VDAC3 and ANT1 in breast cancer cells. Our results suggests
Antalya, Turkey, 2Alanya Alaaddin Keykubat University, Faculty that mOGT-dependent O-GlcNAcylation regulates the expression
of Engineering, Genetic and Bioengineering Department, Antalya, of mega-channel proteins.
Turkey
The aim of the work was to investigate apoptotic effects of essen-
tial oils from cultured and wild form of Origanum acutidens P.10-121-Mon
(Hand.-Mazz.) Ietswaart. (Lamiaceae) on A549 lung cancer cells. Implication of protein kinase CK2 in the
The IC50 values of wild and cultured forms of essential oil on metabolic rewiring of cancer cells
cancer cells were determined by Cell Titer-BlueR Cell Viability F. Zonta1,2, I. Masgras2, A. Rasola2, C. Borgo2, M. Salvi2,
assay. Lactate dehydrogenase activity were measured by Lactate L. A. Pinna1,2, M. Ruzzene2
1
dehydrogenase Activity Assay Kit. Caspase 3/7 activity in essen- CNR Institute of Neuroscience, Padova, Italy, 2Department of
tial oil treated cells were determined by ApoTox-GloTM Triplex Biomedical Sciences, Padova, Italy
Assay Caspase-3/7 assay kit. IC50 values (concentration that kills
Protein kinase CK2 is a Ser/Thr kinase overexpressed in cancer
50% of cells) of wild and cultured forms of essential oil in A549
cells were calculated as 98.1 lg/ml and 136.2 lg/ml respectively cells. A body of knowledge defines its supporting role in an
for 24 h. Cytotoxic effect of essential oil increased depend on ample spectrum of signal transduction pathways whose dysregu-
concentrations. The lactate dehydrogenase (LDH) activities, lation leads to malignancy; however, very little is known about
its involvement in the metabolic rewiring occurring in cancer.
which is a cytoplasmic enzyme released as a result of the disrup-
tion of the cell membrane integrity, were found higher in wild Since CK2 regulates key transcription factors and signal trans-
and cultured forms of essential oil (IC50) treatment in A549 cells duction pathways with well-known functions on cancer metabo-
than control cells. Caspase 3/7 activity, which is one of the key lism (e.g. PI3K/Akt pathway, HIF-1a, c-Myc and p53), we
hypothesize that CK2 targeting can counteract aerobic glycolysis
enzymes of apoptotic pathway, increased in A549 cells respec-
tively 2 and 4 times in comparison to control cells after wild and and other tumor specific metabolic pathways. The aim of this
cultured forms of essential oil (IC50) treatment for 24 h. As a work is a detailed dissection of CK2 contribution to the estab-
lishment of a metabolic onco-phenotype. To this purpose, besides
result, essential oil from wild and cultured O. acutidens caused
canonical studies of cell treatments with CK2 inhibitors, we
cell death inducing apoptosis.
exploit neuroblastoma SK-N-BE(2) and osteosarcoma U2OS cell
lines, permanently depleted of an individual CK2 catalytic iso-
P.10-120-Wed form (a or a’), by CRISP/Cas9 technology. Results obtained
The impact of glucose and mOGT-dependent from cell growth curves, soft agar clonogenic assays and spheroid
formation assays suggest that both CK2 a and a’ contribute to
O-GlcNAcylation on expression of mega- cell proliferation, survival and tumorigenicity of these cells.
channel proteins in breast cancer cells Metabolic features are investigated by assessing the expression of
ozwiak, E. Forma, P. Ciesielski, O. Kuzmycz, M. Brys
P. J several glycolytic enzymes and other regulatory proteins, the
Department of Cytobiochemistry, Faculty of Biology and mitochondria content by NAO fluorescent probe, and mitochon-
Environmental Protection, University of Lodz, Lodz, Poland drial respiration and glycolysis flux using Seahorse XF24 Ana-
Cancer cells energy metabolism have been reported to be strongly lyzer. The effects of combined treatments with CK2 inhibitors
dependent on glucose in comparison to normal cells. This charac- and metabolic inhibitors are also evaluated on wild type cell via-
teristic feature is associated with mitochondrial dysfunction. bility. Our study represents the first comprehensive analysis on
Mitochondria are described as “the powerhouse of the cell” the role of CK2 in tumor metabolism, with the additional advan-
because they supply most cells with most of their ATP. The rate tage of dissecting the separate contribution of each CK2 isoform,
of ATP production in mitochondria is regulated by activity of whose knowledge will be fundamental for future therapeutic
mitochondrial mega-channels that are localized to contact sites strategies based on CK2 targeting.
between the inner and outer mitochondrial membranes. Due to
mitochondrial dysfunction cancer cells shift the burden of energy
metabolism to cytoplasmic fermentation for ATP synthesis.
Within a cell, small fraction of glucose is converted to uridine
diphosphate N-acetylglucosamine (UDP-GlcNAc) which is sub-
strate for O-GlcNAc transferase (OGT). This enzyme is responsi-
ble for the attachment of O-GlcNAc moieties to cellular
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 333
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
334 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 335
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
distinction in 5-mdC level was also observed. Personalized treat- products can be circumvented by the merging of conventional
ment of breast cancer patients may be associated with epigenetic natural product drug discovery paradigms with more modern
changes in DNA, which could have been a valuable significance approaches. A good example of such is the screening and bio-
in predicting of therapy outcome in the future. The work was activity guided isolation of natural metabolites using High Con-
supported by the Polish National Science Center [2015/17/B/ tent Analysis (HCA) equipment. Phytochemical investigation of
NZ5/00640]. Albuca setosa Jacq., a neglected species from the Asparagaceae
family, led to the isolation and chemical characterisation of a
novel C-glucosylflavonoid-O-glucoside with cytotoxic activity
P.10-128-Tue against HeLa cervical cancer cells. The A. setosa bio-mass under-
Impact of TET3 and OGT on the endometrial went soxhlet extraction to obtain a crude extract which was suc-
cancer progression and metastasis cessively partitioned using Hx, EtOAc and n-BuOH. The n-
ozwiak, E. Forma, P. Machała, K. Słapek,
P. Ciesielski, P. J BuOH partition was then chromatographed on Diaion HP-20
A. Zaczek, A. Krzeslak resin with further separation of the most active fraction on
Department of Cytobiochemistry, Faculty of Biology and Sephadex LH-20 gel to obtain 12 sub-fractions. Sub-fractions
Environmental Protection, University of Lodz, Lodz, Poland SF-7 to SF-12 were combined to afford 26.7 mg of the novel
pure compound. Structural elucidation using various spectro-
Ten-eleven translocation (TET) methylcytosine dioxygenases, scopic methods led to the characterization of the compound as
especially TET3 can interact with O-GlcNAc transferase (OGT). (2S)-6-C-[-apio-a-D-furanosyl-(1?6)-b-glucopyranosyl]-40 , 5, 7-
OGT is an enzyme frequently overexpressed in cancers, responsi- trihydroxyflavone. Dual staining with bisBenzamide H33342
ble for protein modifications by addition of a single N-acetylglu- trihydrochloride/propidium iodide (Hoechst/PI) was used to
cosamine moieties to serine or threonine residues of intracellular determine the anti-proliferative activity of the A. setosa extract,
proteins. It is known that TET3 can affect the recruitment of fractions and pure compound. At its IC50 of 2.5 lM the isolated
OGT to chromatin where OGT can regulate the expression of pure compound was further found to induce cell cycle arrest in
certain genes. Previous studies conducted by us on clinical sam- M-phase in HeLa cells. Image cytometry confirmed apoptosis
ples of endometrial cancer have shown that the high expression induction as evidenced by changes in morphological and bio-
of TET3 is associated with more advanced and aggressive chemical markers of apoptosis.
tumors, which may indicate a significant contribution of TET3 to
the progression of endometrial cancer. Therefore, the aim of the
study was to evaluate the effect of altered TET3 expression on P.10-130-Mon
the expression and cellular location of OGT and on the expres- Activation of both pro-survival and pro-
sion of genes that are key regulators of epithelial-mesenchymal
apoptotic signaling pathways in response to
transition (EMT) in two endometrial cancer cell lines. TET3
expression was down-regulated by the RNA interference, whereas KIT downregulation in leukemia cells
TET3 overexpression was performed by transfecting cells with E. Vagapova1,2, P. Spirin1, T. Lebedev1, N. Poymenova1,
expression vector. Although the impaired TET3 expression had A. Buzdin1, P. Rubtsov1, C. Stocking3, V. Prassolov1,2
1
no effect on the OGT gene expression, protein level and cellular Engelhardt Institute of Molecular Biology, Russian Academy of
location (cytoplasmic/nuclear), differences in the amount of OGT Sciences, Moscow, Russia, 2Moscow Institute of Physics and
in the chromatin fraction were observed. Moreover, ChIP analy- Technology (State University), Dolgoprudny, Moscow Region,
ses revealed changes in the amount of OGT in the promoter Russia, 3Department of Stem Cell Transplantation, University
regions of EMT-regulating genes. Expression of tested genes Medical Center Hamburg-Eppendorf, Hamburg, Germany
changed in cells with altered expression of TET3. In addition, Receptor tyrosine kinase KIT is found to be expressed in the
down- and up-regulation of TET3 in endometrial cancer cells high percentage of acute myeloid leukemia (AML) cases. Since
affected their migration and invasion potential. These results sug- that KIT is an attractive target for anti-leukemia treatment, but
gest that the interactions between TET3 and OGT may have a the input of non-mutant KIT up-regulation in AML still remains
significant impact on the process of endometrial cancer progres- unclear. We used RNA interference (RNAi) technique to study
sion. This work was supported by the grant (2015/19/N/NZ3/ the mechanisms responsible for survival of leukemia cells follow-
01311) from the National Science Center, Poland. ing KIT downregulation. We observed significant slowdown of
cell growth when KIT was repressed. Also the alteration in
expression of genes encoding cell cycle regulatory proteins such
P.10-129-Wed as cyclins and cyclin-associated kinases was demonstrated. Fur-
Anti-cancer activity of a novel flavonoid C- ther we performed a genome-wide microarray-based screening of
apioglucoside from Albuca setosa Jacq. gene expression. We showed that KIT downregulation was fol-
(Asparagaceae) lowed by dramatic changes in signaling pathways activation pro-
M. N. Nyambe1, M. van de Venter1, D. R. Beukes2, file. From the one side, most of them are responsible for cell
B. Swanepoel1, N. Smith3, B. Hlangothi4 death and suppression of cell growth. But from the other side,
1
Department of Biochemistry and Microbiology, Nelson Mandela the huge number of activated pathways is associated with the
University, Port Elizabeth, South Africa, 2Department of kinases involved in cell survival, avoiding apoptosis and prolifer-
Pharmacy, University of the Western Cape, Cape Town, South ation stimulation. Among them signaling pathways associated
Africa, 3School of Medicinal Sciences, Nelson Mandela University, with ERK kinases were found to be up-regulated when KIT was
Port Elizabeth, South Africa, 4Department of Chemistry, Nelson suppressed and that could be defined as a compensatory mecha-
Mandela University, Port Elizabeth, South Africa nism. We suggest ERK2 as the key regulator, regarding an exis-
tence of therapy resistant phenotype when anti-KIT drugs are
Albeit the advantages of natural over synthetic compounds, used. The results were obtained within the Program of funda-
recent decades have seen a decline in the approval of natural pro- mental research for state academies for 2013–2020 years (No
duct-based drug candidates due to increased interest in the appli- 01201363823). This work was supported by RSF grant (project
cation of High-Throughput-Screening (HTS) methods to No 14-14-01089-П)
combinatorial synthetic compounds. This challenge to natural
336 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 337
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
338 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 339
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
idarubicin, bromelain, thymoquinone and Nigella Sativa oil were as a potential molecular marker for successful identification and
prepared. MTT and ATP protocol were applied for these sub- treatment for colorectal cancer.
stances with non-cell medium. In addition, idarubicin and brome-
lain were administered at different doses to HL60 and
lymphocyte cells. IC50 value in the HL60 was 5 lM for idaru- P.10-143-Tue
bicin and >20 mg/ml for bromelain. The IC50 value for lympho- Synthesis of new hybrid conjugates based on
cytes was 10 lM for idarubicin and >20 mg/ml for bromelain in amphiphilic porphyrins and nitrilium
MTT method. In ATP assays, the IC50 value in the HL60 was derivatives of boron clusters [B10H10]2-
2.5 lM for idarubicin and 7.5 mg/ml for bromelain. The IC50
A. P. Zhdanov1, K. A. Zhdanova2, K. Y. Zhizhin1
value for lymphocytes was 10 lM for idarubicin and 12 mg/ml 1
Kurnakov Institute of General and Inorganic Chemistry of the
for bromelain. Studies with medium showed no significant
Russian Academy of Sciences (IGIC RAS), Moscow, Russia,
increase in absorbance compared to the control for bromelain 2
Moscow Technological University, Moscow, Russia
(102%) and thymoquinone (107%), while there was a significant
increase in absorbance of idarubicin (%150) and Nigella Sativa Photodynamic therapy (PDT) and boron neutron capture ther-
oil (%121) compared to control. Although bromelain killed the apy (BNCT) are promising alternative methods for treating neo-
cells in the HL60 and lymphocytes, an effective dose for brome- plasms. Boronated porphyrins were suggested as dual sensitizers
lain was not found even at high doses by the MTT method. for both PDT and BNCT because of its tumor affinity of the
When the ATP method was used, it was seen that cells were porphyrin ring; ease of synthesis with a high boron content; low
killed and the IC50 value calculated. In the study with idarubicin, dark cytotoxicity; and desirable photophysical properties, etc.
IC50 value was found by MTT method but lower values were This work is dedicated to the elaboration of synthetic methods
found in ATP method. In particular, plant extracts and active for new boron-porphyrin conjugates receiving. Our approaches
ingredients cause formazan formation in MTT. For this reason, were based on reactions of nucleophilic addition of [2-
we have concluded that the MTT method is not suitable for the B10H9NCMe]- nitrilium derivatives by pendant functional
studies performed with plant extracts and that the ATP method amino- or ethynyl groups of substituted meso-tetraphenylpor-
should be used. phyrins. In received conjugates we varied amounts of porphyrin
moiety (one, two or four). Also, we have investigated interactions
of conjugates with DNA under physiological conditions (pH 7.4,
P.10-141-Wed and ionic strength 0.15 M NaCl). Complexation of the conju-
The influence of SMAD1 gene polymorphisms gates with DNA was confirmed by UV-vis, circular dichroism
on colorectal cancer susceptibility in and fluorescence life-time spectroscopy. It was shown the two
types of complexes with DNA: intercalation (insertion between
Bangladeshi population: A case-control study
DNA bases) and external (attachment to a small groove of
P. F. Karmokar1, M. Asaduzzaman1, M. S. Islam2,
DNA). Type of DNA complexation depends on conjugates struc-
M. Shahriar1, S. Shabnaz1
1 ture. Further studies of these systems might help to understand
University of Asia Pacific, Dhaka, Bangladesh, 2Noakhali Science
the design of anti-cancer drugs with potential biological activity.
& Technology University, Dhaka, Bangladesh
This research was supported by the Russian Foundation for
Various polymorphisms of SMAD1 gene have been found to be Basic Research (grants N16-03-01039, N16-33-60182) and by
linked with elevated colorectal cancer (CRC) risk worldwide. grant “Universitetskiy” (NICH-39).
Genomic analysis has revealed that high incidence of CRC is
caused by altered expression of SMAD1 gene. SMAD1 gene is
responsible for encoding SMAD1 protein which acts as signal P.10-144-Wed
transducers of transforming growth factor- beta (TGF-b) and Investigation of the effects of histone
thus is suggested to be an essential component of the tumor deacetylase inhibitors (HDACi) on apoptotic
growth inhibitory effect of TGF-b. Again, bone morphogenetic pathway in glioblastoma multiforme (GBM)
protein (BMP) is a subgroup of the TGF-b superfamily. BMP-
L. Elmas1, A. Cing€oz2, F. Senbabaoglu2, M. Secme1,
SMAD1 pathway functions as tumor suppressor by stabilizing €
Y. Dodurga1, T. Bagcı-Onder 2
, G. Bagcı3
the p53 tumor suppression activity. So we hypothesize that, 1
Pamukkale University, School of Medicine, Department of
polymorphisms in SMAD1 could alter its antitumor activities
Medical Biology, Denizli, Turkey, 2Koc University, School of
and increase the risk of colorectal cancer. This study aimed to
Medicine, Istanbul, Turkey, 3Pamukkale University, School of
identify the influence of SMAD1 rs11100883 and rs7661162 poly-
Medicine, Department of Medical Genetics, Denizli, Turkey
morphisms on colorectal cancer risk in Bangladeshi population.
A case-control study was carried out on 188 CRC patients and Glioblastoma multiforme is an aggressive brain tumour mainly
180 controls to investigate two allelic variant of SMAD1 gene - occur in adults. Despite surgery, radiotherapy and chemotherapy,
rs11100883 and rs7661162 using Polymerase Chain Reaction survival rate is 14.6 months. Conventional chemotherapy has lim-
(PCR) - Restriction Fragment Length Polymorphism (RFLP). ited effect due to blood-brain-barrier, intrinsic heterogeneity of
An elevated CRC risk was found with heterozygous and tumor, intrinsic GBM resistance and non-specific toxicity.
heterozygous plus mutant variants of rs11100883 and rs7661162 TRAIL which has no effect for normal cells but targets tumour
and the results found were statistically significant. For cells, is a promising agent. It is known that GBM shows resis-
rs11100883 heterozygote GA and combined GA+AA genotypes tance to TRAIL-mediated apoptosis. Therefore, combined treat-
(OR = 1.5472, P = 0.0494 and OR = 1.8559, P = 0.0319 respec- ment agents such as HDACi combined with TRAIL is current
tively) are found to be significantly associated with CRC. Simi- strategy for GBM therapy. The aim of this study is to determine
larly in case of rs7661162, heterozygote AG and combined the best HDACis enhancing the TRAIL-mediated cell death
AG+GG genotypes were also observed to be strongly associated among Vorinostat, MS-275, CBHA, Belinostat and Romidepsin
(P = 0.0128, OR = 1.7720; P = 0.0185, OR = 1.6971) with ele- and to investigate the effects of these HDACis upon apoptotic
vated risk of CRC. The present study indicates that SMAD1 is pathway on GBM. Therefore, cytotoxic effects of single and com-
significantly correlated with increased colorectal cancer risk. The bined treatment of TRAIL and HDACis LN18, T98G, U87MG
result of the investigation point out that SMAD1 could be used and U373 was determined with CellTiter-Glo method and IC50
340 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 341
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
P.10-149-Tue P.10-151-Mon
Sestrin2 protects against cell death in Cisplatin treatment induces autophagy and
response to DNA-damaging drugs through down-regulates E-cadherin in ovarian
regulation of ROS and mitochondrial function carcinoma cells in vitro: a preliminary study
A. Budanov1,2, A. Evstafieva3, A. Dalina1,2, I. Kovaleva3,
A. Mazitova, Y. Topchu, E. Biktagirova, E. Maykova,
A. Zheltukhin1,2, P. Chumakov1, J. Taylor2
1 R. Gabbasov, Z. Abramova
Engelhardt Institute of Molecular Biology, Russian Academy of
Kazan Federal University, Kazan, Russia
Sciences, Moscow, Russia, 2Trinity College Dublin, Dublin,
Ireland, 3Moscow State University, Moscow, Russia It has been demonstrated both in vivo and in vitro that macroau-
tophagy (hereafter autophagy) activation may bolster survival of
Sestrin2 is a protein product of the SESN2 gene that belongs to
cancer cells during acute cisplatin treatment, as well as develop-
the evolutionary-conserved sestrin gene family. The sestrin family
ment of cisplatin resistance. On the other hand, epithelial-
encodes stress responsive proteins responsible for regulation of
mesenchymal transition (EMT) may be yet another process sup-
cell viability, cell growth and metabolism. We have shown previ-
porting survival of cancer cells in these conditions. Here we
ously that Sestrin2 is activated in response to DNA-damage and
demonstrate that cisplatin treatment leads to induction of expres-
is a potential regulator of the DNA-damage response (DDR).
sion of autophagy marker LC3B, as well as reduced of E-cad-
According to our data, the potential mechanism of regulation of
herin expression, the most important marker of EMT, in ovarian
the DDR by Sestrin2 is mediated by the interaction with the
carcinoma cells. These preliminary data has been obtained using
GATOR2 protein complex that controls mTORC1 kinase activity
epithelial ovarian cancer cell line OVCAR3. The cells were incu-
and lysosomal function. Sestrin2 is often downregulated in
bated in RPMI-1640 medium with addition of fetal bovine
human cancers potentially contributing to tumour progression.
serum, insulin, and L-glutamine at 37°C in 5% CO2 atmosphere.
We hypothesised that Sestrin2 can modulate the DNA-damage
Cells were plated onto 6 well plates (300000 cells/well), incubated
response contributing to the regulation of cell viability. Interest-
for 24 h and treated with cisplatin at concentrations of 18–6 lM/
ingly we have demonstrated that Sestrin2 protects cells against
l, or water control. After 24 h of drug treatment, whole cell
DNA-damage induced cells death. This mechanism involves regu-
lysates were collected using RIPA buffer. LC3B and E-cadherin
lation of mitochondrial activities and reactive oxygen species
expression were probed using Western blotting. GAPDH expres-
through a direct control of mitochondrial respiration and mito-
sion was used as loading control. The data was quantified using
phagy. Therefore, Sestrin2 inactivation have an immediate impact
ImageJ software. Cisplatin treatment has led to dose dependent
on the stimulation of cell death, but facilitates mutagenesis to
induction of autophagy marker LC3B expression as well as
support genomic instability and carcinogenesis. This work was
down-regulation of E-cadherin (1.45 and 1.71 folds change
supported by Russian Science Foundation grant 17-14-01420.
342 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
between highest dose-treated and control cells, respectively). Our of primary tumors and matched normal tissues: lung, breast, pros-
preliminary results confirm that both autophagy and EMT induc- tate, kidney, colon, and other cancers. The set of 31 traditionally
tion may occur in ovarian cancer cells in response to cisplatin used reference genes was composed: ACTB, ALAS1, B2M,
treatment. Our future work will be dedicated to further under- CDKN1A, G6PD, GAPDH, GUSB, HBB, HMBS, HPRT1,
standing the roles autophagy and EMT play in survival of cancer HSP90AB1, IPO8, LDHA, NONO, PGK1, POP4, PPIA, PPIH,
cells in presence of cisplatin, including development of cisplatin PSMC4, PUM1, RPL13A, RPL30, RPLP0, RPS17, RPS18,
resistance, as well as investigating the interaction between autop- SDHA, TBP, TFRC, UBC, YWHAZ, TUBB. Besides, we included
hagy and EMT in context of cisplatin-treated cancer cells. Work in analysis RPN1 that was previously identified by us as a promis-
supported by Program of Competitive Growth of KFU. ing reference gene in several cancer types. We developed the scor-
ing that assessed the stability of expression of each gene in each
cancer type examined. PUM1 was one of the most stable genes in
P.10-152-Tue most cancer types. While GAPDH, which is widely used as a refer-
Investigation of seven european colorectal ence gene, was highly variable and inappropriate for expression
cancer related SNPs in Turkish sporadic evaluation by quantitative PCR in more than half of the examined
colorectal cancer population cancer types. RPN1, proposed by us earlier as a reference gene,
€ Cumao showed high score in lung, kidney, colon, liver, thyroid, and pros-
O. gulları1,2, O. Ilk Dag3, F. Rajabli4, A. Kuzu5,
€ tate cancers. For each examined cancer type, we suggested pairs of
H. Ozda g2
1 genes which are the most suitable for use as reference genes. These
Eastern Mediterranean University Dr. Fazil K€ uc€
uk Faculty of
genes are characterized by high scores and absence of correlation
Medicine, Famagusta, Cyprus, 2Ankara University, Biotechnology
between their expression levels. This work was financially sup-
Institute, Ankara, Turkey, 3Middle East Technical University,
ported by the Russian Science Foundation, grant 17-74-20064.
Department of Statistics, Ankara, Turkey, 4University of Miami,
Miami, United States of America, 5Ankara University, School of
Medicine, Department of General Surgery, Ankara, Turkey P.10-155-Tue
Colorectal cancer (CRC) is one of the leading causes of cancer- Insufficient cancer cell elimination by p53-
related death in the developed countries. According to 2014 inducer Idasanutlin® (RG7388) leads to the
WHO report new incidence rate of CRC in Turkey is %6.5
generation of secondary drug-resistance
among other cancer types and %75 of CRC cases are sporadic.
L. Skalniak1, J. Kocik1, M. Rak2, T. A. Holak1
CRC is one of the most well studied cancer type. Owing to diffi- 1
Faculty of Chemistry, Jagiellonian University, Krakow, Poland,
culty of the low allele frequency variations detection genetic asso- 2
Faculty of Biochemistry, Biophysics and Biotechnology,
ciation profile of CRC have not been entirely identified.
Jagiellonian University, Krakow, Poland
Significance of these variations may change according to popula-
tion. To date several GWASs (Genome wide association studies) The protein p53 is activated in response to DNA damage. The
conducted in European CRC population revealed CRC related active protein induces cell cycle arrest and DNA repair program
SNPs (Single Nucleotide Polymorphism). From the findings of and thus protects the organism against carcinogenic events. Inac-
these studies, we selected and analyzed 7 GWAS validated SNPs tivation of p53 gives the cancer cell a profit of genetic instability,
namely rs1035209, rs6691170, rs11169552, rs7136702, rs4925386, resulting in enhanced cancer cell microevolution. Overexpression
rs6983267. rs3217810, with a P value of <1 9 E8 in Turkish of a negative regulator of p53, Mdm2 protein, is one of the most
sporadic CRC cases. The 7 SNPs which resides at 1q41, 8q24, common methods utilized by cancer to keep p53 inactive. Mdm2
10q24.2, 12p13.32, 12q13, 20q13.33 were studied using KASP binds and blocks p53 and directs p53 to degradation. Re-activa-
(Kompetitive Allele Specific PCR) in 1967 cases and healthy con- tion of p53 protein using Mdm2 antagonists is believed to be a
trols. Statistical analysis is carried out by Cochran-Armitage chi- promising therapeutic strategy of the treatment of cancers
square test. According to statistical analysis rs4925386 was found expressing wild-type p53. Several representatives of this class of
associated with Turkish CRC population in our study molecules, such as Idasanutlin (RG7388), are currently evalu-
(P = 0.0018). (This work was supported by TUBITAK _ Grant ated in clinical trials. However no data concerning the generation
no.: 112S634). of secondary resistance is available for these modern, optimized
compounds. In our study four p53wt human cell lines, SJSA-1,
U-2 OS, MCF-7 and HCT116 were treated with Idasanutlin,
P.10-153-Wed followed by the analysis of viability, p53 activation, the induction
Evaluation of expression stability of traditional of apoptosis, cell cycle distribution and cancer cell elimination.
reference genes in different cancer types on The treatment of all four tested p53wt cell lines with Idasanutlin
the basis of TCGA project data leads to a strong activation of p53. As a result Idasanutlin
induces tremendous cell cycle arrest. However the apoptosis is
G. Krasnov, A. Beniaminov, G. Puzanov, R. Novakovskiy,
induced only in SJSA-1 cells, which are highly sensitive to Mdm2
N. Melnikova, A. Dmitriev
antagonists. Idasanutlin-treated U-2 OS cells can be cultured
Engelhardt Institute of Molecular Biology, Russian Academy of
for long time periods in the presence of the drug. This prolonged
Sciences, Moscow, Russia
treatment of U-2 OS cells leads to de novo generation of sec-
Quantitative PCR remains the most widely used technique for gene ondary resistance. Although modern Mdm2 antagonists have
expression evaluation. To obtain reliable data using this method, much improved activities compared to their precursors (i.e. nut-
stable reference genes are needed. Such gene must have unchanged lin-3), they suffer from similar weaknesses, which are limited
expression under experimental and control conditions. This issue is elimination of cancer cells and the generation of p53-mutated
especially crucial in cancer studies because each tumor has unique drug resistant subpopulations. This research was supported by
molecular portrait. We evaluated expression stability of tradition- Sonata grant UMO-2016/21/D/NZ7/00596 from the National
ally used reference genes on the basis of The Cancer Genome Atlas Science Centre, Poland.
(TCGA) data. TCGA provides omics data for thousands of sam-
ples of dozens of cancer types. We included in our study those can-
cer types, for which omics data are available for representative sets
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 343
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
P.10-156-Wed cytometry, and we found that only a small fraction of the cell pop-
ulation underwent apoptosis. We determined the expression of
Identification of DNA methylation markers of
p21, p53, pRb, Bcl-2 and Bax by PCR, and the activation of p53
CIMP+ epithelial tumors on the basis of The and Rb by flow cytometry. We measured senescence with negative
Cancer Genome Atlas data results. The expression of Bax was not affected however the expres-
G. Krasnov, R. Novakovskiy, N. Melnikova, A. Dmitriev sion of Bcl-2 increased at 96 h. The expression of p53 and p21
Engelhardt Institute of Molecular Biology, Russian Academy of increased in HeLa and INBL cells but the expression of Rb did not
Sciences, Moscow, Russia change. Activation of p53 was observed in INBL cells but not in
CIMP+ (CpG island methylator phenotype) tumors were sepa- HeLa cells and activity of Rb was not affected. The effect of IL-2
rated in a distinct molecular group of colorectal cancer more on the cell cycle does not induce the activation of cellular senes-
than two decades ago. Since then, a number of evidences sug- cence, does not activate the pro-apoptotic protein Bax and upregu-
gested the existence of this phenotype in other cancer types also. lates the anti-apoptotic protein Bcl-2 conferring the cells
CIMP+ tumors are characterized by hypermethylation of pro- protection against apoptosis. IL-2 is a pleiotropic cytokine thus a
moter regions of many genes simultaneously. Hypermethylation more detailed inspection of IL-2 signalling in cancer cells will aid
is one of the major mechanism of downregulation of genes our understanding of the specific effects of this cytokine on cervical
including tumor suppressors, which inactivation is necessary for cancer cells.
the transformation of a normal epithelial cell into a malignant
tumor one. It was shown that determination of CIMP status of
P.10-158-Tue
tumors (CIMP+ or CIMP) is important for prognosis of the
disease and prescription of an optimal treatment regimen. How- Effect of IL-2 on the secretion of lactate and
ever, it is hard to implement CIMP status determination in clini- the NADH/NAD+ ratio in cervical carcinoma
cal practice because there are no reliable molecular markers of cell line SiHa
CIMP+ tumors and genome-wide analysis methods are still A. Valle-Mendiola1, M. D. C. Lagunas-Cruz2, B. Weiss-Steider2,
expensive and complicated enough. The Cancer Genome Atlas I. Soto-Cruz2
(TCGA) database, which store omics data for thousands of 1
Facultad de Estudios Superiores Zaragoza, UNAM, Ciudad de
tumors, provides methylation level for up to 485 thousand CpG Mexico, Mexico, 2Facultad de Estudios Superiores Zaragoza,
sites through human genome and represents a good start point UNAM, Mexico City, Mexico
for identification of DNA methylation markers of CIMP+ epithe-
Cervical Cancer is the second cause of death in women worldwide,
lial tumors. Our research was aimed at development of scoring
in Mexico, the highest incidence in gynaecological tumours is
that enabled the search for promising CIMP+ markers of epithe-
breast cancer followed by cervical cancer. The IL-2R has been
lial tumors. To reach the goal, we analyzed TCGA methylomic
found to be expressed on non-haematopoietic cells, especially in
and transcriptomic data as well as ENCODE data on localization
several types of solid tumours. However, the function of this recep-
of functional elements in human genome with our CrossHub
tor on malignant cells has not been defined. We documented the
tool. The criteria for promising CIMP+ markers were developed
expression of the IL-2R and the production of IL-2 in cervical can-
and realized as a scoring. The scoring was applied for more than
cer cells that induces its proliferation. A relevant characteristic of
10 cancer types, for which methylomic and transcriptomic data
many types of cancer is the ability to reprogram energy metabolism
were available in TCGA for representative number of primary
to fuel cell growth and division. The increased uptake of glucose
tumors and matched histologically normal samples, and promis-
and its conversion into lactate is one hallmark of cancer cells, usu-
ing CIMP+ markers were suggested. This work was supported by
ally, in these cells, the excess of lactate is secreted to modify tumour
the Program of fundamental research for state academies for
microenvironment promoting tumour cell invasion and metastasis
2013–2020 years (№ 01201363819).
formation. To analyse the effect of IL-2 on lactate production and
on the NADH/NAD+ redox state we treated cervical carcinoma
P.10-157-Mon cell lines with high (100 UI/ml) or low (10 UI/ml) doses of IL-2.
The cell line SiHa was treated with IL-2 and the metabolites were
Effects of IL-2 on cell proliferation and
measured by using the Lactate Assay Kit or the NADH/NAD+
apoptosis in cervical cancer cell lines quantitation kit. Our results show that the treatment with low
M. D. C. Lagunas-Cruz1, A. Valle-Mendiola2, J. Trejo-Huerta2, doses of IL-2 induces an increase in the secretion of lactate, never-
B. Weiss-Steider2, I. Soto-Cruz2 theless, with high doses the production of lactate decreases. The
1
Facultad de Estudios Superiores Zaragoza, UNAM, Ciudad de NADH/NAD+ ratio was consistent with a high metabolic flux,
Mexico, Mexico, 2Facultad de Estudios Superiores Zaragoza, typical of transformed cells, where a high NADH/NAD+ redox
UNAM, Mexico City, Mexico state is capable of sustaining aerobic glycolysis. The role of IL-2 in
The function and structure of the IL-2 receptor (IL-2R) has been the metabolic switch of cancer cells is poorly studied. Here we
well characterised in normal cells of the immune system but not in show that IL-2 induces a change in lactate secretion and the redox
the normal cervix. The IL-2R has been found to be expressed on state of the cell which is dose-dependent. These results suggest that
several types of tumour cells. We have shown that cervical cancer IL-2 regulates glucose conversion into lactate to increase its con-
cells express this receptor and that low doses of IL-2 induce their centration for intracellular acidification giving cervical cancer cells
proliferation. On the contrary, high doses of IL-2 inhibit the prolif- an advantage to proliferate and to migrate.
eration of cervical cancer cells that could end in cell death. To fur-
ther characterise the role of the IL-2 on cell death we analysed the
expression of cell cycle and apoptosis-related genes in two cervical
cancer cell lines, HeLa and INBL. First, we looked for the ideal
concentration of IL-2 that could affect cell proliferation, using the
crystal violet assay with different concentrations of IL-2. The
results show that 100 UI/ml of IL-2 was the optimal concentration
to decrease proliferation. Then, we analysed apoptosis by flow
344 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Cancer biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 345
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Cancer biology POSTERS
346 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Xenobiochemistry and drug metabolism
Several mechanisms are involved in inducing apoptosis including “Universidad de Malaga. Campus de Excelencia Internacional
ER stress. In our previous study we indicated that MSM induces Andalucıa Tech”].
apoptosis in HCT-116 colon cancer cells regardless of their p53 sta-
tus. The aim of this study is to determine the role of ER stress in
inducing apoptosis in HCT-116 p53 +/+ and HCT p53/ colon P.10-167-Tue
cancer cell lines. HCT-116 p53 +/+ and HCT-116 p53 / colon MicroRNA-206 reduces cell survival of breast
cancer cells were incubated with different doses (200, 400, 600 and cancer cells by targeting nicotinamide
800 mM) of MSM for 24 h. Cells were then collected for RNA iso- phosphoribosyltransferase and reducing
lation, cDNA synthesis and RT- PCR. MSM treatment increased
expression levels of CHOP, ATF6 and IREI genes at 200–400 mM cellular NAD levels
concentrations whereas decreased expression of these genes at 600– M. Nourbakhsh1, Z. Hesari1, S. Hosseinkhani2, Z. Abdolvahabi3,
800 mM concentrations in both cell lines. All concentrations of S. S. Ghorbanhosseini1, M. Tavakoli-Yaraki1, M. Alipour4,
MSM decreased GRP78 gene expression in both HCT-116 p53 +/+ M. Jafarzadeh5
1
and HCT-116 p53 / cells. PERK was downregulated with all Department of Biochemistry, School of Medicine, Iran University
concentrations of MSM except 400 mM in HCT-116 p53 +/+. of Medical Sciences, Tehran, Iran, 2Department of Biochemistry,
MSM treatment increased and decreased PERK gene expression in Faculty of Biological Sciences, Tarbiat Modares University,
HCT-116 p53 / colon cancer cells at 200–400 mM and 600– Tehran, Iran, 3Department of Biochemistry, School of Medicine,
800 mM concentrations respectively. This study was designed to Iran University of Medical Sciences, Tehran, Iran, 4Department of
disclose the role of ER stress in the induction of apoptosis by Nanobiotechnology, Faculty of Biological Sciences, Tarbiat
MSM. Our results showed that different doses of MSM may exhi- Modares University, Tehran, Iran, 5Department of Molecular
bit opposite effects on the expression of ER stress related genes. Genetics, Faculty of Biological Sciences, Tarbiat Modares
According to our data CHOP gene expression increased signifi- University, Tehran, Iran
cantly by 400 mM MSM treatment in both cell lines. In conclu- Background: Nicotinamide adenine dinucleotide (NAD) is a criti-
sion, our results show that modulation of PERK, ATF6, IRE1 and cal coenzyme for redox reactions as well as cellular processes that
CHOP may play important roles in the induction of apoptosis by are important cancer development, including chromatin structure,
MSM in HCT-116 colon cancer cell lines. DNA repair, stress responses, transcription, signaling, apoptosis,
differentiation, and improved life span. Nicotinamide phosphori-
bosyltransferase (NAMPT) is the key enzyme and regulator of the
P.10-166-Mon intracellular nicotinamide adenine dinucleotide (NAD) biosynthe-
Interplay between glucose and palmitate sis. Cancer cells, have higher rate of NAD consumption and there-
uptake in breast carcinoma in vitro fore NAMPT is essential for their survival. Thus the aim of this
M. D. C. Oca~ na Farfan1, P. Carrillo1, B. Martınez-Poveda1, study was to investigate the effect of NAMPT inhibition by miR-
1,2
A. R. Quesada , M. A. Medina1,2 206 on breast cancer cell survival. Material & Methods: MCF-7
1 and MDA-MB-231 breast cancer cells were transfected with miR-
Universidad de M alaga, Andalucıa Tech, Departamento de
Biologıa Molecular y Bioquımica, Facultad de Ciencias e IBIMA 206 mimic, inhibitor and their negative controls. NAMPT levels
(Instituto de Biomedicina de M alaga), M alaga, Spain, 2Unidad were assessed by real-time PCR as well as western blotting. WST-1
741, CIBER de Enfermedades Raras (CIBERER), M alaga, Spain test was used for the assessment of cell survival. Quantification of
NAD levels was performed by enzymatic methods. Apoptosis
One of the most studied tumor cells lines in vitro is the breast carci- assay was performed by labeling cells with Annexin V-FITC and
noma MDA-MB-231 cell line. Several studies have proved its gly- propidium iodide followed by flow cytometric analysis. Bioinfor-
colytic profile, namely known as the Warburg effect. Glutamine
matics analysis was done to assess whether NAMPT 30 -UTR is a
oxidation is also important for its metabolism. Nevertheless, the
direct target of miR-206 and the results were confirmed by the luci-
use of fatty acids for obtaining energy in these cells is still rising. ferase reporter assay. Results: NAMPT levels were higher in MCF-
Palmitic acid is the most common saturated fatty acid, containing 7 and MDA-MB-231 cell lines compared to MCF-10 cells. Upregu-
sixteen carbons in its structure. However, the use of palmitate for
lation of miR-206 reduced NAMPT expression at the protein level,
metabolic studies in MDA-MB-231 is not very extended due to its leading to a significant decrease in the cellular NAD levels.
pro-apoptotic effect in this cell line after certain time exposure. NAMPT 30 -UTR was shown to be a direct target of miR-206.
Nonetheless, in this work we used palmitate as a metabolic fuel for miR-206 reduced cell survival and significantly induced apoptosis.
just 30 min in order to see the almost immediate response of the
Conclusion: Targeting of NAMPT-mediated NAD salvage path-
cells to its presence, after a 30 min fast period. Our results show way by miR-206 and the subsequent decline in cell survival might
that MDA-MB-231 cells are not able of oxidizing palmitate nor provide a new approach for breast cancer therapy.
producing lactate from it. Simultaneous presence of palmitate with
glucose or with glutamine does not affect glucose nor glutamine
uptake in these cells. However, we observed that even low concen- Xenobiochemistry and drug metabolism
trations of glucose increase palmitate uptake in MDA-MB-231
after a 30 min incubation. Treatment with 5 mM 2-deoxyglucose P.11-001-Mon
also for 30 min counters this rise, since 2-deoxyglucose diminishes
Gut hormone GPCRs as potential off-targets
palmitate uptake. Increasing glucose concentration to the same
doses of 2-deoxyglucose leads to a prevalence of the glucose effect for beta-adrenoreceptor blocking agents
on palmitate uptake. The exact role of glucose and glucose deriva- D. Latek1, P. Pasznik1, E. Rutkowska1, S. Niewieczerzal1,
tives should be further studied in order to know more about palmi- J. Cielecka-Piontek2, S. Filipek1
1
tate metabolism in this cell line. [Our experimental work is Faculty of Chemistry, University of Warsaw, Warsaw, Poland,
2
supported by grants BIO2014-56092-R (MINECO and FEDER) Faculty of Pharmacy, Poznan University of Medical Sciences,
and P12-CTS-1507 (Andalusian Government and FEDER) and Warsaw, Poland
funds from group BIO-267 (Andalusian Government). The Type 2 diabetes mellitus (T2DM) is one of the most severe side
“CIBER de Enfermedades Raras” is an initiative from the ISCIII effects induced by pharmacotherapy. Among many drug classes
(Spain). This communication has the support of a travel grant
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 347
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Xenobiochemistry and drug metabolism POSTERS
348 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Xenobiochemistry and drug metabolism
through the body without inducing any resistance from the P.11-006-Wed
immune system of the patient. Furthermore, apoferritin can be
Tyrosine kinase inhibitors vandetanib,
modified with recognition ligands to achieve tumour-specific tar-
geting. The simple-to-use encapsulation protocol (creating lenvatinib and cabozantinib affect oxidation of
ApoElli) was developed and the prepared nanocarrier was char- an anticancer drug ellipticine by cytochromes
acterized. The nanocarrier exhibits narrow size distribution, P450 but not by peroxidases
which suggests being suitable for entrapping of the hydrophobic R. Indra, M. Kolarik, M. Stiborova
molecule of ellipticine. The release of ellipticine at acidic (6.5) Department of Biochemistry, Faculty of Science, Charles
and neutral (7.4) pH was studied. Ellipticine is gradually released University, Prague 2, Czech Republic
from its ApoElli form into the water environment under acidic
Vandetanib, lenvatinib and cabozantinib are tyrosine kinase inhi-
pH; more than 80% ellipticine was released after 48 hrs incuba-
bitors (TKIs) targeting VEGFR subtypes 1 and 2, EGFR and
tion at pH 6.5. In contrast, at pH 7.4 only less than 20% was
the RET-tyrosine kinase, thus considered as multiple TKIs. These
released. ApoElli is also stable after its storage at physiological
TKIs have already been approved for treating patients suffering
pH (7.4) up to 2 month at 4°C. Microsomal cytochrome P450
from thyroid cancer and renal cell carcinoma, and further clinical
incubated with free ellipticine and/or its ApoElli nanocarrier
trials are ongoing for prostate cancer and glioblastoma multi-
form in the presence of NADPH were capable of oxidizing ellip-
forme. Ellipticine and its derivatives are other anticancer agents
ticine to its metabolites and generating ellipticine-derived DNA
that are effective against certain tumors of the thyroid gland
adducts, both under pH 6.5 and 7.4. The ApoElli is toxic to
(anaplastic thyroid carcinoma), ovarian carcinoma, breast cancer
UKF-NB-4 neuroblastoma cancer cells. On the contrary, cyto-
and osteolytic breast cancer metastasis. Ellipticine anticancer effi-
toxicity of this nanocarrier is significantly lower for non-malig-
ciencies are dependent on its metabolism leading both to the acti-
nant cells (non-malignant fibroblasts, HDFn cells) than for these
vation metabolites causing DNA damage (covalent DNA
cancer cells. Supported by GACR 17-12816S and GAUK
adducts) and their detoxification to products that are excreted.
998217.
Ellipticine is oxidized by microsomal cytochrome P450 (CYP)
enzymes and peroxidases. Oxidative activation leads to formation
P.11-005-Tue of 12-hydroxy- and 13-hydroxyellipticine, reactive metabolites
that dissociate to ellipticine-12-ylium and ellipticine-13-ylium,
Dianthus superbus attenuates angiotensin II-
binding to DNA, while formation 9-hydroxyellipticine and the
induced renal dysfunction in glomerular ellipticine dimer are considered to be detoxification products cat-
mesangial cells alyzed by CYPs and peroxidase, respectively. A number of stud-
J. J. Yoon1,2, Y. J. Lee1,2, H. Y. Kim1,2, S. Y. Eun1,2, ies testing the effectiveness of individual anticancer drugs alone
B. H. Han1,2, C. O. Son1,2, D. G. Kang1,2, H. S. Lee1,2 or in a combination with other cytostatics demonstrated that
1
Wonkwang University, Iksan, South Korea, 2Hanbang Cardio- such combination can have additive and/or even synergistic
Renal Syndrome Research Center, Iksan, South Korea effects on treatment regimen. The aim of this study was to study
Glomerular fibrosis is caused by accumulation of extracellular the effect of TKIs vandetanib, lenvatinib and cabozantinib on
matrix (ECM) proteins in the mesangial interstitial space result- oxidative metabolism of ellipticine. All tested TKIs inhibit oxida-
ing in fibrosis manifested by either diffuse or nodular changes. tion of ellipticine catalyzed by hepatic microsomes, but not by
Dianthus superbus belongs to the Caryophyllaceae family and has peroxidases (horseradish peroxidase, lactoperoxidase and
been used in traditional medicine as a diuretic, a contraceptive, myeloperoxidase). The mechanism of these effects is studied in
and an anti-inflammatory agent. The aim of this study was to details. The study might provide a rationale for the clinical evalu-
investigate the effects of Dianthus superbus on angiotensin II ation of the combination of TKIs and DNA-damaging anticancer
(Ang II)-stimulated glomerular fibrosis in human renal mesangial drugs. Supported by GACR 18-10251S.
cells. Dianthus superbus pretreatment attenuated inflammatory
factors such as intracellular cell adhesion molecule-1 (ICAM-1)
P.11-007-Mon
and monocyte chemoattractant protein-1 (MCP-1). Additionally,
Dianthus superbus suppressed transforming growth factor b The hepatoprotective capacity of natural
(TGF-b)/Smad signaling pathway. Collagen type IV, glomerular products from South Africa
fibrosis biomarker, was significantly decreased by Dianthus super- S. Reddy1, T. Koekemoer1, A. Hattingh1, K. Rashed2, M. van de
bus. Moreover, Dianthus superbus inhibited translocation of Venter1
1
nuclear factor kappa B (NF-jB) in Ang II-stimulated mesangial Department of Biochemistry and Microbiology, Nelson Mandela
cell. This study further revealed that Dianthus superbus signifi- University, Port Elizabeth, South Africa, 2National Research
cantly improved Ang II-induced reactive oxygen species (ROS) in Centre, Dokki, Cairo, Egypt
a dose dependent manner. These findings suggest that Dianthus Drug induced liver injury (DILI) is recognised as a significant
superbus have protective effect against renal inflammation, fibro- clinical problem that accounts for up to 50% of all cases of acute
sis and oxidative stress. Therefore, Dianthus superbus may be liver failure. DILI has an approximate annual incidence of 20
potential therapies targeting glomerulonephritis and glomeru- cases per 100 000 people exposed to medications or dietary sup-
losclerosis leading to diabetic nephropathy. plements. Natural products are well established in traditional
medicine, however the therapeutic value of several species has
not been thoroughly explored. The aim of this study was to
develop and characterize a suitable and cost effective in vitro hep-
atoprotection screening assay. High content analysis measured
the hepatoprotective capacity of mushroom (G. lucidum,
R. capensis, P. ostreatus and L. elegans) and plant extracts
(C. intermedia, O. ficus indica and K. africana). All extracts were
screened against HepG2 cells using Hoechst- PI dual staining to
evaluate the cytotoxicity of tested extracts. Only the ethanolic
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 349
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Xenobiochemistry and drug metabolism POSTERS
350 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Xenobiochemistry and drug metabolism
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 351
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Xenobiochemistry and drug metabolism POSTERS
P.11-013-Mon these two drugs. Cytotoxicity of drugs were determined with Ala-
mar Blue Assay and IC50 was calculated. The effects of drugs on
Effect of quercetin on mRNA and protein
Hexokinase II mRNA and protein expressions were determined
expressions of vitamin D metabolizing by qRT-PCR and western immunoblotting techniques, respec-
CYP27B1 and CYP24A1 in human embryonic tively. The intensity of each band was analyzed by Image J soft-
kidney cell line (HEK-293) ware program. IC50 value of cisplatin on PC3 cell line was found
M. Akkulak1, E. Evin1, O.€ Durukan1, G. Ozhan
€ 2
, O. Adali1 as 30 lM. Both alone or combination of drugs were inhibited the
1
Metu Biological Sciences, Ankara, Turkey, 2Izmir International proliferation of PC3 cells in a concentration dependent manner.
Biomedicine and Genome Institute, Dokuz Eylul University, Izmir, Hexokinase II mRNA and protein expressions were significantly
Turkey downregulated in metformin/cisplatin treated cells compared to
control groups. Epidemiological and experimental researches
Vitamin D is essential compound for life having role not only in
indicates that men with abdominal obesity is a risk factor for
the regulation of calcium metabolism but also in the regulation
prostate cancer. Parallel with our results, the adjuvant role of this
of cell proliferation, immune responses, cardiovascular homeosta-
type of antidiabetic drug must be investigated for new cancer
sis and nervous system. These wide range of actions occur with
treatment options.
the enzymatic conversion of vitamin D to 1a,25-dihydroxyvita-
min D by cytochrome P450 enzymes (CYPs). While CYP27B1
provides the synthesis of hormonally active form of vitamin D, P.11-015-Wed
1,25(OH)2D, CYP24A1 involves in catabolism of vitamin D.
In silico prediction of antidepressant-binding
Quercetin, as one of the important member of polyphenols, is
widely studied due to its abundant consumption with a diet, sites on human placental glutathione
important role in human health as activators or inhibitors for S-transferase pi
biochemical reaction, antioxidant and anticancer activities. K. Terali, O. Dalmizrak, H. Ogus, N. Ozer
Recently, interaction between vitamin D receptor and quercetin Department of Medical Biochemistry, Faculty of Medicine, Near
has been reported at molecular level in a few studies. Quercetin East University, Nicosia, Mersin 10, Turkey
may affect the expressions of vitamin D metabolizing CYP Antidepressants are commonly used treatments for major depres-
enzymes. The aim of this study was to investigate the effect of sive disorder worldwide. The lipophilic nature of antidepressants
quercetin on mRNA and protein expressions of vitamin D means that they can cross the placental barrier. Glutathione S-
metabolizing CYP27B1 and CYP24A1 in embryonic kidney transferases (GSTs; EC 2.5.1.18) belong to a family of related
HEK293 cell line. The effects of quercetin on CYP27B1 and homodimeric enzymes that are involved in phase II detoxification
CYP24A1 mRNA and protein expressions in HEK-293 cell line reactions and in non-substrate ligand binding and sequestration.
were determined by qPCR and western blotting techniques, Here, using protein–ligand docking and interaction profiling, we
respectively. Results showed that, quercetin inhibited the prolifer- aim at predicting the mode of interaction between human placen-
ation of HEK-293 cells in a concentration dependent manner. tal GST pi and two tricyclic antidepressants (namely, amitripty-
IC50 value of quercetin on HEK-293 cell line was found as line and clomipramine) as well as between the enzyme and two
60.72 lM. Quercetin treatment did not significantly affect the selective serotonin reuptake inhibitors (namely, fluoxetine and
mRNA expressions of CYP27B1 and CYP24A1. While sertraline) based on in vitro results from several enzyme kinetic
CYP24A1 protein expression was upregulated significantly (1.38 studies previously conducted and reported by our research group.
fold), CYP27B1 protein expression was not affected significantly Amitriptyline and clomipramine appear to interact, through their
with quercetin treatment compared to 0.35% DMSO containing ring system, with a conserved tyrosine residue (Tyr79) tradition-
untreated cells. These results suggested that quercetin may affect ally participating in tocopherol binding, with their hydrophilic
vitamin D metabolism negatively in HEK293 cells due to increase portions protruding into an electrostatic region at the dimer
in CYP24A1 protein expression. interface. On the other hand, fluoxetine and sertraline seem to
favorably occupy both the G- and H-subsites of the enzyme’s
active-site cleft at the same time. Accordingly, these interactions
P.11-014-Tue
may reduce the therapeutic effect of the prescribed antidepressant
Alteration of protein and gene expressions of on the pregnant mother and also leave the fetus prone to various
Hexokinase II in PC3 cell lines by cisplatin- electrophilic substrates with potentially toxic effects. The present
metformin combination treatment study may additionally serve as a starting point for the structure-
€ Durukan1, M. Akkulak1, E. Evin1, S. Arslan2, O. Adalı1
O. based design of anticancer drugs, since GST pi is overexpressed
1
Metu Biological Sciences, Ankara, Turkey, 2Pamukkale in various human cancers and contributes to multidrug resistance
University Department of Biology, Ankara, Turkey by neutralizing chemotherapeutics.
Metformin is an antidiabetic drug with anticancer properties. Cis-
platin is known as one of the most potent chemotherapeutics for P.11-016-Mon
treatment of various types of cancer. In order to overcome cis-
Membrane-bound cytochrome P450s of 1A
platin resistance and toxicity, the drug can be combined with
other drugs that sensitize tumour cells to cisplatin. The ability of subfamily features similar structural pattern of
metformin to potentiate cisplatin-mediated killing of cancer cells the trans-membrane segment
in vitro, makes it a plausible candidate for combination with cis- P. Jerabek1, J. Florian2, V. Martınek1
1
platin-based therapy. The aim of this study is to examine the Charles University, Prague, Czech Republic, 2Department of
combined effect of these drugs on protein and mRNA expres- Chemistry and Biochemistry, Chicago, United States of America
sions of Hexokinase II, a regulatory enzyme participating in gly- Cytochrome P450 (P450) enzymes are components of a mixed-
colysis as well as cancer promotion. The effects of drugs on function oxidase system located in the membrane of endoplasmic
prostate cancer cell lines were studied using androgen indepen- reticulum. Using multiscale computational methods, we investi-
dent PC3 cell line. Cells were treated with either metformin alone gated the structure and dynamics of the full-length membrane-
in the range of 1–10 mM, cisplatin alone or a combination of anchored P450 1A2 enzymes. The absence of the structural
352 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Gaseous molecules
information on the trans-membrane (TM) domain of these two of information is embedded in 30 UTRs of mRNAs in the form of
proteins was surpassed by employing a spontaneous self-assembly RNA structure. Double-stranded RBPs can bind structures in
molecular dynamics (MD). The simulation was performed in ran- 30 UTRs and then exert their function based on dsRNA target
domized dilauroylphosphatidylcholine (DLPC)/water/salt mixture recognition through a combination of structure and sequence.
and also in randomized palmitoyloleoylphosphatidylcholine Staufen1 is a dsRBP involved in mRNA transport and localiza-
(POPC)/water/salt mixture. The resulting membrane-bound full- tion, translational control and mRNA decay by a staufen-
length structures of P450 1A2 in different membranes were then mediated mRNA decay (SMD) pathway. The Staufen1 binding
optimized using coarse-grained and all-atom MD. The resulting site (SBS) within human ADP-ribosylation factor 1 (ARF1)
models show that, despite of the different membrane thickness, 30 UTR is one such target and Staufen1 binding to the SBS regu-
the upper part of the TM helix in both cases directly interacts lates ARF1 cytoplasmic mRNA levels by the SMD pathway.
with a conserved and highly hydrophobic N-terminal proline-rich However, how Staufen1 recognizes specific mRNA targets is still
segment of the catalytic domain. The shallow membrane immer- unknown. To reveal how Staufen1 binds specific dsRNA targets,
sion of the catalytic domain appears to induce a depression in we determined the solution structure of the ARF1 SBS – Stau-
the opposite intact layer of phospholipids, which may help in sta- fen1 complex by NMR spectroscopy. Our structure reveals that
bilizing the position of the TM helix directly beneath the catalytic Staufen1 is indeed a sequence-specific dsRNA binding protein.
domain. The phospholipid membrane thickness has a direct Staufen1 dsRBD4 recognizes consecutive pyrimidines in the
impact on the TM domain tilt being more inclined in case of the minor groove via helix a1, b1b2 loop anchors the dsRBD at the
thinner DLPC membrane. Supported by GACR 18-10251S. end of the dsRNA in the minor groove and lysines in helix a2
Access to the MetaCentrum and CERIT-SC computing facilities bind to the phosphodiester backbone from the major groove side.
provided under the programs LM2015042 is highly appreciated. Staufen1 dsRBD3 displays the same conserved binding mode and
specifically recognizes GC base pairs. While dsRBD4 alone can
bind in a single binding register on the ARF1 SBS, dsRBD3
P.11-017-Tue requires interactions with dsRBD4 to display its sequence speci-
Computer prediction of the drug-drug ficity. Staufen1 mutation of the amino acids involved in minor
interactions severity groove recognition of ARF1 SBS impairs SMD leading to a 2.4-
A. Dmitriev, D. Filimonov, A. Lagunin, A. Rudik, D. Karasev, fold increase in ARF1 mRNA levels as compared to wt Staufen1.
K. Murtazalieva, V. Poroikov Further mutagenesis data and the biological implications will be
Institute of Biomedical Chemistry, Moscow, Russia discussed. This project is funded by a grant from the Czech
Science Foundation to P.J.L. (P305/18/08153S).
When several drugs are co-administrated, a drug-drug interaction
(DDI) phenomenon may appear. Many DDIs are due to changes
in the metabolism of drugs. In this case, DDI is manifested by Gaseous molecules
the effect of one drug on the biotransformation of other drugs,
its slowdown (in case of inhibition of drug-metabolizing enzymes) P.12-001-Mon
or acceleration (in case of induction of drug-metabolizing Challenges in analyzing catabolic products of
enzymes), which leads to a change in the pharmacological action
of co-administrated drugs. The severity of DDIs can be classified
hydrogen sulfide oxidation
by applying different classification systems. One of the most M. Krızkova, J. Sokolova, J. Krijt, P. Jesina, V. Kozich
Department of Pediatrics and Adolescent Medicine, Charles
advanced is OpeRational ClassificAtion (ORCA) system for the
University, First Faculty of Medicine and General University
classification of DDI, created for physicians to assess the risk of
co-administration of two drugs. ORCA divides DDI into five Hospital in Prague, Prague 2, Czech Republic
classes: contraindicated (class 1), provisionally contraindicated Hydrogen sulfide (H2S) is an important endogenous signaling
(class 2), conditional (class 3), minimal risk (class 4), no interac- molecule and its determination is complicated by its volatility,
tion (class 5). For computer prediction of the severity of DDI, propensity to oxidize and dynamic interconversions between free
we collected a training set consisting of about 4000 pairs of 500 and bound forms. Catabolism of H2S may be assessed by analyz-
drugs that belong to classes 1–3 of DDI in case of co-administra- ing the more stable products of its oxidation. We optimized an
tion. The prediction of DDI classes is based on a combination of HPLC method that uses monobromobimane (mBrB) to fluores-
modified MNA descriptors – PoSMNA (Pairs of Substances cently label the H2S catabolites – sulfite, thiosulfate and thio-
Multilevel Neighbourhoods of Atoms), and a classification algo- cyanate. Here we report on challenges in determining these
rithm implemented in the PASS (Prediction of Activity Spectra analytes, especially in the preanalytical phase. Firstly, sulfite is
for Substances) software. The average invariant accuracy of pre- known to be unstable at low pH and oxidizes rapidly on air (e.g.
diction, calculated in the leave-one-out and 20-fold cross-valida- urinary sulfite decreased by 20% in 2 h). Secondly, we examined
tion procedures, is about 0.9. The Russian Science Foundation the effect of blood collection tubes on analyte concentrations;
(grant No. 17-75-20250) has supported the work. EDTA and heparin-lithium (Hep-Li) plasma, and serum were
collected simultaneously from 3 subjects. Serum yielded 3-times
lower concentrations of thiosulfate than Hep-Li plasma, while
P.11-018-Wed EDTA collection tubes were contaminated with thiosulfate and
Staufen1 reads out structure and sequence sulfite (3.3, 0.1 lmol/l respectively) preventing their use in prac-
features in ARF1 SBS for specific target tice. Thirdly, matrix effects were observed necessitating the use of
recognition plasma- and urine-based calibration standards. Using the opti-
P. J. Lukavsky mized mBrB method, we determined typical values in 13 healthy
CEITEC – Central European Institute of Technology, Masaryk control. In Hep-Li plasma the median concentration of sulfite,
University, Brno, Czech Republic thiosulfate and thiocyanate was 0.32 lmol/l (range 0.22–0.59),
0.59 lmol/l (range 0.30–1.07) and 76 lmol/l (range 14.5–165.5),
Most posttranscriptional regulation of gene expression is based respectively. Median urinary concentrations of sulfite, thiosulfate
on RNA elements in mRNAs recognized by RNA-binding pro- and thiocyanate were 0.07 lmol/mmol creatinine (range 0.02–
teins (RBPs). Besides primary sequence elements, a second layer 0.17), 1.24 lmol/mmol creatinine (range 0.80–2.91) and
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 353
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Redox biochemistry and signalling POSTERS
5.0 lmol/mmol creatinine (range 1.2–17.2), respectively. This causing photoreceptor loss and irreversible vision deterioration.
study shows that the novel mBrB method enables simultaneous Here, we demonstrate that oxidative stress of photoreceptors is
determination of three markers of H2S oxidation and that metic- associated with alterations in redox status of neuronal calcium sen-
ulous preanalytical measures have to be observed. This work was sors (NCSs), signaling proteins regulating various aspects of neu-
supported by the grant AZV 16-30384A and the project RVO- ronal function. Western blotting and mass-spectrometry analysis
VFN 64165. revealed accumulation of oxidized monomers, disulfide dimers and
multimers of NCSs involving their conserved cysteine residue
under cellular, ex vivo, and in vivo conditions. NCSs oxidation
Redox biochemistry and signalling precedes and accompanies light-induced damage of photoreceptors
and results in decline in retinal content of some of these proteins.
P.13-001-Mon Disulfide dimerization of NCSs is also observed in intact retina
Expression levels of peroxiredoxins in small suggesting their redox regulation. As exemplified by recoverin and
intestinal mucosa of children with celiac NCS1, NCSs exhibit different susceptibility to oxidation depending
on intracellular conditions. The results of in vitro oxidation assay
disease
and Ellman’s test demonstrate that Ca2+ binding potentiates thiol
F. Aydın Kose1, A. Pabuccuoglu1, M. Karakoyun2, S. Aydogdu2
1 oxidation of recoverin, but inhibits it in the case of NCS1 that
Ege University Faculty of Pharmacy Department of Biochemistry,
instead oxidizes in apo- or Zn2+-bound states. Furthermore, the
Izmir, Turkey, 2Ege University Faculty of Medicine Department of
thiol oxidation differently affects structural features and function
Pediatric Gastroenterology, Izmir, Turkey
of these proteins. Using disulfide dimers and oxidation mimicking
Celiac disease (CD) is a chronic inflammatory and immune- mutants it was demonstrated that the oxidation inhibits photore-
mediated disorder triggered by the ingestion of gluten. The ceptor membrane association of recoverin, but not of NCS1. In
pathophysiological mechanism of CD has not yet been fully elu- addition, it produces constitutively active recoverin and enhances
cidated; however, recent studies suggest that oxidative stress may activity of Ca2+-NCS1 in regulation of rhodopsin desensitization
play an important role in the pathogenesis of CD. Peroxiredoxins by GRK1. Overall, NCSs represent redox-sensitive proteins that
(PRXs) are a family of antioxidant enzymes which detoxify may undergo redox regulation in normal photoreceptors and exhi-
hydrogen peroxide, organic hydroperoxides, and peroxynitrite. bit pronounced structural and functional changes in oxidative
Six isoforms of PRXs (PRX-1-6) are expressed in mammalians in stress, what possibly contributes to pathogenesis of neuro-ophthal-
different cell compartments. In addition to antioxidant functions, mological disorders. The study was supported by Russian Founda-
PRXs have also been associated with significant cellular processes tion for Basic Research (grant #18-04-01250).
such as redox homeostasis, cell survival and proliferation, apop-
tosis and tissue repair. Although it is shown that PRXs play
important role in various inflammatory diseases, the role of P.13-003-Wed
PRXs in the generation of CD is yet to be known. Therefore, this Different responses of hepatocellular
study aims to investigate the involvement of PRXs in the patho- carcinoma cell lines to oxidative stress
genesis of CD. For this purpose, small intestinal biopsy speci- induced by non-thermal plasma
mens were taken from 7 children with CD and 7 children with B. Smolkova1, M. Lunova2, A. Lynnyk1, O. Churpita1,
histologically normal small intestinal biopsies, by endoscopy. The Kubinova1,3, O. Lunov1, A. Dejneka1
S.
mRNA and protein expression levels of six PRXs were deter- 1
Institute of Physics of the Czech Academy of Sciences, Prague,
mined by quantitative real-time polymerase chain reaction and Czech Republic, 2Institute for Clinical and Experimental Medicine,
Western blot, respectively. It was found that in small intestinal Prague, Czech Republic, 3Institute of Experimental Medicine of
mucosa with CD, mRNA and protein expression levels of the Czech Academy of Sciences, Prague, Czech Republic
PRDX-4 were significantly decreased while that of PRDX-5 were
significantly increased, compared to control group (P < 0.05). Reactive oxygen species (ROS) are well-known mediators of
Our preliminary results reveal that PRXs, in particular PRX-4 oxidative stress, and playing role in activation of several cellular
and PRX-5, may have a role in the pathogenesis of CD. redox processes and signalling pathways, including specific cell
Acknowledgment: This study was supported by TUBITAK death pathways. Recently, non-thermal plasma (NTP), containing
(215S650) and Ege University (03/BIL/2016). chemically active species, has been demonstrated to be a potential
tool in anticancer therapy. Previously, many studies have
reported that NTP can induce cell death in cancer cells by ROS
P.13-002-Tue formation, however, the molecular mechanisms of the interaction
Redox status of neuronal calcium sensor between eukaryotic cells and NTP remain elusive. In our study,
proteins in photoreceptor system we assessed the influence of plasma-generated ROS on two
V. Baksheeva1, N. Gotmanova1, N. Tikhomirova1, human hepatocellular carcinoma cell lines (Huh7 and HepG2).
M. Serebryakova1, A. Nazipova2, O. Gancharova1, Interestingly, NTP showed greater selective anti-proliferative
V. Vladimirov3, D. Zinchenko3, I. Senin1, P. Philippov1, activity against Huh7 cells relative to HepG2, suggesting that
S. Permyakov2, A. Zamyatnin Jr4, E. Zernii1,4 plasma-triggered signalling cascades might be grossly different
1
Belozersky Institute of Physico-Chemical Biology, Lomonosov between cell lines. Our data revealed that air-NTP treatment in
Moscow State University, Moscow, Russia, 2Institute for Huh7 cells results in increased mitochondrial ROS production
Biological Instrumentation of the Russian Academy of Sciences, and mitochondrial depolarization. Subsequently, ROS accumula-
Pushchino, Moscow Region, Russia, 3Branch of Shemyakin- tion and accompanied mitochondrial dysfunction led to apoptotic
Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy cell death in Huh7 cells. Additionally, Caspase-3 activation and
of Sciences, Pushchino, Moscow Region, Russia, 4Sechenov First phosphatidylserine expression confirmed ROS-mediated apoptosis
Moscow State Medical University, Institute of Molecular pathway activation in Huh7 cells. In contrast, in HepG2 NTP
Medicine, Moscow, Russia treatment leads to activation other specific mechanisms, which
resulted in greater degree of resistance to oxidative stress medi-
In the retina, high metabolic activity and abundance of photosensi- ated by plasma in comparison to Huh7. This study elucidates the
tized reactions contribute to elevated risk of oxidative stress mechanism linking physicochemical signals from the NTP
354 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Redox biochemistry and signalling
cascade to the intracellular cancer death signaling pathway, pro- interacting partners. We demonstrated that all studied proteins
viding physical, chemical and biological insights into the develop- co-precipitate with caveolin-1 from rod outer segment mem-
ment of therapeutic techniques to treat cancer related diseases. branes, and can directly interact with caveolin-1. Pull-down
assay, surface plasmon resonance spectroscopy and isothermal
titration calorimetry data indicate that there is interaction with
P.13-004-Mon full-length caveolin-1 N-terminal domain (1-101) and caveolin-1
Mapping the morphological and functional scaffolding domain (81-101). Interestingly that this interaction
alterations of the red blood cells in active occurs only in absence of calcium ions, what is supported by sur-
face localization of caveolin-1 interaction site in Ca2+-free NCS
maternal smoking during pregnancy
proteins state. Caveolin-1 increase Ca2+-sensitivity of recoverin,
P. Chakraborty, K. N. Dugmonits, S. Zahoran, E. Hermesz
and as a consequence, makes its inhibitory activity to rhodopsin
University of Szeged, Department of Biochemistry and Molecular
kinase more pronounced, but not interfere with recoverin-rho-
Biology, Szeged, Hungary
dopsin kinase interaction. Amount of free Ca2+, required for this
In today’s scenario, maternal smoking during pregnancy is quite process, consider caveolin-1 influence, become in good agreement
prevalent which risks to adverse pregnancy outcomes and abnor- with physiological conditions of photoreceptor cell. GCAP-2 is
mal fetal development. The umbilical cord lacks innervations and upregulated by caveolin-1 in Ca2+-free state, which increase
thereby the main regulator of its vascular tone and blood flow is guanylate cyclase activity. It seems that there is a common mech-
the nitric oxide (NO) signaling molecules produced by the anism of interaction between caveolin-1 and NCS proteins in
endothelial nitric oxide synthase (NOS3). Recent evidences show Ca2+-free state. For recoverin increasing of Ca2+-sensitivity is
that red blood cells (RBCs) also possess a functional NOS (NOS3 due to the stabilization of the open conformation of its second
like) that produce and release bioactive NO which maintain the Ca2+-binding domain EF2. Obtained data suggest that at low
rheological properties of RBCs and may play a compensatory role calcium NCS proteins are compartmentalized in photoreceptor
in case of any dysfunction in the cord endothelium. The aim of rafts via binding to caveolin-1, what enhances their activity or
this study is to evaluate the morphological and functional alter- ensures their faster responses on Ca2+-signals. The study was
ations in RBCs of heavy smoker mothers and their neonates’ cord supported by RFBR (grant # 15-04-07963).
blood. Our findings on RBCs originated from the smoker’s sam-
ples that represents (1) distinct morphological variations, followed
with microscopic and software analysis (2) significant changes in P.13-006-Wed
the activation of NOS, based on immunohistochemistry and flow Effect of polyphenols from Silybum marianum
cytometry data (3) macromolecular damages, developed by 4- L. and UVA radiation on Nrf2 signalling
hydroxy-2-trans-nonenal staining and lipidomic measurements.
pathway in skin cells
(4) Furthermore, atomic force microscopy dissections are done on
A. Rysava1, D. Biedermann2, B. Zalesak3, J. Ulrichova1,
the RBCs to evaluate their rheological alterations like Young’s
J. Vostalova1, A. Rajnochova Svobodova1
Modulus to derive on the deformability index, viscosity etc. Our 1
Department of Medical Chemistry and Biochemistry, Faculty of
molecular and biophysical data indicates that maternal smoking
Medicine and Dentistry, Palack y University in Olomouc, Olomouc,
can exert marked effects on the outcome of in-utero development
Czech Republic, 2Institute of Microbiology of the CAS, v.v.i.,
because of the insufficient supply of O2 which may results in long-
Laboratory of Biotransformation, Prague, Czech Republic,
lasting (partly related to- epigenetic alteration) health conse- 3
Department of Plastic and Aesthetic Surgery, Faculty Hospital
quences on the developing fetus.
Olomouc, Olomouc, Czech Republic
Exposure to solar radiation is one of the most important envi-
P.13-005-Tue ronmental factors affecting skin physiology. The adverse biologi-
Caveolin-1 as a regulator of neuronal calcium cal effects of sunlight are mainly associated with ultraviolet (UV)
sensor proteins in phototransduction system wavelengths. Sunlight is primarily an UVA (315–400 nm) source
V. Vladimirov1,2, E. Zernii3, K. Koch4, V. Baksheeva3, comprising 90–95% of solar UV rays. UVA penetrates through
H. Wimberg4, A. Zamyatnin Jr5, S. Permyakov6, A. Kazakov6, epidermis deep into the dermis and thus affects both major skin
N. Tikhomirova3, I. Senin3, D. Zinchenko1 cells, keratinocytes and fibroblasts. UVA initialises a massive
1
The Branch of Shemyakin-Ovchinnikov Institute of Bioorganic production of reactive oxygen or nitrogen species (ROS, RNS)
Chemistry, Russian Academy of Sciences, Pushchino, Moscow leading to oxidation of lipids, proteins and DNA lesions. ROS/
Region, Russia, 2Pushchino State Institute of Natural Sciences, RNS and unstable oxidised products can affect various cellular
Pushchino, Moscow Region, Russia, 3Department of Cell pathways. One of them is the Nrf2 (Nuclear factor erythroid-2
Signaling, Belozersky Institute of Physico-Chemical Biology, related factor 2) signalling pathway. Nrf2 protein, a redox-sensi-
Lomonosov Moscow State University, Moscow, Russia, tive transcription factor is normally bound in the complex with
4
Department of Neurosciences, Biochemistry Group, University of the Keap1 protein (Kelch-like ECH-associated-protein-1). Due to
Oldenburg, Oldenburg, Germany, 5Institute of Molecular Medicine, the oxidative stress, the affinity of Keap1 to Nrf2 decreases, the
Sechenov First Moscow State Medical University, Moscow, Keap1/Nrf2 complex decays and Nrf2 is translocated from the
Russia, 6Protein Research Group, Institute for Biological cytosol to the nucleus where it binds to the DNA into promoter
Instrumentation of the Russian Academy of Sciences, Pushchino, sequence of the antioxidant responsive element and stimulates
Moscow Region, Russia expression of detoxication and antioxidant enzymes. The Nrf2
pathway has been described to be activated by several naturally
Caveolin-1 is the major regulatory protein of detergent resistant occurring compounds including polyphenols. Here we have stud-
membranes (DRM), which associating with regulation of signal- ied effect of silymarin, a standardized extract of polyphenols
ing activity in different organism systems. Rod cell membranes from Silybum marianum L., its flavonolignans silybin, silychristin,
contain cholesterol-rich DRM, which accumulate visual cascade silydianin, isosilybin, 2,3-dehydrosilybin and flavonoids taxifolin
proteins. In this study, photoreceptor Ca2+-binding proteins and quercetin on the Keap1/Nrf2/ARE signalling pathway in
recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal human skin cells, primary dermal fibroblasts and epidermal ker-
calcium sensor (NCS) family, were recognized as novel caveolin-1 atinocytes and human keratinocyte cell line HaCaT. Effects were
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 355
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Redox biochemistry and signalling POSTERS
determined by Western Blot and immunocytochemistry. The modifications of protein molecules and DNA damage. Tyrosine
results will be discussed in a poster presentation. This work was molecules are less resistant to the action of plasma radiation,
financially supported by grants GACR 15-10897S, which determines the oxidative modification of the erythrocyte
IGA_LF_2018_012 and institutional resources of Palacky proteins of intact animals and animals with experimental sar-
University in Olomouc – RVO 61989592. coma. Exposure to plasma radiation causes a modification of the
tertiary and secondary structure of erythrocyte proteins, disrupt
the functional activity of the cell as whole. The obtained data
P.13-007-Mon allow developing effective gas-discharge devices for biomedical
Idh2 deficiency accelerates renal dysfunction research and can be used to introduce the innovative plasma
in aged mice technologies in medicine.
J. Park, S. J. Lee, H. Kim, S. H. Kim
Kyungpook National University, Daegu, South Korea
P.13-009-Wed
The free radical or oxidative stress theory of aging postulates Functional analysis of type VI sulfide:quinone
that senescence is due to an accumulation of cellular oxidative
oxidoreductase in photosynthetic purple sulfur
damage, caused largely by reactive oxygen species (ROS) that are
produced as by-products of normal metabolic processes in mito- bacterium
Duzs1, N. Miklovics2, F. Balogh1, B. Nemeth2, G. Rakhely1,2,
A.
chondria. The oxidative stress may arise as a result of either
increased ROS production or decreased ability to detoxify ROS. A. T oth1,2
1
The availability of the mitochondrial NADPH pool is critical for Institute of Biophysics, Biological Research Centre Hungarian
the maintenance of the mitochondrial antioxidant system. The Academy of Sciences, Szeged, Hungary, 2University of Szeged,
major enzyme responsible for generating mitochondrial NADPH Department of Biotechnology, Szeged, Hungary
is mitochondrial NADP+-dependent isocitrate dehydrogenase Despite its toxicity, sulfide plays essential physiological role in
(IDH2). Depletion of IDH2 in mice (idh2/) shortens life span eukaryotes and prokaryotes as neurotransmitter or electron
and accelerates the degeneration of multiple age-sensitive traits, donor. Sulfide detoxification, homeostasis and sulfide-dependent
such as hair grayness, skin pathology, and eye pathology. Among energy conversation processes via electron transfer from sulfide
the various internal organs tested in this study, IDH2 depletion- to the membrane quinone pool is catalyzed by sulfide:quinone
induced acceleration of senescence was uniquely observed in the oxidoreductases (SQR). SQRs are ancient membrane bound
kidney. Renal function and structure were greatly deteriorated in flavoproteins, members of disulfide oxidoreductase enzyme fam-
24-month-old idh2/ mice compared with wild-type. In addition, ily. For catalysis SQRs require FAD cofactor and redox-active
disruption of redox status, which promotes oxidative damage disulfide bridge formed between conserved essential cysteines.
and apoptosis, was more pronounced in idh2/ mice. These data Based on phylogenetic and structure-based classifications there
support a significant role for increased oxidative stress as a result are six types of SQR proteins. Different type SQRs have diverse
of compromised mitochondrial antioxidant defenses in modulat- catalytic mechanisms might due to the variances in cysteines.
ing life span in mice, and thus support the oxidative stress theory Thiocapsa roseopersicina, the photosynthetic purple sulfur bac-
of aging. terium possesses a type VI SQR enzyme (SqrF). Biochemical and
enzyme kinetic analysis of homologously-expressed and purified
recombinant SqrF showed significant differences between SqrF
P.13-008-Tue and other type SQRs indicating different catalytic mechanism
Electric discharge redox technology for compared to the known models. To identify the role of cysteine
biomedical investigation residues of SqrF in the catalytic process cysteine mutant enzymes
I. Piskarev1, I. Ivanova2 were created by site-directed mutagenesis, expressed in T. roseop-
1
Skobeltsyn Institute of Nuclear Physics, Moscow, Russia, 2Nizhny ersicina and purified by affinity chromatography. Characteriza-
Novgorod State Medical Academy, Nizhny Novgorod, Russia tion of biochemical and catalytic properties of cysteine mutant
SqrF variants, the effect of sulfhydryl-blocking agents on the
The possibility to use a plasma radiation of an electric discharge
enzyme activity and FAD cofactor binding of wild type and
in air for biomedical research was investigated. A pulse plasma
mutant enzymes identified the reacting disulfide pair of the pro-
radiation generator with a discharge power of 0.6 J/s was used.
tein and revealed the function of cysteines in catalytic process of
Under irradiation and products formed in the discharge itself,
T. roseopersicina SqrF. Here, we report the first circumstantial
long-lived active species are formed in aqueous solution. Active
characterization and a model for the sulfide oxidation site and
species, generated in the liquid under the action of a discharge,
mechanism of type VI SQR enzyme.
have both oxidative and reducing properties. The formation of
nitrous, peroxynitrous acids and peroxynitrite was identified in
liquid. The reducing properties were identified by means of free P.13-010-Mon
stable radical DPPH and the reduction of methaemoglobin. In
Bach1, a heme-containing mammalian
albumin the reduction of –S-S- groups to –SH has been estab-
lished. Oxidative modification of tryptophan, albumin and hemo- transcription factor: study on structure and
globin molecules in solution after exposure to gas discharge function relationship
plasma radiation is caused by the formation of complexes with A. Lengalova1, J. Vavra1, P. Mihalcin1, M. Watanabe-Matsui2,
products of plasma irradiation: compounds of nitrogen, nitrogen K. Igarashi2, T. Shimizu2, M. Martınkova1
1
radicals and hydroperoxides. It was shown, that due to a long Department of Biochemistry, Faculty of Science, Charles
lifetime, active species penetrate the skin of the rat. The gas dis- University, Prague, Czech Republic, 2Department of Biochemistry,
charge plasma and its radiation have a cytotoxic effect on lym- Tohoku University Graduate School of Medicine, Sendai, Japan
phoid and epithelial neoplastic cells. The cytotoxic effect is seen The Bach1 protein is the first mammalian transcription factor
by disturbances in the structure of the cytoplasmic and nuclear found to bind heme. Bach1 senses the heme concentration and in
membranes, intracellular contents, in the increase of cytoplasmic
the case of heme accumulation it stimulates transcription of heme
membranes fluidity, in the accumulation of oxidative oxygenase, an enzyme which is responsible for free heme
356 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Redox biochemistry and signalling
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 357
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Redox biochemistry and signalling POSTERS
stress response (YAP1, TRX2), multidrug resistance (PDR1, analyzed molecular forms of selenium compounds. K562 cells
YRR1, SNQ2), cysteine, methionine and hydrogen sulfide biosyn- were cultured in the Ham’s F12 medium containing 10% FBS
thesis (CYS3, MET3, MET5, NFS1, and others). Several of these with 0–10 lM selenoneine. Oxidative stress was induced by tert-
genes are directly regulated by Rpn4 while others are regulated butyl hydroperoxide (t-BHP), 2, 20 -azobis (2-amidinopropane)
by proteasome subcomplexes both in proteolysis dependent and - dihydrochloride (AAPH), pyocyanin and S-nitrosoglutathione
independent manner. Consistently, we found that activity of (GSNO), and cell viability was measured. Molecular forms of
homologous recombination is increased in the mutant strain, and selenium compounds in the cells and medium were analyzed by
it shows hyper-resistance to oxidative damaging agents like ICP-MS. Selenoneine slightly but significantly improved cell via-
hydrogen peroxide and heavy metals. Our data imply that yeast bility in each four types of oxidative stress groups. By addition
treated with 4-NQO activates the complex cellular response that of 10 lM selenoneine in the culture medium, cell viabilities
is controlled by Rpn4 and proteasome subcomplexes. This work showed an increase of 14.4% for 0.2 mM t-BHP, 9.2% for
was supported by Russian Science Foundation (project #17-74- 50 mM AAPH, 8.3% for 0.1 mM pyocyanin, 10.4% for 2 mM
30030) and the Program of fundamental research for state acade- GSNO, as compared with selenoneine free medium. The oxida-
mies for 2013–2020 years (# 01201363822). tive stress conditions induced by these four chemicals can be
repressed by antioxidant effects of selenoneine in the cells. ICP-
MS analysis showed that almost all selenoneine in the medium
P.13-014-Tue was incorporated into K562 cells. Large amounts of selenium
Mitochondrial NADP+ dependent isocitrate were detected in high molecular weight protein fraction by sepa-
dehydrogenase deficiency aggravates high fat ration with the GPC column Ultrahydrogel 120, and these sele-
nium compounds were detected in the cellular fraction in the
diet-induced hypertension
presence of 0.2 mM t-BHP. Small amounts of selenium was due
J. I. Kim1, M. R. Noh2, K. M. Park2
1 to selenoneine. The treatment of high molecular weight fractions
Department of Molecular Medicine and MRC, Keimyung
in the K562 cell with protease or hydrochloride showed the
University School of Medicine, Daegu, South Korea, 2Department
release of selenoneine from the protein fraction. Therefore, sele-
of Anatomy and BK21 Plus, School of Medicine, Kyungpook
noneine may bind to other intracellular protein and form high
National University, Daegu, South Korea
molecular weight complex to protect from the oxidative stress
Obesity is a major risk factor for essential hypertension. ROS conditions.
and oxidative stress is known as a mediator of obesity-induced
hypertension. Mitochondrial NADP+-dependent isocitrate dehy-
drogenase (IDH2) is a producer of mitochondrial NADPH which P.13-016-Mon
is an essential factor in glutathione (GSH) and thioredoxin The relationship of Escherichia coli Hyd
antioxidant systems in the mitochondria. Therefore, we investi- enzymes with the FOF1-ATPase during
gate the role of IDH2 on the obesity-induced hypertension. Obe-
fermentation of mixture of carbon sources
sity in Idh2 gene-deleted (Idh2/) mice and wild-type (Idh2+/+)
H. Gevorgyan, A. Poladyan, A. Trchounian, K. Trchounian
littermates was induced through feeding of a high fat diet
Yerevan State University, Yerevan, Armenia
(HFD). HFD accelerated increase in body weight and mean
blood pressure (MBP) compared to those in the normal diet Escherichia coli is able to produce molecular hydrogen via rever-
(ND) in both mice. HFD-induced increase of MBP was higher in sible membrane-associated four [Ni-Fe]-hydrogenases (Hyd). In
Idh2/ mice than in Idh2+/+ mice. Mitochondrial disruption of this study it was first investigated overall ATPase activity and its
kidney tubule cell and oxidative stress in the kidney tissue were inhibition by N,N0 -dicyclohexylcarbodiimide (DCCD) in E. coli
observed after HFD feeding in both mice. These HFD-induced wild type and hypF mutant lacking all Hyd enzymes during
changes were greater in the Idh2/ mice than Idh2+/+ mice. mixed carbon sources (glucose, glycerol, formate) fermentation at
mRNA levels of renin, angiotensin converting enzyme, and different pHs (7.5, 6.5, 5.5). ATPase activity was higher in wild
angiotensin II receptor type I increased in the HFD mouse kid- type at pH 7.5 than pH 6.5 and pH 5.5. DCCD-inhibited
neys and these increases were higher in Idh2/ mice than in ATPase activity was 89% and 78% of overall ATPase activity in
Idh2+/+ mice. In conclusion, these results demonstrate that wild type and hypF mutant accordingly at pH 7.5. Overall and
IDH2 gene deletion exacerbates HFD-induced hypertension with F0F1-ATPase activity of hypF mutant was decreased compared
greater mitochondrial oxidative stress and activation of RAS sys- to wild type 1.4 fold and 1.6 fold respectively at alkaline pH.
tem in the kidneys, suggesting that mitochondrial redox balance Potassium ions had low effect on overall ATPase activity in wild
plays an important role in obesity-induced hypertension. type and didn’t have influence on hypF mutant at pH 7.5, but
they stimulated DCCD-inhibited ATPase activity in wild type
and hypF mutant. Effect of DCCD was higher in hypF mutant
P.13-015-Wed compared with wild type at slightly acidic pH. Potassium ions
Antioxidative effects of selenium containing didn’t have role in promotion of overall ATPase activity, but
imidazole compound, selenoneine, on human they had stimulating effect on DCCD-inhibited ATPase activity
leukemic K562 cells in wild type and hypF mutant at pH 6.5. Overall and F0F1-
ATPase activity of hypF mutant was decreased compared to wild
T. Seko1, S. Imamura1, K. Ishihara1, Y. Yamashita1,
type at pH 5.5. DCCD had almost the same effect in wild type
M. Yamashita2
1 and hypF mutant at acidic pH. These results establish that alka-
National Research Institute of Fisheries Science, Yokohama,
line pH is the most optimal condition for operation of F0F1-
Japan, 2National Fisheries University, Shimonoseki, Japan
ATPase in E. coli during mixed carbon fermentation and deletion
Selenoneine is a selenium containing imidazole compound discov- of hypF gene is negatively affected not only on overall ATPase
ered in the blood of bluefin tuna. People often eating fish also activity, but also on F0F1-ATPase activity at pH 7.5. Taken
have higher level of selenoneine in their erythrocyte. Since it has together it might be concluded that proton ATPase activity is
strong radical scavenging activity, it is expected to decrease highly dependent on Hyd enzymes and this relationship could be
oxidative stress in erythrocyte. This study verified the effect of due to maintaining intracellular pH and thus proton motive force
selenoneine on oxidative stress in human leukemic K562, and generation.
358 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Redox biochemistry and signalling
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 359
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Redox biochemistry and signalling POSTERS
gated sodium channel activity play a role in the response of can- P.13-022-Mon
cer cells to oxidative stress. We used the strongly metastatic
Autophagy protects neurons and astrocytes
human breast cancer cell line MDA-MB-231, which expresses
functional voltage-gated sodium channel, as a model. Both basal from bilirubin-induced cytotoxicity
and H2O2-induced oxidative stress were measured using 2,7- M. Qaisiya1, P. Mardesic2, B. Pastore3, C. Tiribelli2,
dichlorofluorescein diacetate (DCF-DA). voltage-gated sodium C. Bellarosa2
1
channel activity was blocked by tetrodotoxin or ranolazine. Hebron University, Hebron, Palestinian Territories, 2Fondazione
Application of tetrodotoxin or ranolazine (both 10 lM) to rest- Italiana Fegato ONLUS, Trieste, Italy, 3International School for
ing cells significantly increased ROS levels by 61% and 86%, Advanced Studies (SISSA), Trieste, Italy
respectively, consistent with there being (i) a basal level of oxida- Unconjugated bilirubin (UCB) neurotoxicity involves oxidative
tive stress and (ii) ongoing voltage-gated sodium channel activity. stress, calcium signaling and ER-stress. The same insults also
Application of H2O2 (200 mM) for 30 mins significantly elevated induce autophagy, a process of “self-eating”, with both a pro-sur-
the ROS level by 100%. Co-application of H2O2 with tetrodo- vival or a pro-apoptotic role. Our aim was to study the outcome
toxin or ranolazine significantly increased ROS production fur- of autophagy activation by UCB in the highly sensitive neuronal
ther, by 154% and 131%, respectively. Functional voltage-gated SH-SY5Y cells and in the resistant astrocytoma U87 cells. Upon
sodium channel expression (in particular the persistent current – treatment with a toxic dose of UCB, the conversion of LC3-I to
INaP) endowed metastatic breast cancer cells with significant LC3-II was detected in both cell lines. Inhibition of autophagy
advantage in resisting oxidative stress. Thus, the effectiveness of by E64d before UCB treatment increased SH-SY5Y reduction of
therapeutic regimens (e.g. chemotherapy or radiotherapy), aiming cell viability from 40% to 60% and made U87 cells sensitive to
to kill cancer cells, can be boosted by combination with voltage- UCB. In SH-SY5Y cells autophagy related genes ATG8(5 folds),
gated sodium channel/INaP blockers. ATG18 (5 folds), p62 (3 folds) and FAM 129A (4.5 folds) were
induced 8 h after UCB treatment while DDIT4 up-regulation (13
folds) started at 4 h. mTORC1 inactivation by UCB was con-
P.13-021-Wed firmed by phosphorylation of 4-EBP1. UCB induced LC3-II con-
Glutathione S-transferase; moonlighting version was completely prevented by pre-treating the cells with
protein in haem homeostasis of tick digest the calcium chelator BAPTA and reduced by 65% using the ER-
cells stress inhibitor 4-PBA. Pre-treatment with the PKC inhibitor
J. Perner1, J. Kotal1, P. Sojkova1, P. Brophy2, P. Kopacek1 reduced LC3 mRNA by 70% as compared to cells exposed to
1
Institute of Parasitology, Biology Centre of the Academy of UCB alone. Finally, autophagy induction by Trifluoroperazine
Sciences of the Czech Republic, Ceske Budejovice, Czech Republic, (TFP) increased the cell viability of rat hippocampal primary
2
Aberystwyth University, Aberystwyth, United Kingdom neurons upon UCB treatment from 60% to 80%. In SH-SY5Y
cells, TFP pre-treatment blocked the UCB-induced cleaved cas-
Haem and iron homeostasis in most eukaryotic cells is based on pase-3 protein expression, decreased LDH release from 50% to
a balancing flux between the opposing pathways of haem synthe- 23%, reduced the UCB-induction of HO1, CHOP and IL-8
sis and haem degradation. Ticks, blood-feeding arthropods, mRNAs by 85%, 70% and 97%. Collectively these data indicate
encode neither haem biosynthetic nor haem-degrading enzymes. that the activation of autophagy protects neuronal cells from
Excess of acquired host haem thus needs to be disposed of by UCB cytotoxicity. The mechanisms of autophagy activation by
selective accumulation, export, and scavenging. RNA-seq analy- UCB involves mTOR/ER-stress/PKC/calcium signaling.
ses of the Ixodes ricinus midguts from blood- and serum-fed
females identified an abundant transcript of glutathione S-trans-
ferase (gst) to be substantially up-regulated in the presence of red P.13-023-Tue
blood cells in the diet (blood-meal). We determined the full Recovery of NAD+ level and DNA repair
sequence of this encoding gene, ir-gst1, and found that it is
efficiency by NAD+ precursors in cultivated
homologous to the delta/epsilon-class of GSTs. We have con-
firmed that ir-gst1 expression is induced by dietary haem(oglo- human cells after genotoxic stresses
bin), and not by other components of red blood cells. Kinetics M. Svetlova, O. Lublinskaya, L. Solovjeva, A. Nikiforov
evaluation of recombinant IrGST1 was performed by model and Institute of Cytology, Saint-Petersburg, Russia
natural GST substrates, conforming its ancestral catalytic proper- NAD is critical for the regulation of cell response to genotoxic
ties. The enzyme was also shown to bind haemin in vitro as evi- stresses where it is used as a substrate for poly (ADP-ribose)
denced by inhibition assay, VIS spectrophotometry, gel filtration, polymerases (PARPs). PARP1 is hyperactivated and auto-poly
and affinity chromatography. In the native state, IrGST1 forms a (ADP-ribosyl)ated in response to DNA strand breaks. It accumu-
dimer which further polymerises upon binding of excessive lates poly (ADP-ribose) (PAR) on chromatin at the sites of DNA
amount of haemin molecules. Phylogenetic analysis revealed that damage and facilitates recruitment of DNA repair factors. In this
homologous group of IrGST1 is formed only in tick species, and study we examined the effect of administration of NAD+ precur-
not in other mites or blood-feeding insect. It suggests that these sors on the level of NAD+ and DNA repair after the treatment
enzymes exaptated from its ancestral enzyme only after forma- of human dermal fibroblasts (HDF) and HEK293 cells with
tion of ticks during Arthropods evolution and gained their haem H2O2 or X-ray irradiation. Using immunofluorescence micro-
binding properties. Due to susceptibility of ticks to haem as a scopy and immunoblotting, it has been shown that hyperactiva-
signalling molecule, we speculate that the expression of IrGST1 tion of PARP1 in response to oxidative stress leads to a very fast
in tick midgut functions as intracellular buffer of labile haem and intensive accumulation of PAR in HEK293 cells and to a
pool to ameliorate its cytotoxic effects upon haem liberation of less PAR accumulation in HDF. Significant drop of NAD+ was
haemoglobin hydrolysis. Acknowledgements: The project is sup- observed 30 min after H2O2 treatment in both cell lines, and, at
ported by Czech Science Foundation No. 18-018132S (PK). 24 h, the content of NAD+ was recovered to the control level.
Administration of NAD+ precursors (nicotinamide riboside, NR
or nicotinic acid riboside, NAR) before and during treatment
with H2O2 led to a partial recovery of NAD+ level. Effectiveness
360 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Redox biochemistry and signalling
of DNA single strand break (SSB) repair after H2O2 treatment P.13-025-Mon
was estimated by immunofluorescence microscopy using an anti-
Ultrasound-based imaging of reactive oxygen
body to phosphorylated H2AX (gH2AX), the marker of DNA
double-strand breaks (DSBs) formed from unrepaired SSBs species overproduction associated with
located in opposite DNA strands. Administration of NAR dimin- atherosclerosis in hypercholesterolemic
ished gH2AX staining indicating that SSB repair is more efficient apolipoprotein E-deficient mice
in the presence of NAD+ precursor. Induction of DSBs after X- S. Manea, M. Antonescu, D. Stan, A. Lazar, M. Calin,
ray irradiation of HDF at the dose of 5 Gy only slightly A. Manea
decreased the level of NAD+. The rate of DSB elimination was Institute of Cellular Biology and Pathology “Nicolae Simionescu”,
not changed after pretreatment with FK866, an inhibitor of the Bucharest, Romania
main pathway of cellular NAD+ synthesis. These data suggest
Imaging of critical pathological processes such as oxidative stress,
that intensive poly ADP-ribosylation is not necessary for the effi-
which is typically correlated with robust inflammatory reactions
cient DSB repair. This work was supported by the Russian
and the severity of atherosclerosis is challenging. The aim of this
Science Foundation grant 16-14-10240.
study was to develop at preclinical level an ultrasound-based
imaging method to assess reactive oxygen species (ROS) overpro-
P.13-024-Wed duction in atherosclerosis employing sterically stabilized targeted
liposomes (Lp) encapsulating allylhydrazine (ALH). ALH reacts
Phragmitis Rhizoma attenuates hydrogen
with ROS and results in the generation of echogenic gas (nitro-
peroxide induced oxidative stress and gen and propene)-filled microbubbles. Fluorescent labeled, steri-
apoptosis by regulation of the Nrf2 signaling cally stabilized Lp entrapping the ALH were prepared using the
pathway in Chang liver cells extrusion method. Peptides that recognize the vascular cell adhe-
Y. H. Choi1,2, S. H. Hong1, J. Jeong3, C. Park4 sion molecule-1 were conjugated to the surface of Lp. Male
1
Department of Biochemistry, Dongeui University College of ApoE/ mice were randomized to receive normal (ND) or
Korean Medicine, Busan 47340, Republic of Korea, Busan, South high-fat cholesterol-rich diet (HD) for 10 weeks. The mice were
Korea, 2Anti-Aging Research Center and Blue-Bio Industry RIC, treated via retro-orbital injection with Lp incorporating ALH.
Dongeui University, Busan 47340, Republic of Korea, Busan, After 10 min, the changes in echo intensity triggered by ROS-
South Korea, 3Freshwater Bioresources Utilization Bureau, induced microbubbles were detected by scanning vascular territo-
Nakdonggang National Institute of Biological Resources, Sangju ries prone to atherosclerosis (aortic arch, thoracic aorta) by high
37242, Republic of Korea, Sangju, South Korea, 4Department of frequency, high resolution imaging (Vevo 2100). Pre-formed,
Molecular Biology, College of Natural Sciences, Dongeui nitrogen- and perfluorobutane-filled microbubbles were used as
University, Busan 47340, Republic of Korea, Busan, South Korea positive control. High resolution fluorescence imaging (IVISTM
Caliper) indicated the high uptake of Lp in the aortic atheroscle-
Phragmitis Rhizoma (PR), the roots of Phragmites communis Tri-
rotic lesions and in organs (liver, lungs, kidney). Significant
nius, has long been used as a traditional medicinal herb to
increases in acoustic impedance were detected in the aortic arch
remove heat, promote the production of body fluids, induce
territories of ApoE/ (HD) mice compared to ApoE/ (ND)
excretion of urine and suppress melanogenesis. However, the
animals. ROS-induced ALH echo signals correlated with the
molecular mechanisms of its anti- oxidative activity have not
severity of atherosclerotic lesions (Oil red O) and ROS overpro-
been clearly elucidated yet. In the present study, we investigated
duction (dihydroethidium) in ApoE/ mice. Acoustic detection
the potential therapeutic efficacy of the PR against hydrogen per-
of ROS via ALH-induced chemically-generated gas microbubbles
oxide (H2O2)-induced oxidative stress and apoptosis in Chang
may be a reliable strategy to routinely diagnose early and
liver cells. It was found that exposure of Chang liver cells to
advanced atherosclerosis. Work supported by UEFISCDI (PN-
H2O2 caused a reduction in cell viability by generation of intra-
III-P2-2.1-PED-2016-1308, PN-III-P4-ID-PCE-2016-0665).
cellular reactive oxygen species (ROS), induction of DNA dam-
age, disruption of mitochondrial membrane permeability and
apoptosis. However, pretreatment of PR before H2O2 exposure P.13-026-Tue
effectively attenuated these changes, suggesting that PR pre-
Regulation of autophagy by mitochondrial
vented H2O2-induced oxidative stress and mitochondria-depen-
dent apoptosis. Furthermore, the increased expression and electron transport chain complex III
phosphorylation of nuclear factor erythroid 2-related factor 2 A. V. Tokarchuk, A. A. Panteleeva, K. G. Lyamzaev,
(Nrf2) and up-regulation of heme oxygenase-1 (HO-1), a phase II B. V. Chernyak
antioxidant enzyme, were detected in PR-treated Chang liver Lomonosov Moscow State University, Belozersky Institute of
cells. We also found that zinc protoporphyrin IX (ZnPP), a Physico-Chemical Biology, Moscow, Russia
HO-1 inhibitor, attenuated the protective effects of PR against We have shown that mitochondrial complex III inhibitors
H2O2-induced oxidative stress and apoptosis. Collectively, these decrease autophagy activity, however mechanism of this phenom-
findings indicate that PR enhances cellular antioxidant defense ena remains unclear. We assumed that in this process an impor-
capacity through the inhibition of ROS generation and the acti- tant role is played by dihydroorotate dehydrogenase (DHODH)
vation of the Nrf2 signaling pathway, thus protecting Chang liver that could be inhibited by myxothiazol. DHODH is localized in
cells from H2O2-induced oxidative stress and apoptosis. [This the inner mitochondrial membrane and catalyzes the oxidation of
research was supported by Basic Science Research Program dihydroorotate to orotate that is used for biosynthesis of pyrim-
through the National Research Foundation of Korea (NRF) idines that important for further phospholipids biosynthesis. The
grant funded by the Korea government (2015R1A2A2A01004633 activity of this enzyme is associated with the activity of complex
& 2017R1D1A1B03032689)]. III as DHODH uses ubiquinone as the electron acceptor. We
demonstrated that incubation of cells with complex III inhibitor
myxothiazol as well as with DHODH inhibitor leflunomide leads
to significant pyrimidines depletion, p53 activation and reduces
both basal and rapamycin induced autophagy in Rko and Hela
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 361
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Redox biochemistry and signalling POSTERS
cells, while adding to the cells incubation medium of uridine, is the key regulator of its activity under hypoxia. Na,K-ATPase
cytidine or cytidine diphosphate choline neutralizes these effects. is also the receptor for cardiotonic steroids, in particular, oua-
Adding of phosphatidylethanolamine or phosphatidylcholine bain. One of the main ways of signal transmission upon binding
does not affect the level of pyrimidines in the cell, but prevents of ouabain is the activation of Src kinase, which forms a complex
the inhibitory effect of myxothiazol on autophagy. We assume with Na,K-ATPase. Earlier we observed that under hypoxia
that low levels of pyrimidines could influence the autophagy at receptor function of Na,K-ATPase is inverted: binding of oua-
least in two ways: by p53 protein activation and by inhibition of bain leads to decrease of ROS and GSH and decrease of Src
biosynthesis of a number of key phospholipids in the cell that kinase activating phosphorylation. In this study, to reveal the
may play a signaling role in the mechanism of autophagy. Our mechanism of redox regulation of Na,K-ATPase receptor func-
results could be important for the clinical application, as tion, we exposed murine fibroblast cells to hypoxia or ouabain,
DHODH inhibitors are actively investigated for the use in treat- monitored the level of ROS and GSH and studied the influence
ment of autoimmune and immune abnormalities, multiple sclero- of Na,K-ATPase glutathionylation on its interaction with Src.
sis and some cancers, however the influence of these inhibitors We have found that ROS growth occurs within 10 min after
on autophagy is disregarded. hypoxia or ouabain treatment, while GSH level – only after
30 min of affection, which may be an adoptive response to
oxidative stress, caused by hypoxia or ouabain. We have
P.13-027-Wed observed that activation of Src coincides in time with the growth
AQP5-mediated hydrogen peroxide of ROS under hypoxia or ouabain treatment. We have demon-
permeation is altered by nutritional strated that glutathionylation of Na,K-ATPase can be caused not
only by hypoxia, but also by ouabain treatment, and it is accom-
compounds and induces cell resistance
panied by Src activation. The proposal mechanism of redox regu-
C. Rodrigues1, A. F. M osca1, D. Lopes2, P. A. Pedersen3,
lation of Na,K-ATPase receptor function is as follows: exposure
M. Henriques , F. Antunes4, G. Soveral1
1
1 to hypoxia or binding of ouabain to Na,K-ATPase causes growth
Research Institute for Medicines, Faculty of pharmacy,
of ROS, which leads to glutathionylation of Na,K-ATPase. Glu-
Universidade de Lisboa, Lisbon, Portugal, 2Faculty of pharmacy,
tathionylation, in turn, prevents the interaction of Na,K-ATPase
Universidade de Lisboa, Lisbonne, Portugal, 3Department of
with the kinase domain of Src, resulting in the activation of Src.
Biology, university of Copenhagen, Copenhagen, Denmark, 4Centro
Under hypoxia ouabain binding decreases the level of ROS and
de Quımica e Bioquimica e departamento de quimica e bioquimica,
induces deglutathionylation of Na,K-ATPase with the consequent
Faculdade de Ci^ encias, Universidade de Lisboa, Lisbon, Portugal
deactivation of Src. The study was supported by Russian Science
Aquaporins (AQPs) are a family of transmembrane proteins pre- Foundation (grant #14-14-01152).
sent in all types of organisms and involved in the selective trans-
port of water and small solutes such as glycerol. A few isoforms
can also permeate H2O2 (peroxiporins). So far, 13 aquaporins P.13-029-Tue
(AQP0-12) were identified in humans and reported to be involved Utilization of Se-containing imidazole
in a wide range of physiological functions, as water/salt home- compound, selenoneine, extracted from tuna
ostasis, exocrine fluid secretion and epidermal hydration, and
and fisheries wastes
human diseases including glaucoma, cancer and obesity. High
M. Yamashita1, Y. Yamashita2
level of H2O2 misbalance cell redox reactions and may induce 1
National Fisheries University, Shimonoseki, Japan, 2National
tumorigenesis. Recently, we assessed AQP-mediated hydrogen
Research Institute of Fisheries Science, Yokohama, Japan
peroxide permeation of mammalian AQPs individually expressed
in yeast and reported the rat AQP5 can mediate H2O2 transport. Selenoneine, 2-selenyl-Na,Na,Na-trimethyl-L-histidine, is a
Sequence alignment of human and rat AQP5 isoforms show a strong antioxidant found in the tissues of tuna and other fish that
sequence identity of 91%. Thus, we next investigated H2O2 per- can mediate the prevention of autoxidation of heme proteins,
meation by human AQP5 and related cell resistance to oxidative such as hemoglobin and myoglobin. We measured concentrations
stress. The results indicate that, similarly to AQP3 and AQP8, of selenoneine, total selenium, and total mercury in the red mus-
human AQP5 also permeates H2O2 and importantly, induces cell cles of tuna species. Red muscles of yellowfin tuna contained the
resistance under oxidative stress. In addition, the effect of anti- high levels of selenium at 5.6–21.2 mg/kg, and almost all sele-
oxidant compounds on hAQP5-mediated H2O2 accumulation nium in tuna muscles was identified to be selenoneine by HPLC-
and cell growth was investigated. Curcumin and naringenin ICP-MS analysis. We developed selenium-rich surimi products
enhanced cell resistance in AQP5-transformed yeast cells and from the red muscles of yellowfin tuna. The surimi product con-
anti-proliferative properties known for these compounds were tained selenoneine at 0.28 mg Se/kg. We also concentrated and
attenuated in yeast cells expressing human AQP5. These data purified selenoneine from the waste water and internal organs of
propose the AQP5 isoform as a key player in oxidative stress the surimi processing of fish. After filtration of the boiling water
resistance and point to a novel mechanism of AQP5 involvement by reverse osmosis membrane and column chromatography with
in tumorigenesis. cation exchange and Sepabeads resins, selenoneine was purified
by reverse-phase HPLC. The tuna surimi products and the puri-
fied selenoneine concentrate can be applied to be the selenium-
P.13-028-Mon rich functional food and feed to enhance selenium-mediated
The role of glutathionylation in redox antioxidant functions.
regulation of Na,K-ATPase receptor function
V. Lakunina, I. Petrushanko, V. Mitkevich, A. Anashkina,
A. Makarov
Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences, Moscow, Russia
Na,K-ATPase maintains the homeostasis of Na+ and K+ in ani-
mal cells. Na,K-ATPase is redox–sensitive, and glutathionylation
362 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemical processes at cellular membranes
Biochemical processes at cellular membranes AQP1 and AQP2 mediate water reabsorption from primary urine
across the apical membrane of proximal and distal nephron seg-
ments. Studies indicate that renal functional/structural changes in
P.14-001-Mon humans and experimental animals develop with age. Aging pro-
STIM1 deficiency triggers mitochondrial motes fluid loss in tissues which could reflect changes in the
impairment and senescence by upregulation of expression of AQPs along the nephron. We investigated the pos-
L-type voltage operated Ca(2+) entry in SH- sible age-ameliorating effects of two potential geroprotector sub-
SY5Y cells stances, melatonin (endogenous hormone) and resveratrol (plant
flavonoid), on AQP1 and AQP2 expressions in the rat model of
C. Pascual-Caro, A. M. Lopez-Guerrero, E. Pozo-Guisado,
experimental aging. Three month old rats of both sexes were
F. J. Martin-Romero
given melatonin or resveratrol for 21 months via drinking water
University of Extremadura, Badajoz, Spain
(~1 mg/kg b.w./day), whereas control animals drank vehicle
STIM1 is a transmembrane protein located at the endoplasmic (0.01% ethanol in drinking water). 24-h urine was collected from
reticulum (ER), with a role in Ca2+ mobilization and signaling. animals using metabolic cages. The renal expression of AQP1
As a sensor of the Ca2+ level within the ER, STIM1 modulates and AQP2 was determined using immunofluorescence cytochem-
plasma membrane (PM) Ca2+ channels to regulate the store- istry tissue cryosections and Western blot analysis in isolated
operated Ca2+ entry (SOCE). However, STIM1 also inhibits the total cell membranes. Results showed that melatonin and resvera-
voltage-operated calcium channel Cav1.2 by direct binding of trol had no effect on renal AQP1 expression in both sexes. AQP2
STIM1 to the channel and by enhancing the channel internaliza- expression in male and female kidneys of resveratrol-treated rats
tion from the PM. Besides, it has been shown that the presenilin- also did not change compared to controls. Melatonin exerted a
1 (PS1)-associated c-secretase interacts with STIM1 in neuroblas- sex-dependent AQP2 upregulation in the female kidney inner
toma SH-SY5Y cells, familial Alzheimer’s disease patient fibrob- stripe and papilla. However, no differences were found in the 24-
lasts, and mouse primary cortical neurons, and that STIM1 is h urine outputs between treated and control animals, indicating
cleaved at the transmembrane domain, where the protein shows a that melatonin and resveratrol did not impact the renal AQP
target sequence for c-secretase. To understand better the role of expression to significantly alter urine production in aging rats.
STIM1 in neurodegenerative diseases, we designed a strategy to (Croatian Science Foundation project IP-2013-11-1481).
knock-out the expression of STIM1 gene in SH-SY5Y neuroblas-
toma cell line, as an ex vivo model to study the phenotype of
STIM1-deficient cells. As a result, we proved that STIM1, which P.14-003-Wed
was silenced by CRISPR genome editing, is not required for dif- Stress related changes in Na+/K+-ATPase
ferentiation but it is essential for cell survival of differentiating activity and way of action of creatine
cells. Differentiated cells showed inner mitochondrial membrane G. Burjanadze, N. Dachanidze, K. Menabde, M. Chachua,
depolarization, measured with tetramethyl-rhodamine, as well as M. Koshoridze, N. Koshoridze
senescence, measured with the substrate of the senescence-asso- Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia
ciated b-galactosidase C12FDG. In parallel, STIM1-KO cells
showed an upregulation of CACNA1C gene transcripts, which Negative influence of psycho-social stress is widely common. In a
correlates with a potentiated Ca2+ entry in response to depolar- recent years, Creatine (Cr) that has been known for its involve-
ization with KCl. The stable knocking-down of CACNA1C tran- ment in the high energy phosphate shuttle has been actively dis-
scripts with specific shRNA restored mitochondrial function, and cussed for its wide range of impact on different cellular systems
dropped senescence to basal levels, confirming the essential role and amongst them membrane pumps. The Na+/K+-ATPase is
of Cav1.2 channels in the cell death triggered by STIM1 defi- one of the enzymes that is influenced by various factors and
ciency. Funded by grants BFU2014-52401-P and IB16088 of the works for maintaining balance in the cell and thought to be
Spanish Ministerio de Economia and Junta de Extremadura. tightly functionally bound to the NMDA Receptor in neural sys-
tem. Although the data show no significant changes in Na+/K+-
ATPase activity in hippocampus of control group rats under the
P.14-002-Tue 30-days intraperitoneal injection (i.p.) of Cr (140 mg/kg), the
Effect of melatonin and resveratrol on renal valuable results were taken for the stressed ones, were the enzyme
expression of aquaporins 1 and 2 in 2-year-old activity was taken back to the normal value. In addition to the
normalization Na+/K+-ATPase activity, the 30-days i.p. injec-
male and female rats tion of Cr had positive influence on stress induced increase in
D. Karaica1, I. Vrhovac Madunic1, V. Micek2, M. Ljubojevic1, Ca2+ levels. Such changes were predicted to be due to the modu-
M. Geric3, G. Gajski3, D. Rasic4, M. Peraica4, T. Orct5, lation of NMDA Receptor as no changes were seen in control
J. Jurasovic5, I. Novak Jovanovic4, L. Nanic6, I. Rubelj6, group. The idea was strengthened by in vitro experiments with
I. Sabolic1, D. Breljak1 Glutamate/Glycine and Ketamine and co-addition them with the
1
Institute for Medical Research and Occupational Health, Cr in the reaction media of hippocampal slices. Cr was seen as a
Molecular Toxicology Unit, Zagreb, Croatia, 2Institute for blocker of Glutamate binding site and prevent from the stimula-
Medical Research and Occupational Health, Laboratory Animals tion of NMDA receptor. Finally it can be concluded that there is
Unit, Zagreb, Croatia, 3Institute for Medical Research and no direct relations between Cr and Na+/K+-ATPase, but under
Occupational Health, Mutagenesis Unit, Zagreb, Croatia, the certain pathological conditions it can maintain the homeosta-
4
Institute for Medical Research and Occupational Health, sis of the system by indirect influence on the Na+/K+-ATPase
Toxicology Unit, Zagreb, Croatia, 5Institute for Medical Research via NMDA receptor, thus retrieving the membrane potential and
and Occupational Health, Analytical Toxicology and Mineral diminishing the negative influence of psycho-emotional stress.
Metabolism Unit, Zagreb, Croatia, 6Ruder Boskovic Institute, However, the exact mechanisms of this interaction need more
Division of Molecular Biology, Laboratory for Molecular and detailed research and clarification.
Cellular Biology, Zagreb, Croatia
Water channels (aquaporins; AQPs) facilitate the passive move-
ment of water across cell membranes. In mammalian kidneys,
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 363
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemical processes at cellular membranes POSTERS
364 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemical processes at cellular membranes
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 365
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemical processes at cellular membranes POSTERS
comprehensive knowledge of the processes going on within a programme under the Marie Sklodowska-Curie grant agreement
synapse, since the processes in question determine the coordinated no. 665735 (Bio4Med) and by the funding from Polish Ministry
integral activity of the central nervous system. In spite of the fact of Science and Higher Education within 2016–2020 funds for the
that much in this respect is already known, many problems still implementation of international projects (agreement no 3548/
remain undefined, or insufficiently studied. In order to settle them, H2020/COFUND2016/2).
it is necessary to study the individual processes taking place within
a synapse and to define their role in the complex sequence of pro-
cess chain during synaptic transmission. Ca2+ and respectively its P.14-012-Wed
transport systems take one of the important places in these pro- Length of the cytoplasmic C-terminal part of
cesses. One of the significant transportable systems of Ca2+ is yeast Nha1 Na+/H+ antiporter influences its
Mg2+-depending Ca-ATPase. Activity of this latter is controllable. plasma-membrane targeting in cells lacking
Study of regulation Ca-ATPase activity is especially important
with neurotransmitters, in our case with dopamine, for the neuro- Erv14 COPII cargo receptor
transmitters are directly involved in synaptic transmission Study of M. Hraskova, K. Papouskova, H. Sychrova, O. Zimmermannova
the effect of dopamine and synaptosomal factor (factor localized in Department of Membrane Transport, Institute of Physiology
the synaptic cytosol) on the Ca-ATPase activity, localized in the ASCR, Prague, Czech Republic
albino rat brain synapses and microsomal fraction revealed: 1) All living cells spend a lot of energy to maintain their intracellu-
Dopamine activates Ca-ATPase activity, localized in synapses; 2) lar alkali-metal-cation homeostasis. In yeasts, various trans-
Dependence of Ca,MgATPase activity on dopamine concentration porters work in plasma and intracellular membranes to ensure
has bell like shape, maximum at 0.05 mM concentration; 3) Dopa- proper alkali-metal-cation concentrations both in the cytoplasm
mine does not effects Ca,MgATPase (localized in albino rat brain and in the organelles. In the model yeast Saccharomyces cere-
synaptosomal membrane) transport stoichiometry; 4) Synaptoso- visiae, the plasma-membrane Na+/H+ antiporter Nha1 interacts
mal factor does not affect CaATPase activity; 5) Together with with the COPII cargo receptor Erv14. The lack of Erv14 results
dopamine and synaptosomal factor inhibits Ca-ATPase activity, in intracellular stacking of Nha1 and affects its function. Erv14
localized in synapses; 6) Dopamine and synaptosomal factor do binds the transmembrane domains of Nha1, but, interestingly, no
not have effect on Ca-ATPase activity localized in microsomal intracellular stacking of Nha1 in erv14D cells can be observed
fraction. when Nha1 lacks its long hydrophilic C-terminal part. To reveal
the role of Nha1’s C-terminus in its intracellular stacking in
erv14D cells, we first studied the function and localization of
P.14-011-Tue Nha1 homologues from various yeast species produced in
Exit of SLC6A14, an amino acid transporter B S. cerevisiae cells either lacking or possessing Erv14. Studied Nha
(0,+), from the endoplasmic reticulum depends antiporters significantly differ in the lengths of their C-terminal
on the interaction with a cargo recognizing parts and our results suggest that Erv14 is especially important
for proper plasma-membrane delivery of antiporters with long C-
COPII element SEC24C
termini. Second, we prepared several mutated versions of ScNha1
V. Kovalchuk1, L. Samluk1, B. Juraszek1, D. Jurkiewicz1,
with truncated C-terminal part and studied their function and
S. Sucic2, M. Freissmuth2, K. A. Nałecz1
1 localization in cells with or without Erv14 protein. This approach
Nencki Institute of Experimental Biology, Warsaw, Poland,
2 allowed us to reveal which region of Nha1’s C-terminus is
Institute of Pharmacology, Medical University of Vienna, Vienna,
responsible for the antiporter’s requirement of Erv14 in its proper
Austria
plasma-membrane targeting. This work was supported by a
The solute carrier 6 (SLC6) gene family encodes for transporters GACR grant 17-01953S.
of neurotransmitters, amino acids, osmolytes, and energy
metabolites. SLC6A14 [ATB(0,+)] is a Na+/Cl--dependent trans-
porter for neutral and cationic amino acids. Moreover, its expres- P.14-013-Mon
sion is increased in certain cancers. In the initial step of Trk transporters mediate potassium uptake
trafficking to the plasma membrane, it has to interact with the and contribute to pH homeostasis in Candida
cargo recognizing protein SEC24 within the coatomer II (COPII) species
complex to exit the endoplasmic reticulum (ER). Trafficking of
H. Elicharova1, V. Llopis-Torregrosa1, H. Sychrova2
rat SLC6A14 to the plasma membrane was studied in a heterolo- 1
Institute of Physiology CAS, Prague 4, Czech Republic, 2Institute
gous expression system in HEK293 cells to understand, which of
of Physiology, The Czech Academy of Sciences, Prague, Czech
the four paralogues of SEC24 was required for its ER export.
Republic
Immunofluorescence and biotinylation analyses showed that
more than half of SLC6A14 reached the plasma membrane in its The regulation of ion and pH homeostases is an essential process
fully glycosylated form after 48 h. Experiments with dominant critical for cell viability. The maintenance of high intracellular
negative mutants of COPII component Sar1A-T39N verified that concentrations of potassium and neutral pH is important for a
the export from the ER was COPII-dependent. In fact it was variety of cellular functions including cell volume, DNA integ-
exclusively dependent on a direct interaction with SEC24C, as rity, protein modification and trafficking. It is becoming increas-
shown by immunoprecipitation, immunofluorescence and the ingly evident that the coordination between primary H+-
proximity ligation assay. Interestingly, co-transfection with a ATPases and transport systems involved in the influx and efflux
dominant mutant of SEC24C did not only decrease the surface of potassium allows this pH maintenance to occur. Genes encod-
levels of SLC6A14 but also its total cellular amount, a finding ing three types of potassium uptake systems have been found in
also seen after co-transfection with Sar1A-T39N. This suggested genomes of pathogenic Candida species. In silico studies revealed
that a portion of ER-resident SLC6A14 was subject to ER-asso- that Candida spp. differ in the number and type of potassium
ciated degradation, a conjecture, confirmed by experiments with transporters. Whereas C. albicans possesses genes for all three
the proteasome inhibitor bortezomib. This work was supported known types of transporters (Trk1, Hak1, Acu1), C. glabrata
by the European Union’s Horizon 2020 research and innovation and C. krusei genomes contain only genes encoding a putative
366 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemical processes at cellular membranes
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 367
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemical processes at cellular membranes POSTERS
368 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemical processes at cellular membranes
sequences around the signal-anchor and C-terminus. The Golgi protein-interacting theory is not able to explain all of the phar-
and plasma membrane proteins could be discriminated with high macological and toxicological features of these agents. To study
accuracy (> 90%) using the PSSM created by the signal-anchor the molecular mechanisms of the influence of LAs on ionic chan-
sequences. The trans-Golgi membrane proteins are well distin- nels we used pore-forming antimicrobial agents reconstituted into
guished from plasma membrane proteins using the PSSM created planar lipid bilayers. We found that the effects of LAs on the
by the C-terminal sequences. These results suggested that the conductance of single ionic channels induced by well-known pep-
amino acid propensities around signal-anchor were related to the tide antibiotic, gramicidin A, and by antifungal cyclic lipopep-
Golgi to plasma membrane localization mechanism. The C-term- tide, syringomycin E, were caused by their influence on
inal sequence was considered to related to the trans-Golgi-plasma membrane electrostatic potentials; the regulation by amide-linked
membrane transport. To verify this presumption by experimental compounds, in particular, lidocaine, prilocaine, and mepivacaine,
methods, the GFP fusion proteins with signal-anchors of the rep- was related to the membrane surface potential, whereas modula-
resentative proteins selected from each group were designed. The tion by ester-bound LAs, in particular, tetracaine, was predomi-
subcellular localization of these GFP fusion proteins expressed in nantly due to changes in a dipole potential. Moreover, at high
HeLa cells were observed by confocal laser fluorescence micro- electrolyte concentration in the membrane bathing solution
scope. amide-linked LAs block ion passage through syringomycin chan-
nels; they permeate a pore from its wider lipid mouth; this leads
to a sharp drop in channel lifetime and conductance at negative
P.14-020-Tue transmembrane voltages. The cooperativity of interaction
Comparative analysis of GPI modification observed with lidocaine indicated the existence of specific binding
mechanisms between human and rice plant sites. LAs affecting lipid packing, especially, tetracaine, led to a
proteins significant increase in the steady-state membrane conductance
induced by antifungal polyene macrolides, nystatin and ampho-
K. Etchuya1,2, T. Kikegawa3, H. Sugita3, H. Kaku4, Y. Mukai3
1 tericin B, syringomycin E, and antimicrobial peptides, magainin
Biomedical Research Institute, National Institute of Advanced
and melittin. The results were consistent with a lipid-mediated
Industrial Science and Technology, Tsukuba, Japan, 2Dept. Electr.
mode of action of the LAs via decreasing a curvature stress at
& Bioinform., Sch. Sci. & Tech., Meiji University, Kawasaki,
the formation of toroidal pores. The work was supported by the
Japan, 3Dept. Electr, Grad. Sch. Sci. & Tech., Meiji University,
Russian Science Foundation (# 14-14-00565-P).
Kawasaki, Japan, 4Dept. Lifesci., Grad. Sch. Agr., Meiji
University, Kawasaki, Japan
Glycosylphosphatidylinositol (GPI) is one of the protein post- P.14-022-Mon
translational modification molecules. GPI-anchored proteins RNA-binding to negatively charged membrane
(GPI-APs) in mammalian cells are known to localize themselves surfaces affects the structure of the membrane
to raft domain via the endoplasmic reticulum (ER). GPI-APs
and of the RNA
have two signal sequences, signal-peptide (SP) and GPI-attach-
S. Pannwitt, K. Slama, M. Helm, D. Schneider
ment signal (GPI-AS), which are N-terminal ER localization
Institute of Pharmacy and Biochemistry, Johannes Gutenberg-
sequences and C-terminal signal sequences for GPI modification,
University, Mainz, Germany
respectively. Plant proteins also have GPI modification systems
as well as mammalian proteins, can translocate to the cell wall or Based on the RNA world hypothesis, early RNA molecules are
the raft region on the cell membrane. However, because few the origin of life. However, this idea begs the question of what
plant proteins have been isolated as GPI-APs, the plant GPI actually occurred in primordial cells when RNA was encapsu-
modification mechanisms have not been clarified. In this study, lated within a membranous compartment. Did the RNA anyhow
GPI modification mechanisms were compared between plant and “interact” with the lipid bilayer? And did such an interaction
human proteins using bioinformatics and experimental anyhow impact the structure and integrity of the membrane or
approaches. By the bioinformatics analysis, some differences the RNA molecule? Recently, RNA molecules were identified
between plant and human GPI-APs were found in the physico- that were suggested to interact with phosphatidylcholine (PC)
chemical characteristics, especially in SPs. Furthermore, the wild- bilayers, which are net uncharged. We now have synthesized
type and chimera CEBiP (Chitin Elicitor Binging Protein) were these RNA molecules in vitro and have analyzed their interaction
expressed in the HeLa cells, and the subcellular localization of with model membranes in great detail. While we did not observe
the wild-type and chimera CEBiP was observed by the immunos- any impact of the RNA molecules on the organization of PC
taining method. The results indicated that one of the differences membranes, we detected formation of liposome aggregates. How-
of the GPI modification mechanisms between human and plant ever, in presence of the RNA molecules the phase transition tem-
proteins is the protein transport system which depends on SPs. perature of phosphatidylglycerol (PG) liposomes was shifted to
higher temperatures. Since NaCl exterminated this effect, binding
of RNA to the PG surfaces appears to be electrostatically driven.
P.14-021-Wed Yet, this was a rather unexpected finding, as PG membranes
Dualism of action of local anesthetics on ionic carry a negative surface charge, and the negatively charged RNA
channels formed by antimicrobial agents appears to interact specifically with negatively charged membrane
O. Ostroumova, S. Efimova, A. Zakharova, E. Chulkov, surfaces. Furthermore, interaction with the negatively charged
L. Schagina, V. Malev PG, but not with the zwitterionic PC, does affect the RNA sta-
Institute of Cytology of the Russian Academy of Science, St. bility and decreases the RNA melting temperature. In summary,
Petersburg, Russia we could show that RNA molecules can interact with membrane
surfaces, and such an interaction does affect the physico-chemical
Despite a long history of an application, the precise mechanisms properties of one other.
of action of local anesthetics (LAs) are still under debate. The
question is whether the effects are induced by acting on func-
tional proteins, on membrane lipids, or on both. It is generally
recognized that LAs block sodium channels, but membrane
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 369
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemical processes at cellular membranes POSTERS
370 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemical processes at cellular membranes
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 371
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Signalling through membranes and receptors POSTERS
a key step of autophagy, however our knowledge about its provide basic data to understand and estimate the effects on the
genetic regulation is still limited. During the autophagic process, human beings.
the cellular endomembrane system undergoes extensive transfor-
mation. Small GTPases of the Rab protein family are major reg-
ulators of membrane dynamics. The nucleotide bound state of P.14-032-Tue
Rab proteins is dependent of several cofactors, including the acti- Negative charge and membrane tethered viral
vator GEFs (guanine nucleotide exchange factor) and inactivator 3B cooperate to recruit viral RNA-dependent
GAPs (GTPase-activating protein). Our research focuses on the RNA polymerase 3Dpol
Rab3GAP2 protein that forms a heterodimeric complex with
A. Dubankova1, J. Humpolickova2, E. Boura2
Rap3GAP1. Rab3GAP1/2 complex is multi-modal Rab cofactor 1
Institute of Organic Chemistry and Biochemistry IOCB Research
as on one hand it can function as a GAP for Rab3, but on the
Centre & Gilead Sciences, Academy of Sciences of the Czech
other, it can also act as a GEF for Rab18. In our study using
Republic, Flemingovo n am. 2, 166 10, Prague, Czech Republic,
Drosophila melanogaster, we found that the loss of Rab3GAP2
Prague, Czech Republic, 2IOCB, Prague, Czech Republic
inhibits autophagic degradation. In addition, we showed that loss
of Rab3GAP2 leads to the severe alteration of lysosome mor- Most +RNA viruses replicate at replication organelles (ROs)
phology and function leading to autophagosome accumulation. known as replication factories that are derived from the host
We also found that knocking-down Rab18 inhibits autophagy in membranes (Golgi or ER most often). ROs provide microenvi-
a similar way to Rab3GAP2. Finally, we found that aged Rab3- ronment needed for efficient viral replication and also serve as a
GAP2 mutants show neuromuscular disorders, which is a well- shelter from innate intracellular immunity. The key role in
known hallmark of autophagy defect. Taken together our results +RNA viral replication plays viral RNA dependent RNA poly-
suggest that Rab3GAP-Rab18 module acts as a novel regulator merase 3Dpol. This enzyme replicates RNA at (ROs) and the
of autolysosome maturation. lipid hallmark of the Golgi – the phosphatidylinositol 4-phos-
phate (PI4P) lipid – was implied in recruitment of 3Dpol to ROs.
However, +RNA viruses do not possess any phosphatidylinositol
P.14-031-Mon 4-kinase (PI4K), instead they hijack the human PI4Ks. Many
The genotoxic and antioxidant effects of picornaviruses use the Golgi resident acyl-CoA-binding domain-
environmental pollutant sodium omadine on containing protein-3 (ACBD3) to hijack the lipid kinase PI4KB1.
Here, we show using biomimetic model membranes, hijacking of
zebra fish (Danio rerio)
PI4KB by the nonstructural viral 3A protein via the ACBD3
R. Tural1, I. Yilmaz2, A. C. Gunal3, A. Sepici Dincel4
1 protein. Upon hijacking, PI4KB hyperphosphorylates membranes
Vocational School of Health Services, University of Sinop, Sinop,
overproducing the PI4P. However, we found that not PI4P but
Turkey, 2Department of Environmental Sciences, Graduate School
rather the negative charge is responsible for the recruitment of
of Natural and Applied Sciences, University of Gazi, Ankara,
3Dpol to the membrane as PS is as efficient as PI4P2. Addition-
Turkey, 3Division of Biology Education, Department of
ally, we show that membrane tethered 3B protein cooperates with
Mathematics and Science Education, Faculty of Education,
the negative charge to increase the efficiency of 3Dpol recruit-
University of Gazi, Ankara, Turkey, 4Gazi University Faculty of
ment.
Medicine, Department of Medical Biochemistry, Ankara, Turkey
References
Growing of living organisms such as bacteria, protozoans, algae, 1. Klima, M., Chalupska, D., Rozycki, B., Humpolickova, J.,
and crustaceans can accumulate in large numbers on surfaces like Rezabkova, L., Silhan, J., Baumlova, A., Dubankova, A. &
pipes, tanks, and ships’ hulls, resulting in corrosion, clogging and Boura, E. (2017) Kobuviral Non-structural 3A Proteins Act as
contamination, known as biocides. The control of biofouling in Molecular Harnesses to Hijack the Host ACBD3 Protein, Struc-
water systems is achieved through the avoidance of natural ture. 25, 219–230.
recruitment, physical removal and the use of antifoulants. 2. Dubankova, A., Humpolickova, J., Klima, M. & Boura, E.
Sodium omadine (NaoM) is a commonly used disintegrant and (2017) Negative charge and membrane-tethered viral 3B cooper-
biocide for micro-organisms. The aim of the present study is to ate to recruit viral RNA dependent RNA polymerase 3D (pol),
determine the effect of environmental pollutants (NaoM) on Scientific reports. 7, 17309. The Project was supported by Inter-
aquatic toxicology and the possible negative effects on humans BioMed LO1302 and RVO: 61388963.
by evaluating DNA/RNA oxidative damage, and free radicals.
To generate tissue damage six groups of zebrafish (length 3–
Signalling through membranes and receptors
4.5 cm) were exposed to NaoM 1 lg/l and 5 lg/l for the planned
time intervals as 24, 72 and 96 h. To evaluate the DNA/RNA
oxidative damage a total zebra fish was homogenised for DNA P.15-001-Mon
isolation, hydrolysed and damage was measured by commercial Molecular mechanisms of vitamin D3 action
kit as EIA. In addition, catalase (CAT) and superoxide dismutase on bone resorption/formation balance in Type
(SOD) levels were measured by commercial spectrophotometric
1 diabetes
enzyme assay kits. DNA/RNA oxidative damage as 8-hydroxy-
A. Mazanova, I. Shymanskyi, O. Lototska, O. Makarova,
20 deoxyguanosine (pg/ml) and SOD activity (U/ml) was statisti-
M. Veliky
cally significantly different compared to 24 h (1 lg/l) group
Palladin Institute of Biochemistry of the National Academy of
(P < 0.05). Zebrafish (Danio rerio) is becoming a more widely
Sciences of Ukraine (NASU), Kyiv, Ukraine
used and increasingly powerful model organism for many fields
of modern biomedical research. In conclusion, as marine pollu- Type 1 diabetes (T1D) is endocrine disorder, accompanied by a
tion and deterioration of ecosystems are directly affect the number of side effects, one of which is secondary osteoporosis.
humans. the results presents awareness among environmental T1D-induced osteoporosis can be attributable to significant dis-
pollution, marine pollution and health problems. The toxic turbances in osteoblast to osteoclast balance caused, in particu-
effects of environmental pollutants on DNA damage and antioxi- lar, by changes in the activity of such proinflammatory
dant systems on different stages of organisms on the food web transcription factors as the nuclear factor kB (NF-kB) and the
372 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Signalling through membranes and receptors
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 373
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Signalling through membranes and receptors POSTERS
374 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Signalling through membranes and receptors
maintained significantly higher (Trk2-less strain) or lower (Trk1- (Tag7-LA) express membrane FasL, which interacts with the Fas
less strain) Ca2+ concentration. We also measured the transient receptor on the surface of the target cell and triggers the mecha-
increase of cytosolic Ca2+ during an osmotic shock and found nisms of cell death in HLA-negative tumor cells. It was shown
that the strain lacking both Trk systems had the highest amount that Tag7-LA induce in tumor cells 2 types of programmed cell
of Ca2+ released into the cytosol after the osmotic shock. On the death (PCD): apoptosis and necroptosis. The apoptosis was
other hand, a strain lacking all K+ importers and exporters induced by Tag7-LA after short time of incubation through cas-
exhibited the lowest amount of released Ca2+ in the cytosol and pases activation. However, some tumor cells inhibit caspases and
the slowest reabsorption of Ca2+ back to the vacuole. Alto- prevent development of caspase-depended apoptosis. We have
gether, our data revealed mutual interconnection between potas- detected necroptosis in target cells after long-time incubation
sium and calcium homeostasis in yeast cells. This work was Tag7-LA with tumor cells. Using the methods of inhibitory
supported by GACR (17-01953S). assay, western blot analysis and flow cytometry, we have demon-
strated the main stages of necroptosis. We have approved that at
the initiation of Fas-mediated necroptosis, a necrosome consist-
P.15-008-Tue ing of RIP1 kinase and RIP3 kinase is assembled. We have
Octarphin, a nonopioid peptide of opioid shown the participation of lysosomes enzymes cathepsin B and
origin D, ROS-generation and P-STAT3 translocation in mitochondria.
E. Navolotskaya The results of this study confirm the fact that induction of
Branch of Shemyakin and Ovchinnikov Institute of Bioorganic necroptosis occurs after the activation of the Fas-receptor by a
Chemistry of the Russian Academy of Sciences, Pushchino, FasL and it’s characterized by recruitment of necrosome and
Moscow Region, Russia involvement of lysosomes and mitochondria and it’s accompa-
nied by ROS accumulation. This work was supported by Russian
The peptide octarphin (TPLVTLFK) corresponding to the Science Foundation grant N 15–14–00031.
sequence 12–19 of beta-endorphin, was synthesized. It has been
shown that it is a selective agonist of nonopioid (insensitive to
the opioid antagonist naloxone) beta-endorphin receptor, Using P.15-010-Mon
tritium labeled octarphin the distribution of the receptor in the L-type voltage-gated calcium channels
body was studied. It was found that it is available on immune
hyperactivity as a result of calcium store
cells (peritoneal macrophages, T and B lymphocytes of spleen
and blood), endocrine (adrenal cortex, pituitary), and cardiovas- overload in neuronal AD models
cular (cardiomyocytes) systems. Characterization of the binding E. Kaznacheyeva, M. Ryazantseva, E. Mikhaylova, K. Skobeleva
specificity showed that only unlabeled beta-endorphin was able Institute of Cytology, Russian Academy of Sciences, Saint-
to compete with the labeled octarphin for the binding to recep- Petersburg, Russia
tor, tested in parallel alpha-endorphin, gamma-endorphin, enke- Today there is still no effective treatment for Alzheimer’s disease
phalins and a number of peptide hormones were inactive. (AD) and scientific society is in intense search for novel therapeu-
Investigation of the octarphin effect on the target cells was tic targets. Nearly a half of Familiar AD cases are connected
showed that it increases the mitogen-induced proliferation of with mutation in presenilin-1 (PS1) gene. It has long been known
human and mouse T and B lymphocytes in vitro, activates mur- that calcium homeostasis disturbances can mark a starting point
ine peritoneal macrophages in vitro and in vivo, stimulates growth for neurodegeneration. Numerous studies have demonstrated
of human T-lymphoblast cell lines Jurkat and MT-4, inhibits changes in activity of different calcium channels in plasma mem-
adenylate cyclase activity of rat adrenal cortex membranes and brane or endoplasmic reticulum (ER). It is also known that activ-
suppresses the secretion of glucocorticoids from the adrenal gland ity of L-type voltage-gated calcium channels (L-VGCC) is
into the blood. Data on the molecular mechanism of octarphin suppressed by STIMs (calcium ER sensors). This study is aimed
action were obtained. It was shown that in a concentration range to investigate the activity of voltage-gated calcium channels in
of 1–1000 nM the peptide increases the activity of inducible NO- connection with ER calcium store content in 5xFAD hippocam-
synthase (iNOS), and the content of NO and cGMP in pal neurons (HNs) and HNs expressing M146V PS1. Calcium
lipopolysaccharide-activated murine peritoneal macrophages. The imaging, fluorescent microscopy, HNs from 5xFAD/C3HA mice,
octarphin activity was depended on the dose and was maximal at lipofectamine/lentiviral transfections. We have previously shown
a concentration of 100 nM. Taking into account that NO acts as that SH-SY5Y cells bearing M146V PS1 were able to overload
a primary activator of soluble guanylate cyclase (sGC), it can be calcium stores and disturb translocation of STIM1 calcium sen-
assumed that the activating effect of octarphin on macrophages sors. Here we show that HNs bearing PS1 M146V mutant and
is realized in the following way: increase in th iNOS expression HNs from 5xFAD mice had higher calcium ER content, abated
? increase in the NO production ? increase in the sGC activity store-operated entry, and elevated L-VGCC entry. L-VGCC
? increase in intracellular levels of cGMP. entry in neurons after calcium store depletion equals to controls.
Additionally, in M146V PS1 expressing cells nuclear factor of
activated T-cells (NFAT1) tended to have nuclear localization,
P.15-009-Wed STIMs knock-down converted this changes. L-VGCC channels
Tag7-activated lymphocytes induce are a possible source of pathological neuronal calcium in FAD
necroptosis via FasL-Fas interaction models. These channels can serve as therapeutic targets for future
T. Sharapova, O. Ivanova, E. Romanova, L. Sashchenko, drug development. This work was supported by the RSF № 14-
D. Yashin 14-00720.
IGB RAS, Moscow, Russia
A lot of tumor cells can avoid immune attack by depletion MHC
complex on their surface. These cells are invisible for effector
immune cells. Tag7 (PGLYRP1) is innate immune protein that
can activate classical cytotoxic T cells after 6 days of treatment
in 109 M concentration. Tag7-activated cytotoxic lymphocytes
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 375
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Signalling through membranes and receptors POSTERS
376 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Signalling through membranes and receptors
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 377
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Signalling through membranes and receptors POSTERS
signaling pathways and histological assays were also evaluated. transcription polymerase chain reaction and Western blot analy-
The results show that blood glucose, serum triglycerides and total sis suggested that the expression of KATP channel subunits such
cholesterol of HFD-fed db/db mice were higher than those in as Kir6.1, Kir6.2, and SUR2B, were decreased in the GDM
Control group (the mice fed with normal diet), but the levels group relative to the normal group. The application of forskolin
were not reduced by chymostatin and captopril treatment. Uri- and adenosine, which activate protein kinase A (PKA) and
nary albumin level was significantly increased in HFD-fed db/db thereby KATP channels, elicited KATP current in both the normal
mice and significantly decreased after chymostatin treatment. and GDM groups. However, the current amplitudes were not dif-
After HFD feeding, renal ACE, chymase, Ang II, AT1R levels ferent between the normal and GDM groups. In addition, the
were significantly increased, and only ACE level was significantly expression levels of PKA subunits were not altered between the
decreased after captopril treatment. Chymase, Ang II and AT1R two groups. These results suggest that the reduction of KATP cur-
levels show a reduction in the mice treated with chymostatin. rent and KATP channel-induced vasorelaxation are due to the
Increased p-STAT3 level was observed in HFD-fed groups and decreased expression of KATP channels, not to the impairment of
were significantly decreased after chymostatin treatment. Patho- KATP-related signaling pathways.
logical results display severe leukocyte infiltration in HFD-fed
groups and chymostatin could significantly ameliorate the leuko-
cyte infiltration. By PAS staining, HFD-fed groups show slight P.15-020-Tue
mesangial matrix expansion compare with that in Control group, Blockade of voltage-dependent K+ current in
but mesangial matrix expansion were significantly reduced by rabbit coronary arterial smooth muscle cells
chymostatin treatment. In conclusion, chymase might play a role by the tricyclic antidepressant clomipramine
in DN pathogenesis, and chymase-Ang II-AT1R axis exacerbat-
J. R. An, W. S. Park
ing DN might via STAT3 signaling pathway in db/db mice.
Kangwon National University, Chuncheon, South Korea
We investigated the effect of the tricyclic antidepressant clomipra-
P.15-018-Wed mine on voltage-dependent K+ (Kv) channels in native rabbit
Inhibitory effect of tricyclic antidepressant coronary arterial smooth muscle cells. Our results showed that clo-
desipramine on voltage-dependent K+ current mipramine inhibited vascular Kv channels in a concentration-
dependent manner, with an IC50 value of 8.61 4.86 lM and a
in rabbit coronary arterial smooth muscle cells
Hill coefficient (n) of 0.58 0.07. The application of 10 lM clo-
H. Li, W. S. Park
mipramine did not affect the activation curves of the Kv channels;
Kangwon National University, Chuncheon, South Korea
however, the inactivation curves of the Kv channels were shifted
We describe the effect of a tricyclic antidepressant drug, desipra- toward a more negative potential. The clomipramine-induced inhi-
mine on voltage-dependent K+ (Kv) currents in freshly isolated bition of Kv currents was not changed by the application of train
rabbit coronary arterial smooth muscle cells using a conventional pulses (1 or 2 Hz), which demonstrated that clomipramine inhib-
whole-cell patch clamp technique. Application of desipramine ited Kv current in a state (use)-independent manner. Pretreatment
rapidly decreased the Kv current amplitude in a concentration- with the Kv1.5 and Kv2.1 inhibitors, DPO-1 and guangxitoxin,
dependent manner, with an IC50 value of 5.91 0.18 lM and a respectively, did not affect clomipramine-induced inhibition of Kv
Hill coefficient of 0.61 0.09. The steady-state inactivation currents. Therefore, we concluded that clomipramine inhibited
curves of the Kv channels were not affected by desipramine. vascular Kv channels in a concentration-dependent, but state
However, desipramine shifted the steady-state inactivation curves (use)-independent manner, regardless of its own function.
toward a more negative potential. Application of train pulses (1
or 2 Hz) slightly reduced the Kv current amplitude. Such reduc-
tion of the Kv current amplitude by train pulses increased in the P.15-021-Wed
presence of desipramine. Furthermore, the inactivation recovery Vildagliptin, an anti-diabetic drug of the
time constant was also increased in the presence of desipramine, dipeptidyl peptidase-4 inhibitor class, induces
suggesting that desipramine-induced inhibition of the Kv current vasodilation via Kv channel and SERCA pump
was use-dependent. Application of a Kv1.5 inhibitor (DPO-1)
and/or a Kv2.1 inhibitor (guangxitoxin) did not change the inhi- activation in aortic smooth muscle
bitory effect of desipramine on Kv currents. Based on these M. S. Seo, W. S. Park
results, we concluded that desipramine directly inhibited the Kv Kangwon National University, Chuncheon, South Korea
channels in a dose- and state-dependent manner, but the effect This study investigated vildagliptin-induced vasodilation and its
was independent of norepinephrine/serotonin reuptake inhibition. related mechanisms using phenylephrine induced precontracted
rabbit aortic rings. Vildagliptin induced vasodilation in a concen-
tration-dependent manner. Pretreatment with the large-conduc-
P.15-019-Mon tance Ca2+-activated K+ channel blocker paxilline, ATP-sensitive
Alterations of ATP-sensitive K+ channels in K+ channel blocker glibenclamide and inwardly rectifying K+
human umbilical arterial smooth muscle channel blocker Ba2+ did not affect the vasodilatory effects of vil-
during gestational diabetes mellitus dagliptin. However, application of the voltage-dependent K+ (Kv)
H. Li, W. S. Park channel inhibitor 4-aminopyridine significantly reduced the
Kangwon National University, Chuncheon, South Korea vasodilatory effects of vildagliptin. In addition, application of
either of two sarcoplasmic/endoplasmic reticulum Ca2+-ATPase
We investigated the alterations of ATP-sensitive K+ (KATP) (SERCA) inhibitors, thapsigargin or cyclopiazonic acid, effectively
channels in human umbilical arterial smooth muscle cells during inhibited the vasodilatory effects of vildagliptin. These vasodila-
gestational diabetes mellitus (GDM). The amplitude of the KATP tory effects were not affected by pretreatment with adenylyl
current induced by application of the KATP channel opener pina- cyclase, protein kinase A (PKA), guanylyl cyclase, or protein
cidil (10 lM) was reduced in the GDM group than in the control kinase G (PKG) inhibitors, or by removal of the endothelium.
group. Pinacidil-induced vasorelaxation was also predominant in From these results, we concluded that vildagliptin induced
the normal group compared with the GDM group. Reverse
378 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Signalling through membranes and receptors
vasodilation via activation of Kv channels and the SERCA pump. most important tumor suppressor genes. In about 50% of human
However, other K+ channels, PKA/PKG-related signaling cas- tumors, it can be observed in the occurrence of mutations. The
cades associated with vascular dilation, and the endothelium were most common mutations is missense mutations and base substi-
not involved in vildagliptin-induced vasodilation. tution. In this study, roles of p53, AP-1, cyclin D1 cyclin-depen-
dent kinase 4 (CDK4) and E2F-1 were investigated in B(a)P
transformed human embryo lung fibroblast (T-cells). The stable
P.15-022-Mon transfectants, HELF AP-1 (H-AP), HELF p53 siRNA (H-p53),
Site-directed mutagenesis of the TRPV1 T-cell AP-1 (T-AP) and T-cell p53 siRNA (T-p53) were estab-
reveals amino acid residues crucial for lished. Our current analysis showed that in T-AP cells expres-
receptor gating sions of cyclin D1 and p53 were higher than H-AP, but not
CDK4 and E2F-1/4. AP-1 can regulate the expression of cyclin
K. Lubova1, A. Chugunov1, Y. Andreev1,2
1 D1 in T-AP, but not in p53. p53 can regulate the expression of
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
p21 and E2F-1, but not the import of p21 into nucleus, the AP-1
Russian Academy of Sciences, Moscow, Russia, 2Sechenov First
activities and cyclin D1 in T-AP. The combinations of cyclin D1-
Moscow State Medical University, Institute of Molecular
p21 and cyclin D1-CDK 4 were significantly higher than H-AP,
Medicine, Moscow, Russia
and p53 effects the combination of cyclin D1-p21 in T-AP. In
Pain is an universal protective neural response to noxious stimuli. summary, present studies demonstrate that in T-AP cells expres-
Painful stimuli activate different receptors on the membrane of sions of cyclin D1 and p53 were higher than H-AP, but not
sensory neurons. One of the most important is nonselective CDK4 and E2F-1/4. AP-1 can regulate the expression of cyclin
cation channel TRPV1. An improper functioning of TRPV1 D1 in T-AP, but not in p53. p53 can regulate the expression of
leads to pain syndromes with quite challenging treatment. Under- p21 and E2F-1, but not the import of p21 into nucleus, the AP-1
standing of the relationship between the structure and function activities and cyclin D1 in T-P. The combinations of cyclin D1-
of TRPV1 may promote new therapies development. Existing p21 and cyclin D1-CDK 4 were significantly higher than H-AP,
cryo-EM 3D models do not provide a complete picture of the and p53 effects the combination of cyclin D1-p21 in T-AP.
receptor gating mechanism as well as contradict each other in
many ways. We identified several amino acid residues crucial for
pore domain functioning by site-directed mutagenesis. “Hot P.15-024-Wed
spot” residues were proposed by molecular dynamics. TRPV1 Cav1.2 mediated nuclear signaling via the
mutants were tested in two-microelectrode voltage-clamp experi- H-Ras-ERK-CREB pathway
ments of Xenopus laevis oocytes. We found two residues critical E. Servili, M. Trus, D. Atlas
for rTRPV1 gating. Mutations of Ile679, Ala680 within the Department of Biological Chemistry, Alexander Silberman
inner-pore region were found to inactivate the channel. Molecu- Institute of Life Sciences, The Hebrew University of Jerusalem,
lar dynamics model suggests Asn676 being involved in channel Jerusalem, Israel
desensitization by calcium ions. We replaced Asn by Ser because
of similar physical properties. This mutant appeared to be non- Voltage-gated calcium channels are transmembrane cell surface
functional that proves the importance of this residue. In addition, proteins responsible for multifunctional signals. The mechanisms
replacing conservative Lys688 with Gly increases the flexibility of linking neuronal activity to the excitation-transcription (ET) cou-
the TRP domain collocated with the pore region making the pling is not fully understood. The L-type Cav1.2 channel has
whole receptor more sensitive to activation. Identification of con- been shown to activate a large number of different neuronal-spe-
crete amino acid residues involved in the pore domain function- cific genes. The prevailing view of the L-type Ca2+ channel acti-
ing may allow us to take a completely different look at the vation of the downstream signaling is via mobilization of
present approach of development of new TRPV1-targeted drugs calmodulin (CaM) and the CaM-dependent protein kinase kinase
for the inflammation and pain syndromes treatment. This work (CaMKK) cascade responsible for the CREB phosphorylation.
was supported by Russian Science Foundation Grant 16-15- Other proposed pathways involve the calcium-regulated phos-
00167. phatase calcineurin and the activation of the Ras/MAPK signal-
ing pathways. To investigate the underlying mechanism of ET
coupling, we transiently transfected HEK293 cells with wild-type
P.15-023-Tue (wt) Cav1.2 channel and monitored phosphorylation of ERK1/2,
p53-E2F pathway plays different roles in benzo RSK, and CREB. We found that expressing dominant-negative
(a)pyrene induced transformed and normal H-RasS17N mutant together with wt Cav1.2 channel significantly
inhibited ERK phosphorylation, while the dominant negative
human embryo lung fibroblasts RasS17N only partially inhibited CREB. This negative impact sug-
W. Hu1, M. Ye2, X. Zhang3, Y. Qian4 gests a functional role of H-Ras in mediating nuclear signaling.
1
National Institute for Occupational Health and Poison Control, We tested the role of Ca2+ and showed that Ca2+-impermeable
Chinese Center for Disease Control and Prevention, Beijing, mutant channel a11.2L745P/a2d/b2b expressed in HEK293 cells
China, 2National Institute for Occupational Health and Poison triggered ERK1/2 and CREB phosphorylation, albeit smaller
Control, Chinese Center for Disease Control and Prevention, compared to wt channel. These results indicate that there is an
Beijing, China, 3Chinese Center for Disease Control and additional ERK-independent nuclear activation pathway. The
Prevention,, Beijing, China, 4Chinese Center for Disease Control Fluo-4 fluorescence assay results showed elevated intracellular
and Prevention, Beijing, China Ca2+ ([Ca2+]i) under depolarizing conditions in HEK293 cells
B(a)P is one of the polycyclic hydrocarbon environment pollu- expressing wt channel, while cells expressing the Ca2+-imperme-
tants, existed in cigarette smoke and diesel. B(a)P is an indirect able mutant channel exhibited no [Ca2+]i rise. Taken together,
carcinogen which transformed into in the carcinogen BPDE our data suggest the existence of a specific mechanism by which
under action of the cytochrome p450 enzyme system. BPDE can Cav1.2 can activate nuclear signaling independently of Ca2+
form DNA adducts, then cause DNA damage, lead to the disor- entry, and provide insight into the role of the Cav1.2 Ca2+-
der of cell cycle. In mammalian cells, proliferation is controlled bound pore in triggering ET coupling.
by a series of positive and negative regulatory factors. p53 is the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 379
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Signalling through membranes and receptors POSTERS
P.15-025-Mon astrocytes from newborn rats were obtained from brain cortices
by conventional procedures. Cells were cultured 12 days before
A novel regulatory role of RGS4 in neuronal
experiments in either DMEM with 5 mM glucose (normal glu-
cell proliferation and sprouting mediated via cose level, NG) or in media with 22.5 mM glucose (long-term
STAT5B transcriptional responses high glucose level). In short-term HG model cells, 5 mM glucose
P. Pallaki1, I. Serafimidis2, D. Thomaidou3, M. Gaitanou3, DMEM was switched to 22.5 mM glucose for 48 h before stimu-
Z. Georgoussi1 lations. Astrocytes were stimulated with the lipopolysaccharide
1
National Center for Scientific Research “Demokritos”, Athens, (LPS, a TLR4 agonist) or the poly I:C (PIC, a TLR3 agonist).
Greece, 2Biomedical Research Foundation of the Academy of Analysis of mRNA expression of cyclooxygenase 2 (COX-2),
Athens, Athens, Greece, 3Hellenic Pasteur Institute, Athens, TNF-alpha and IL-10 was performed by qPCR, protein levels of
Greece COX-2 were determined by Western blot, levels of released
The regulator of G protein signaling 4 (RGS4) is a multitask cytokines IL-10, TNF-alpha and prostaglandin E2 (PGE2) were
protein highly expressed in developing neurons associated with determined using ELISA kits. We obtained that inflammatory
various neurological disorders. Despite its initial role as GTPase response to LPS reduced in long-term HG and induced in short-
activating protein, we have previously demonstrated that RGS4 term HG (in comparison with NG). Both types of HG treatments
interacts directly with opioid receptors (ORs) conferring selectiv- induced inflammatory response to PIC. Effects were observed on
ity for G protein coupling, interfering in ORs signaling and accel- mRNA levels of IL-10, TNF-alpha and both protein and mRNA
erating receptor endocytosis. It was also shown that d-opioid of COX-2. Alterations in concentrations of released mediators
receptor (d-OR) forms a signaling complex, consisted of RGS4 IL-10, TNF-alpha and PGE2 were detected. The difference
and the Signal Transducer and Activator of Transcription 5B between PIC and LPS action was observed in a time-dependence
(STAT5B) that leads to neurite outgrowth and neuronal differen- manner. Supported by RSF (№ 16-15-10298).
tiation upon d-ΟR activation. Based on these findings we won-
dered whether RGS4-STAT5B interplay is involved in cellular
P.15-027-Wed
mechanisms controlling neurogenesis through STAT5B transcrip-
tional regulation. Our data demonstrated that primary cortical Effective TRPA1 modulator from sea anemone
neuronal cultures from RGS4/ mice, lacking a functional venom appeared due to a mutation of a
RGS4, exhibit differential neuronal sprouting compared to con- processing site
trols in absence or presence of DSLET administration. Moreover, Y. Logashina1,2, E. Maleeva2, K. Lubova3, Y. Andreev1,2
RGS4/ adult brain extracts, displayed increased levels of p- 1
Sechenov First Moscow State Medical University, Institute of
STAT5B and the expression levels of STAT5B target genes Snx9 Molecular Medicine, Moscow, Russia, 2Shemyakin-Ovchinnikov
and Ptk2, implicated in axonogenesis, were also altered indicating Institute of Bioorganic Chemistry, Russian Academy of Sciences,
that RGS4 is involved in neurite outgrowth possibly via STAT5B Moscow, Russia, 3Lomonosov Moscow State University, Moscow,
mediated transcriptional responses. In contrast, Neuro2A cells Russia
expressing RGS4 exhibited a significant reduction in cell number
Pain is a physiological condition that affects the being quality of
and proliferation rate. Additionally, isolated neural stem cells
life. The transient receptor potential ankyrin-repeat 1 (TRPA1)
showed elevated proliferative properties with concomitant
receptors play the significant role in initiation and propagation of
increases of the mRNA levels of the STAT5B Bcl-2 and Bcl-xl
pain signals. TRPA1 expressed in sensory neurons that innervate
anti-apoptotic target genes. Collectively, these results demon-
different tissues and organs. Receptors activate upon noxious
strate for the first time a novel, non-canonical regulatory role of
chemical, mechanical and thermal (low temperatures) conditions,
RGS4 in STAT5B mediated-transcription thus providing insights
while some TRPA1 agonists expressed in mammalians during
into the involvement of RGS4 in neuronal signaling and synaptic
inflammation. So TRPA1 is an attractive target for the medical
plasticity.
drug development. Animal venoms contain a dozen of bioactive
compounds which includes modulators of different ion channels
P.15-026-Tue and receptors. During the search for TRPA1 modulators among
sea anemone toxins, we identified a novel peptide named Ms 9a-
Activation of TLR3 and TLR4 in long- and
1, which potentiates receptor activity. The bioactive molecule was
short-term cells incubation of astrocytes in isolated from Metridium senile venom by using chromatography
high-glucose medium methods. In electrophysiology experiments on Xenopus laevis
N. Azbukina1, D. Chistiakov2,3, A. Astakhova2, M. Sergeeva2 oocytes expressing TRPA1 peptide increased agonist-induced
1
Lomonosov Moscow State University (MSU), Moscow, Russia, activation of the receptor. However, pretreatment of mice by
2
A. N. Belozersky Institute of physico-chemical biology MSU, intravenous injection of Ms 9a-1 (0.3 mg/kg) significantly
Moscow, Russia, 3Pirogov Russian National Research Medical reduced nocifensive behavior and paw edema induced by TRPA1
University, Moscow, Russia agonist allyl isothiocyanate (AITC), as well as decreased non-spe-
Bacterial and viral agents have different effects on the inflamma- cific inflammation in Freund’s Complete Adjuvant test. Most
tory responses in hyperglycemia. Accumulating studies have probably Ms 9a-1 could potentiate TRPA1 activation by endoge-
shown that hyperglycemia show inflammation in the central ner- nous agonists and produce a depolarization block of sensory neu-
vous system (CNS). Astrocytes play an important role in the rons. Peptide Ms 9a-1 evidently differs from homologous sea
development of inflammation responses in CNS. However, anemone peptides by extended C termini that probably appeared
changes in inflammatory responses at different glucose concentra- due to a mutation of a processing site. Such a mutation could be
tions are not well characterized. The aim of our research was to responsible for the appearance of TRPA1-potentiating activity.
investigate the influence of long- and short-term high glucose To determine the active site of the peptide we analyzed the effects
(HG) cultivation medium on the response of astroglial cells to of the N-terminal and C-terminal domains of Ms 9a-1. As a
inflammatory stimuli. We compare inflammatory responses in result, both parts of Ms 9a-1 were able to potentiate TRPA1, but
course of the activation of Toll-like receptors (TLR) with a bac- with different level of efficiency. This work was supported by
terial-like (TLR4) and viral-like (TLR3) stimuli. Primary Russian Science Foundation Grant 16-15-00167.
380 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Signalling through membranes and receptors
P.15-028-Mon P.15-030-Wed
Peptidoglycan can induce expression of CD44 Protein sorting upon exit from the
on lymphocytes during inflammatory response endoplasmic reticulum
of bovine mammary gland S. Rodriguez-Gallardo1, S. Sabido-Bozo1, A. M. Perez-Linero1,
P. Slama1, L. Kratochvilova1, K. Kharkevich1, J. Y. Kwak2 J. Manzano-Lopez1, K. Funato2, K. Kurokawa3, A. Nakano3,
1
Mendel University in Brno, Brno, Czech Republic, 2Ajou M. Mu~ niz1
1
University School of Medicine, Suwon, South Korea Department of Cell Biology, University of Seville. IBiS (Instituto
de Biomedicina de Sevilla) HVR/CSIC/US, Sevilla, Spain,
CD44 is a proteoglycan that is expressed by different cell types. 2
Department of Bioresource Science and Technology, Hiroshima
CD44 plays a role in leukocyte trafficking to extra lymphoid sites University, Hiroshima, Japan, 3Live Cell Molecular Imaging
of inflammation or as a non-specific accessory adhesion molecule. Research Team, RIKEN Center for Advanced Photonics,
The aim of this study was to inquire development over time of Hirosawa, Wako, Saitama, Japan
the surface expression of CD44 on lymphocytes during an inflam-
matory response of bovine mammary gland induced by peptido- Protein sorting upon vesicular transport through the secretory
glycan. Intramammary instillation of peptidoglycan resulted in pathway is essential to dynamically maintain functional compart-
an increase in the proportion of CD44-positive lymphocytes after mentalization and homeostasis in eukaryotic cells. During many
24 and 48 h. During resolution of the inflammatory response, years it was generally thought that all newly synthetized secretory
there was observed a decrease in the proportion of CD44-positive proteins travel together in the same COPII vesicles from the
lymphocytes. The results suggest that the cell surface receptor endoplasmic reticulum (ER) to the Golgi, where they are sorted
CD44 can play an important role in the inflammatory response to different destinations. However, we found in yeast that glyco-
of bovine mammary gland to bacteria and their components. sylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), a
special category of lipid-anchored secretory proteins, are segre-
gated from transmembrane cargos at the level of the ER and
P.15-029-Tue then incorporated into distinct COPII vesicles. We have devel-
Expression and functional activity of specific oped in yeast a genetic assay that allows visualization of cargo
membrane transport of serotonin in the sorting in ER exit sites (ERES) by using super-resolution confo-
cal live imaging microscopy. Our data suggest that GPI-AP sort-
mouse ovary ing into specific ERES is driven by the acquisition of ceramide
D. Nikishin1,2, N. Alyoshina1,2, Y. Shmukler1 C:26 (a very long and saturated lipid) by the GPI anchor. GPI-
1
Koltzov Institute of Developmental Biology, Russian Academy of ceramide remodeling might induce a strong hydrophobic mis-
Sciences, Vavilov Street 26, 119334, Moscow, Russia, 2M.V. match in the ER membrane leading to the clustering of GPI-APs
Lomonosov Moscow State University, Faculty of Biology, into specific lipid domains. We also show that the specific ER
Moscow, Russia export machinery is recruited after GPI-AP sorting at ERES to
Along with the classical function of serotonin as the neurotrans- produce specialized COPII vesicles.
mitter, it performs the regulation of female reproductive function.
Serotonin is present in the female reproductive system and affects
the maturation of oocytes, the synthesis of sex steroid hormones, P.15-031-Mon
as well as the processes of early embryonic development, which is Study of the PFNA cluster in bifidobacteria:
shown in a variety of animals, including mammals. However, the structure, evolution and possible functions
mechanisms of serotonergic regulation of the female reproductive M. Chekalina, V. Danilenko
function have been poorly studied. The serotonin transporter Vavilov Institute of General Genetics, Russian Academy of
Sert (Sls6a4) is of particular interest in this respect, which carries Sciences, Moscow, Russia, Moscow, Russia
out the uptake of the serotonin from the extracellular medium.
The mRNA of the Sert gene is expressed in the ovary, including Signaling through STPK appears to be the dominant prokaryotic
signaling system. STPK Pkb2 is the receptor-type kinase, with an
follicular cells and oocytes at different stages of folliculogenesis.
A quantitative study have shown that the expression level of the extracytoplasmic sensor domain and an intracellular kinase
transporter is constant and does not depend on the development domain, that indicates this kinase to transduce the external sig-
stage of the ovarian follicle. Immunostaining revealed that the nals. Using the phylogenetic profiling method, we found an evo-
lutionarily stable cluster of genes linked to pkb2 gene in the
transporter is localized in ovarian follicles, with immunoreactivity
much more pronounced in the oocytes. There was an increase of Bifidobacterium genus. The operon organization of the cluster in
the immunostaining intensity in all compartments of the ovary, B. longum GT15 was confirmed with transcriptional analysis.
including mouse follicles and oocytes after subcutaneous injec- The transcription start site was determined by 50 -RACE method.
The cluster named PFNA was found in most bifidobacterial spe-
tions of serotonin during five days. According to the data
obtained by HPLC, the concentration of serotonin in the ovaries cies obtained from various sources. The cluster contains genes
of mice in the experimental group increased more than 4-fold. that encode, mainly, membrane proteins, among which a protein
with a cytokine receptor motif is annotated. Genes in the PFNA
Thus, the ovary actively seizes exogenous serotonin from the
bloodstream, probably through the membrane transporter Sert. cluster show high sequence divergence between species which
The transmitter was accumulated in oocytes during cultivation of may be an indicator of rapid evolution in response to the rapid
isolated ovarian follicles in vitro in the presence of serotonin but evolution of host’s cytokine genes. To investigate the molecular
evolution of the cluster we tested in silico hypotheses concerning
the addition of a selective serotonin reuptake inhibitor fluoxetine
reverses the effect, which indicates the specificity of membrane positive and relaxed negative selection. The molecular evolution
transport. Thus, expression of the specific membrane serotonin analysis of the cluster showed the presence of episodic positive
transporter Sert in the ovary, and its specific functional activity selection as well as the relaxed negative selection in some phylo-
genetic clades. This allowed us to determine the structurally and
in the oocytes of preantral follicles are revealed.
functionally significant sites under the pressure of selection. The
presence of episodic positive selection can indirectly indicate a
possible role of the cluster in interacting with the host’s immune
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 381
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Signalling through membranes and receptors POSTERS
382 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Extracellular matrix
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 383
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Glycans POSTERS
384 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Glycans
saminoglycan (GAG) chains with soluble mediators and their tumor cells can activate cell death by apoptosis. Previously we
receptors. The initiation of the synthesis of GAG chains of PGs isolated two GalNAc/Gal-specific lectins from marine mussels
is catalyzed by xylosyltransferase I (XT-I). To study the role of Crenomytilus grayanus (CGL) and Mytilus trossulus (MTL). The
XT-I in vivo, we generated XT-I knock-out mice. Embryo analy- establishment of the amino acid sequence showed that CGL and
sis shows that XT-I knock-out mice exhibit pronounced dwarfism MTL have a high degree of homology and represent a new class
and apparent frontonasal hypoplasia, thus reflecting abnormali- of lectins. Glycan array assay revealed that CGL and MTL were
ties in skeletal development. Analysis of PG content revealed a able to bind both a and b anomer of galactose. CGL interaction
strong decrease in PGs synthesis in XT-I knock-out mice. Exami- with the aGal-terminated glycans was stronger. As for MTL,
nation of chondrocytes zones in growth plate of long bones preferable binding with b anomers was observed. CGL showed
revealed extended proliferative zone and short hypertrophic zone. high affinity to Gala1-4Galb1-4GlcNAc motif similar to globo-
To identify the factors and mechanisms underlying skeletal triose structure (Gb3: Gala1-4Galb1-4Glc). CGL recognized Gb3
abnormalities in XT-I KO mice, the expression of several genes on the surface of Burkitt’s lymphoma Raji cells (high Gb3
involved in skeletal development and in the regulation of chon- expression), leading to dose-dependent cytotoxic effect, G2/M
drogenesis were analyzed by in situ hybridization using RNA- phase cell cycle arrest and apoptosis. Lectin had no effect on ery-
scope technique. The results showed a strong expression of throleukemia K562 cells (no Gb3 expression). The activity of
hypertrophic chondrocyte markers in XT-I KO mice compared CGL was specifically blocked by alpha-galactoside. MTL, on the
to wild-type alternates, thus suggesting an early maturation of other hand, had dose-dependent cytotoxic effect on K562 cells,
chondrocytes in the XT-I knock-out mice leading to premature but no activity was observed on Raji cells. TF-antigen (Galb1-
ossification and hence to dwarfism. 3GalNAc) expressed on the membrane of K562 cells was signifi-
cant for MTL effect. Both lectins effectively inhibited the tumor
cells proliferation. Thus despite the high degree of homology,
P.17-003-Wed CGL and MTL have different ligand specificity as lectins inter-
Importance of CH-p stacking interaction in acted with different glycoconjugates expressed on the surface of
carbohydrate-protein complexes tumor cells. Overall, our data indicate that CGL and MTL have
Z. Zufanova1, J. Houser1,2, S. Kozmon2,3, D. Mishra2, the potential to be used in cancer diagnosis and treatment. The
M. Wimmerov a1,2,4, J. Koca1,2 investigation was partially supported by the Program of FEB
1
Masaryk University Brno, Faculty of Science, National centre for RAS “Far East” (project 18-4-007).
Biomolecular Research, Brno, Czech Republic, 2CEITEC – Central
European Institute of Technology, Masaryk University, Brno,
Czech Republic, 3Institute of Chemistry, Slovak Academy of
P.17-005-Tue
Science, Bratislava, Slovakia, 4Department of Biochemistry, Arginase is an enzyme with real tricks in
Faculty of Science, Masaryk University, Brno, Czech Republic antitumor treatment of 7,12-dimethylbenz(a)
CH-p stacking, a special type of hydrogen bonding interaction, anthracene induced breast cancer in rats
plays an important role in carbohydrate-biomacromolecule inter- G. Petrosyan1, N. Avtandilyan2, H. Javrushyan3, A. Trchounian4
1
actions involved in cell recognition, growth, and differentiation, Ph.D. student, senior assistant, Department of Biochemistry,
as well as in many pathological processes. To better understand Microbiology and Biotechnology, Yerevan State University,
this particular type of hydrogen bonding between a CH group Yerevan, Armenia, 2Junior researcher, PhD, Research Institute of
and an aromatic ring, we examined the CH-p stacking interaction Biology, Yerevan State University, Yerevan, Armenia, 3Member of
in carbohydrate-protein complexes in a computational structure- Scientific Group, PhD, Research Institute of Biology, Yerevan
based study. We searched the Protein Data Bank database to State University, Yerevan, Armenia, 4Ph.D., D.Sc., Professor,
examine complexes with carbohydrates in a close proximity of Head, Department of Biochemistry, Microbiology and
aromatic amino acid (tryptophan, tyrosine, phenylalanine, and Biotechnology, Yerevan, Armenia
histidine). CH-p stacking interaction was found in 61% of these Polyamines are vital for cell proliferation and the increased level
complexes, where the complexes between a carbohydrate and of ornithine, due to the elevated arginase activity, linked to the
tryptophan were the most populated. Each aromatic amino acid development of carcinogenesis. The main question was addressed
showed a unique CH-p stacking pattern, demonstrated by a char- in this work, would treating the rats with arginase inhibitor NG-
acteristic orientation, bond distances, and bond angles between hydroxy-nor-L-arginine (nor-NOHA) affect cancer progression
the carbohydrate and a particular amino acid. These results pro- and the oxidative responses (malondialdehyde (MDA), nitrite
vide critical insight into the importance of CH-p stacking in car- and ammonia ions)? A total of 35 adult female Wistar rats
bohydrate-protein interactions. This work was supported by the weighing 90-120 g were used. Rats were divided into five groups
Ministry of Education, Youth and Sports of the Czech Republic (control, saline, nor-NOHA; 7,12-dimethylbenz(a)anthracene
under the project CEITEC 2020 (LQ1601). (DMBA) and DMBA+nor-NOHA, 7 rats per group). Rats in
DMBA and DMBA+nor-NOHA groups were administrated
intragastrically each with a single dose of 20 mg/ml DMBA dis-
P.17-004-Mon solved in 0.5 ml olive oil and 0.5 ml saline. Rats in nor-NOHA
Marine bivalve lectins induce tumor cells and DMBA+nor-NOHA groups were injected by nor-NOHA
death intraperitoneally for 5 weeks (after 10 days of DMBA adminis-
O. Chernikov, A. Kuzmich, I. Chikalovets, V. Molchanova tration, every 3rd day) in dose of 3 mg/kg body-weight in
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far 0.25 ml saline. Rats blood plasma arginase activity, MDA, total
Eastern Branch of the Russian Academy of Sciences, Vladivostok, nitrite anions and ammonia quantities were determined by corre-
Russia sponding methods and with the rules of the Ethical Committee.
At the end of 20th week, rats were sacrificed by decapitation
Glycosylation plays an important role in determining protein
under anesthesia. Histopathological alteration in DMBA group
function, and can play a role in disease development. An alter-
(2–4 tumors, 0.3–0.8 cm) in the 20th week has revealed the inva-
ation in the glycosylation patterns of cell surface proteins is asso-
sive ductal and lobular carcinoma, and in DMBA+nor-NOHA
ciated with tumor progression. Binding of lectins to glycans on
group (0–2 tumors, 0.1–0.3 cm) – only lobular carcinoma in situ.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 385
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Glycans POSTERS
386 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Glycans
occupies in free unbound state predominantly one conformation, growth of mycobacteria and they catalyze reactions involving
which is very similar to the conformation of TDG, and therefore UDP-galactofuranose, a rare sugar nucleotide, which is not pre-
perfectly arranged to fit into the binding site of galectin-1. How- sent in humans. We have developed cell-free enzyme assays, and
ever, the determination of bidding affinity of CDG (Kd = a whole cell labeling assay that can determine whether inhibitors
416 mM) to galectin-1 by isothermal titration calorimetry showed of these enzymes successfully block the synthesis of mycobacterial
repeatedly Kd worse than for lactose (Kd = 327 mM). Moreover, galactan polymer. For the cell-free assays, a fraction of mycobac-
TDG binds to galectin-1 about 6 times stronger than lactose (Kd terial cell walls and membranes is used as a source of the
= 57 mM). This difference in binding affinities of CDG and enzymes for galactan synthesis, and UDP-[14C]Galp is used to
TDG might be therefore attributed to special geometric arrange- monitor the synthesis of galactan intermediates. The addition of
ment in proximity of sulfide bridge. We have tested prepared gly- an active inhibitor of any of the enzymes mentioned, should sig-
comimetics in our optimized viral entry assay using LuSIV cells nificantly reduce the incorporation of the radiolabeled Galf into
infected with HIV-1 and in red blood cells hemagglutination the growing polymers. The whole cell labeling using [14C]-glucose
assay. Both assays show, that CDG has comparable effect as or [14C]-acetate allows us to monitor the effects of the inhibitors
TDG. These biochemical and biological findings, together with on the synthesis of the cell wall in growing mycobacteria. We will
molecular modeling, will serve as a basis for further synthetizing present evaluation of the selected compounds described in the lit-
novel glycomimetics with improved efficiency, stability and erature as inhibitors of the enzymes involved in galactan biosyn-
bioavailability. thesis by these assays in order to assess their potential for further
development. This work was supported by Slovak Research and
Development Agency (project no.: APVV-15-0515).
P.17-009-Wed
Withdrawn
P.17-011-Tue
Heparitinase I and II from Pedobacter
heparinus and their use in the study of
heparan sulfate proteoglycan-dependent
cytoskeleton dynamics
T. Bergonci, A. D. Januzzi, R. P. Cavalheiro, M. A. Lima,
H. B. Nader
Federal University of Sao Paulo, S~
ao Paulo, Brazil
The use of heparitinases has been limited by difficulties in isola-
tion and separation of sufficient quantities of the enzymes and by
contaminating polysaccharide degrading and modifying enzymes,
such as chondroitinases and sulfatases. We cloned and expressed
heparitinases I and II genes from Pedobacter heparinus in Escher-
ichia coli BL21. Expression was induced in the log phase by addi-
tion of 0.5, 1 or 2 mM of IPTG. Three temperatures, 22, 29 and
37°C, and three different times for induction, 4, 12 and 18 h,
were tested. Soluble fraction was purified by Ni2+ affinity col-
umn chromatography. Heparan sulfate and heparin were used as
substrates to the recombinant enzymes. The digestion products of
heparan sulfate and heparin were analyzed by HPLC and agarose
gel electrophoresis in PDA buffer. We found that the ratios of
soluble fraction to inclusion body were influenced by the high
temperatures to heparitinase I and high concentration of IPTG
for both heparitinases I and II. Optimum expression conditions
for heparitinase I was 29°C for 12 h and 1 mM IPTG, whereas
heparitinase II was 37°C for 18 h and 1 mM IPTG. Both
enzymes were active and stable at 37°C. Owing to the fact that
both enzymes target different regions of heparan sulfate chains
P.17-010-Mon that decorate heparan sulfate proteoglycans (HSPGs), they were
Investigating mycobacterial cell wall synthesis used to study cytoskeleton dynamics. Vascular endothelial cells
inhibitors: galactan biosynthesis as a potential from rabbit aorta were transfected with vectors encoding actin
fused with GFP and treated for 6 h with both enzymes sepa-
antituberculosis drug target? rately. Cytoskeleton parameters such as fiber organization, cou-
a, K. Mikusova
Z. Konyarikov pling of actin-associated proteins and focal adhesion points
Department of Biochemistry, Faculty of Natural Sciences, distribution were analyzed using confocal microscopy and the
Comenius University in Bratislava, Bratislava, Slovakia results compared to cells in which HSPGs had been knocked
The synthesis of the cell wall of Mycobacterium tuberculosis, the down. The gathered data shows the pivotal role of HSPGs gly-
causative agent of tuberculosis, is a target for several antibiotics. cosaminoglycan chains and further pinpoint their importance for
However, none of them targets the synthesis of the galactan poly- cell behavior.
mer, a part of the mycolyl-arabinogalactan-peptidoglycan cell
wall core. The three main enzymes participating in the synthesis
of the galactan polymer, UDP-galactopyranose mutase UGM,
galactosyltransferase GlfT1 and galactosyltransferase GlfT2, can
be regarded as potential drug targets. They are essential for the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 387
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
388 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
P.18-004-Mon P.18-005-Tue
The calpain-calpastatin system in human Effects of melatonin and resveratrol on renal
central nervous system tumours: new insight expression of sodium-glucose cotransporters
on its prognostic value SGLT1 and SGLT2 in rat model of aging
A. Martines1, M. Averna1, A. Franchi1, R. De Tullio1, I. Vrhovac Madunic1, D. Karaica2, V. Micek3, M. Ljubojevic2,
M. Pedrazzi1, B. Sparatore1, I. Melloni2, G. Zona2, E. Melloni1 M. Geric4, G. Gajski5, D. Rasic6, M. Peraica6, T. Orct7,
1
University of Genova, Genova, Italy, 2Department of Neurology, J. Jurasovic7, I. Novak Jovanovic8, L. Nanic9, I. Rubelj10,
Rehabilitation, Ophthalmology, Genetics, Maternal and Child I. Sabolic2, D. Breljak1
1
Health, Section of Neurosurgery, University of Genoa, Italy Institute for Medical Research and Occupational Health,
Molecular Toxicology Unit, Zagreb, Croatia, 2Institute for
The calpain/calpastatin proteolytic system regulates several phys-
Medical Research and Occupational Health-Molecular Toxicology
iopathological processes and can play opposing roles in cancer.
Unit, Zagreb, Croatia, 3Institute for Medical Research and
New strategies to control alterations of this system for therapeu-
Occupational Health-Laboratory Animals Unit, Zagreb, Croatia,
tic purposes require a full understanding of the functional role 4
Institute for Medical Research and Occupational-Molecular
played by the single protein members. For this purpose we have
Toxicology Unit, Zagreb, Croatia, 5Institute for Medical Research
evaluated the involvement of calpain-1 in the proliferation of
and Occupational Health-Mutagenesis Unit, Zagreb, Croatia,
human meningioma and glioblastoma cells obtained from brain 6
Institute for Medical Research and Occupational Health-
surgery. We demonstrate that tumour samples with an altered
Toxicology Unit, Zagreb, Croatia, 7Institute for Medical Research
intracellular homeostasis of Ca2+ show increased conversion of
and Occupational Health-Analytical Toxicology and Mineral
the 80 kD inactive calpain-1 to the autolysed 75 kD form, cat-
Metabolism Unit, Zagreb, Croatia, 8Institute or Medical Research
alytically active at mM Ca2+ concentrations in vitro. Intracellular
and Occupational Health-Toxicology Unit, Zagreb, Croatia,
calpain-1 targets, such as spectrin and the natural calpain inhibi- 9
Ruder Bo skovic Institute-6Division of Molecular Biology,
tor calpastatin, result degraded in vivo in these tumour samples.
Laboratory for Molecular and Cellular Biology, Zagreb, Croatia,
Specifically, calpastatin undergoes a limited digestion to discrete 10
Ruder Boskovic Institute-Division of Molecular Biology,
fragments that maintain the inhibitory activity. Tumour samples
Laboratory for Molecular and Cellular Biology, Zagreb, Croatia
with mean or high amounts of 75 kD calpain-1 show a wide
cytosolic diffusion of the inhibitor, while samples characterized Mechanisms of aging are poorly understood. Aging is associated
by low levels of active calpain-1, retain calpastatin in cytosolic with loss of renal function and structure. Elevated tissue concen-
aggregates. Since higher calpain-1 activation does not correspond trations of reactive oxidative species, known to be present in old
to a detectable decrease in the Ki67 cell proliferation index, the humans and experimental animals, may affect the expression
activity of calpain-1 seems to have a low prognostic significance and/or activity of various renal transporters, including those that
in the progression of the brain tumours analysed in this study. mediate reabsorption of glucose, such as SGLT1 and SGLT2.
However, the perturbation of Ca2+ homeostasis promotes an SGLT2 in the proximal tubule S1/S2 segments mediates a bulk
increased synthesis of Matrix Metalloproteinase-9 (MMP-9), (65–90%) glucose reabsorption, whereas SGLT1 in the S3 seg-
known to be involved in tumour invasiveness, suggesting that ment mediates reabsorption of the remains. To test hypothesis
other Ca2+-dependent processes could be related to the malig- that the expression of SGLT1 and SGLT2 could be changed in
nancy of these cells. These evidences suggest that the potentially old age, and corrected with antioxidants, we treated male and
harmful activity of autolysed 75 kD calpain-1 can be efficiently female Wistar rats with melatonin and resveratrol. Starting from
controlled by the low Mr calpastatin produced by the protease their age of 3 months, for the next 21 months the rats were
itself in vivo, allowing cell proliferation. drinking antioxidants in water (~1 mg/kg b.w./day), whereas the
control animals were drinking water with vehicle (0.01% etha-
nol). The expression of renal SGLT1 and SGLT2 was analysed
by Western blotting of isolated total cell membranes and by
immunohistochemistry of tissue cryosections using specific anti-
bodies. Melatonin and resveratrol did not notably change the
expression of renal SGLT1 in both sexes, but in melatonin-trea-
ted males, a slight tendency to SGLT1 upregulation was
observed. Also, melatonin treatment did not affect the SGLT2
expression in both sexes. However, resveratrol significantly
upregulated the SGLT2 expression in male, but not in female
rats. We conclude that in old rats, the melatonin treatment has a
negligible effect on renal SGLTs, whereas the resveratrol effect
on SGLT2 is sex-related, being restricted to males. (Croatian
Science Foundation project IP-2013-11-1481).
P.18-006-Wed
Comparative analysis of PARP1 and PARP2 in
base excision repair context
M. Kutuzov, E. Ilina, M. Sukhanova, E. Belousova,
S. Khodyreva, O. Lavrik
Institute of chemical biology and fundamental medicine,
Novosibirsk, Russia
The genome stability in eukaryotes is provided by functioning of
DNA repair systems. Base excision repair (BER) is one of the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 389
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
main DNA repair systems in eukaryotes. For correct functioning performed in the case of nicking endonuclease (NE) BspD6I.
these systems require precise regulation. PARP1 and PARP2 are This enzyme recognizes a specific sequence in DNA and cuts only
widely considered as regulators of DNA repair processes, includ- one strand nearby the recognition site. No crystal data for the
ing BER. PARP in response to the DNA damage synthesize complex of NE BspD6I with DNA is available to date. To eluci-
polymer of ADP-ribose (PAR) covalently attached to the accep- date the possible contacts of the protein with DNA, the
tor proteins including PARP itself. PAR formation can directly crosslinking reaction was performed. In this case, DNA con-
influence the acceptor properties and also it is an intracellular tained disulfide group in 20 -position of nucleoside that reacted
signal about DNA damage. PARP1 is well characterized protein only with cysteine residues. The obtained data allow suggesting
but both proteins are under careful attention of researchers. In which cysteine residues of NE BspD6I can locate in close prox-
our work we studied interaction of PARP2 with model DNAs imity to DNA during complex formation. The work was funded
containing several key BER intermediates including AP sites. by RFBR grant № 17-54-45126.
Moreover AP-site is one of the most frequent types of DNA
damages. Using reconstituted systems we made a comparative
study of PARP1/PARP2 interaction with several key BER pro- P.18-008-Tue
teins (Pol-beta, FEN1 and APE1) demonstrating inhibitory effect Low homology FRPs from different
of both PARPs. Wherein in contrast to PARP1 the inhibition cyanobacteria functionally interact with
caused by PARP2 is hardly regulated by PAR synthesis. We next
Synechocystis OCP
demonstrated the ability of PARP2 to cross-link with AP site via
Y. Slonimskiy1,2, E. Maksimov3, N. Sluchanko2,3
Schiff base formation like PARP1 does. For both PARPs we 1
M.V. Lomonosov Moscow State University, Department of
determined major role of the N-terminal domains in formation
Biochemistry, Faculty of Biology, Moscow, Russia, 2A.N. Bach
of cross-links with AP DNA. We have also confirmed that DNA
Institute of Biochemistry, Federal Research Center of
binding by PARP2, in contrast to PARP1, is not modulated by
Biotechnology of the Russian Academy of Sciences, Moscow,
autopoly(ADP-ribosyl)ation. Taken together, our results testify
Russia, 3M.V. Lomonosov Moscow State University, Department
to the complicated multilevel regulation of short- and long-patch
of Biophysics, Faculty of Biology, Moscow, Russia
pathways of BER under coordinated action of PARP1 and
PARP2. This work was supported by RSCF project № 17-74- Photosynthesis requires a balance between efficient light harvest-
20075. ing and protection against photodamage. The cyanobacterial
photoprotection system uniquely relies on the functioning of the
photoactive orange carotenoid protein (OCP) that under intense
P.18-007-Mon illumination provides fluorescence quenching of the light-harvest-
Studying of DNA binding proteins using ing antenna complexes, phycobilisomes. The recently identified
crosslinking reaction with the participation of fluorescence recovery protein (FRP) binds to the photoactivated
OCP and accelerates its relaxation into the basal form, complet-
the cysteine residues
ing the regulatory circle. Lately, it was shown that, in addition to
L. Abrosimova1, M. Monakhova2, L. Zheleznaya3, T. Oretskaya2,
the function mentioned above, FRP is able to detach photoacti-
D. Rao4, E. Kubareva5
1 vated OCP from phycobilisomes. At the same time, the molecular
Department of Chemistry, M.V. Lomonosov Moscow State
mechanism of FRP functioning is largely controversial. More-
University, Moscow, Russia, 2Lomonosov Moscow State
over, since the available knowledge has mainly been gained from
University, Chemistry Department and Belozersky Institute of
studying Synechocystis proteins, the cross-species conservation of
Physical and Chemical Biology, Moscow, Russia, 3Institute of
the FRP mechanism remains unexplored. Besides the phyloge-
Theoretical and Experimental Biophysics, Pushchino, Moscow
netic analysis of FRP sequences, we performed a detailed struc-
Region, Russia, 4Department of Biochemistry, Indian Institute of
tural-functional analysis of two selected low-homology FRPs by
Science, Bangalore, India, 5M.V. Lomonosov Moscow State
comparing them with Synechocystis FRP (SynFRP). While
University, A.N. Belozersky Institute Of Physico-Chemical
adopting similar dimeric conformations in solution according to
Biology, Moscow, Russia
the SAXS data and preserving binding preferences of SynFRP
The correction of the errors occurred in the process of DNA toward various OCP variants, the low-homology FRPs demon-
replication is performed by DNA mismatch repair (MMR) sys- strated distinct binding stoichiometries and differentially accentu-
tem. The process of DNA repair is initiated when MutS protein ated features of this functional interaction. By providing clues to
binds to the mismatch. At the next step MutS binds ATP, understand the FRP mechanism universally, our results also
releases the mismatch and starts moving along DNA as a ‘sliding establish foundations for upcoming structural investigations nec-
clamp’. This process results in complex formation of MutS with essary to elucidate the FRP-dependent regulatory mechanism.
other MMR protein – MutL. The existence of this ternary com-
plex activates DNA repair. This multistage process has been
thoroughly studied for E. coli MMR system. We suggest investi- P.18-009-Wed
gating the process of DNA repair by MMR system taking place Analysis of the interaction between 14-3-3 and
in bacterium Neisseria gonorrhoeae, a parasite of mammals. The steroidogenic acute regulatory protein (StAR)
MutL protein in N. gonorrhoeae possesses endonuclease activity K. Tugaeva1,2, D. Sotnikov1, N. Sluchanko1,3
contrary to MutL from E. coli and initiates MMR by itself. Tak- 1
A.N. Bach Institute of Biochemistry, Research Center for
ing into account the conformation variability and dynamic nature Biotechnology of RAS, Moscow, Russia, 2Department of
of the MMR proteins themselves and in the complex with DNA, Biochemistry, School of Biology, M.V. Lomonosov Moscow State
one of the ways to study the interaction of constituents of this University, Moscow, Russia, 3Department of Biophysics, School of
complex is their covalent binding. For this purpose, the crosslink- Biology, M.V. Lomonosov Moscow State University, Moscow,
ing reaction between the MutL cysteine and amino groups of Russia
MutS is performed using N-b-maleimidopropyl-oxysuccinimide
ester in the presence of DNA. The crosslinked biomolecules are 14-3-3 proteins participate in different cellular processes through a
planned to be analyzed by mass spectrometric method. The multitude of interactions with phosphorylated partner proteins.
crosslinking reaction involving cysteine residues was also Recently, 14-3-3 proteins were found as components of the multi-
390 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 391
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
ligands. SPrOS calculates the scores for single positions based of P.18-014-Tue
the similarity of position surroundings in the studied amino acid
Arginyltransferase Ate1 is a target of
sequence and training sequences, classified in accordance with the
protein inhibitor interactions. We used the training sets based on apoptosis-inducing effect of amyloid-beta
publications presenting different experimental studies. The signifi- peptides
cant results were reproduced with the different training sets. Vali- O. Kechko1, I. Petrushanko1, K. Piatkov2, V. Mitkevich1,
dation with 3D structures and molecular dynamics of protein A. Makarov1
1
kinase complexes with their inhibitors was performed, allowing Engelhardt Institute of Molecular Biology, Russian Academy of
to confirm the interaction between predicted positions and the Sciences, Moscow, Russia, 2Skolkovo Institute of Science and
ligand molecule. We also revealed structural characteristics, Technology, Moscow, Russia
which conditioned the interaction of the same proteins with dif- Accumulation of amyloid-beta (Ab) in the form of insoluble
ferent ligands. This work is supported by Russian Foundation of amyloid plaques in specific brain regions is one of the main neu-
Basic Research, Grant no. 16-04-00491. romorphological signs of Alzheimer’s disease (AD). However,
long before the formation of amyloid plaques intracellular Ab
accumulation occurs in the pyramidal neurons of the hippocam-
P.18-013-Mon
pus and entorhinal cortex, that points to the important role of
Structural and functional characterization of intracellular proteolytic systems in AD pathogenesis. Up to 90%
PMGL3 esterase from Siberian permafrost of proteins in mammalian cells degrade through ubiquitin-protea-
microbial community some system (UPS). We have shown that Ab inhibits the activity
M. Kriukova1, K. Boyko2, A. Nikolaeva1, D. Korzhenevskiy1, of the N-end rule proteolytic pathway, which is the part of UPS
L. Petrovskaya3, K. Novototskaya-Vlasova4, E. Rivkina4, and involved in the proteins degradation prior proteasome. Ab
D. Dolgikh5, V. Popov6 impairs the process of proteins arginylation – second step of the
1
National Research Center “Kurchatov Institute”, Moscow, Russia, N-end rule pathway. Arginylation regulates a lot of key physio-
2
Bach Institute of Biochemistry, Research Center of Biotechnology logical events, in particular, degradation of proteins with Asp,
of the Russian Academy of Sciences, Moscow, Russia, Glu, and Cys at N-terminus through UPS. Ab bearing “English”
3
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the H6R familial mutation (H6R-Ab) that causes early-onset AD has
Russian Academy of Sciences, Moscow, Russia, 4Institute of greater inhibition effect on the proteins degradation through the
physicochemical and biological problems of soil science Russian N-end rule pathway than Ab. We have shown that Ab peptides
Academy of Science, Pushchino MR, Russia, 51917 – Shemyakin- interact with both isoforms of arginyltransferase (Ate1-1, Ate1-
Ovchinnikov Institute of Bioorganic Chemistry of the Russian 3). Ab reduces the catalytic rate of Ate1-1. H6R-Ab impairs the
Academy of Sciences, Moscow, Russia, 6A.N. Bach Institute of activity of both Ate1-1 and Ate1-3 significantly more efficient
Biochemistry, Federal Research Center of Biotechnology of the than unmodified Ab. In contrast, Ab peptides do not affect the
Russian Academy of Sciences, Moscow, Russia catalytic power of arginyl-tRNA synthetase. Using flow cytome-
try, it was found that H6R-Ab induces apoptosis and necrosis of
The Siberian permafrost is a unique environment for a variety of
mouse neuroblastoma Neuro-2a cells more efficient than Ab.
microorganisms surviving at low temperatures. The elucidation
Ate1 knockout in Neuro-2a cells completely eliminates the apop-
of the structural and molecular mechanisms of adaptation of
totic effect of both Ab peptides. Thus, suppression of Ate1 activ-
microorganisms provides understanding of the possible limits of
ity by amyloid peptides leads to inhibition of proteins
existence of life and the basis for the development of new biocat-
degradation and apoptosis that play the important role in AD
alysts. As a result of the construction and screening of fosmid
development. Supported by the Russian Scientific Foundation
libraries from permafrost metagenomic DNA, we obtained genes
(grant #14-24-00100).
coding for new esterases belonging to Hormone Sensitive Lipase
(HSL) Family. To determine the molecular basis of microbial
adaptation to permafrost conditions, we studied the functional P.18-015-Wed
characteristics of the enzyme PMGL3 and determined the 3D
It was demon- Biochemical and functional characterization of
structure of a protein with a resolution of 2.1 A.
strated that PMGL3 has a typical a/b-hydrolase fold and forms the yeast dead-box protein Dbp7
dimers in crystals and solution. We have shown that PMGL3 is J. Contreras1, J. Bravo2, J. De la Cruz3, E. Villalobo1
1
an esterase with maximum activity at 30°C toward p-nitrophenyl Departamento de Microbiologıa, Facultad de Biologıa,
octanoate (C8) as a substrate. The half-life time of the protein at Universidad de Sevilla, 41012-Sevilla, Spain, Seville, Spain,
2
40°C was 6 min, and it was completely inactivated by incubation Instituto de Biomedicina de Valencia (CSIC), Seville, Spain,
3
for 6 min at 50°C. Thus PMGL3 is a typical cold-adapted Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario
enzyme with decreased stability. To increase the thermal stability Virgen Del Rocio, Seville, Spain
of the enzyme, we performed site-directed mutagenesis aimed to The RNA helicases are DEAD box proteins involved in ATP-
enhance the cross-domain hydrophobic interactions in the protein dependent double stranded-RNA unwinding and strand anneal-
molecule. For the resulting mutants with increased stability ther- ing. They perform these activities during RNA metabolism ensur-
modynamic parameters were measured and compared with wild ing the correct folding occurs and maintaining the RNA
type PMGL3. For the mutant C207F which demonstrated 4-fold structure. One of the processes in which they are involved is the
increased stability after incubation at 40°C thermodynamic biogenesis of rRNA. Nineteen RNA helicases have been identi-
parameters were measured and compared with wild type fied that are involved in the synthesis of ribosomal RNA in yeast.
PMGL3. The work is supported by RFBR grant 18-04-00491. These include DBP6, DBP7, DBP9 and SPB4. These RNA heli-
cases are required for the correct maturation and assembly of
pre-60S ribosomal particles. To develop their functions in rRNA
biogenesis, the RNA helicases are often regulated by protein
partners. Until now, not too much is known about the function
of DBP6, DBP7, DBP9 and SPB4 in rRNA biogenesis and the
392 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
proteins that can modulate their activities in this process. Conse- P.18-017-Tue
quently, the objective of this project is to characterize the RNA
Characterization of membrane complexes
helicases DBP6, DBP7, DBP9 and SPB4 including its cofactors
and substrates, determining the role that these proteins carry out involved in CFTR stabilization by EPAC1
in rRNA biogenesis. To accomplish this goal the recombinant activation
proteins DBP6, DBP7, DBP9 were overexpressed in E. coli BL21 J. D. Santos1, F. R. Pinto1, M. D. Amaral1, M. Zaccolo2,
Codon Plus and purified by affinity and size exclusion chro- C. M. Farinha1
1
matography. The ATPase activity of these helicases were mea- BioISI-Biosystems and Integrative Sciences Institute, University
sured by a colorimetric assay. The essential kinetic parameters of Lisboa, Faculty of Sciences, Lisboa, Portugal, 2Department of
were determined in Dbp7 including Km and Vmax. In parallel, we Physiology, Anatomy and Genetics, University of Oxford, Oxford,
are analysing the in vivo relevance of different domains of the United Kingdom
Dbp7 protein by site-directed mutagenesis, including specific sub- Cystic Fibrosis is caused by mutations in the gene encoding the
stitutions of single conserved residues and truncations at its N- Cystic Fibrosis Transmembrane conductance Regulator (CFTR)
and C-terminal extensions. Progress on this approach will be pre- protein, a cAMP-regulated chloride channel expressed at the api-
sented. cal surface of epithelial cells. At the plasma membrane (PM),
CFTR membrane stability is regulated by cAMP through activa-
tion of the exchange protein directly activated by cAMP 1
P.18-016-Mon
(EPAC1), a process that involves binding to the PDZ-domain
Properties of MutL protein from the protein NHERF1. However, the mechanism by which EPAC1
Rhodobacter sphaeroides mismatch repair interacts with NHERF1 and consequently regulates CFTR is still
system poorly understood. Here, we aim at identifying the proteins
M. Monakhova1, A. Pavlova2, A. Alekseevski1, M. Milakina2, involved in CFTR stabilization at the PM under EPAC1 activa-
T. Oretskaya2, E. Kubareva1 tion to identify putative therapeutic targets to CFTR PM life-
1
Belozersky Institute of Physico-Chemical Biology, Lomonosov time. Methods: Cystic Fibrosis Bronchial Epithelial (CFBE) cells
Moscow State University, Moscow, Russia, 2Chemistry expressing wt-CFTR were treated with the adenylyl cyclase acti-
department, Lomonosov Moscow State University, Moscow, Russia vator forskolin or with the specific EPAC1 agonist 007-AM.
CFTR Immunoprecipitation followed by NanoLC-Triple TOF
The DNA mismatch repair (MMR) pathway removes errors that
was performed and proteins showing differential interactions
appear during genome replication. MMR corrects DNA by excis-
were selected for validation. Specific siRNAs for 19 genes were
ing an extended single-stranded fragment of the newly synthe-
used to determine their impact in CFTR trafficking. Results and
sized DNA and then filling the resulting gap. The signal for the
Discussion: More than 1000 interacting proteins were identified.
excision is hydrolysis of the phosphodiester bond in one of the
The largest number of specific CFTR interactors was detected in
DNA strands. In c-proteobacteria this break is introduced by the
cells treated with the EPAC1 agonist (>100 proteins) with 15%
MutH. However, most prokaryotes and all eukaryotes lack a
corresponding to proteins involved in cytoskeleton regulation.
mutH gene. Presumably, in these organisms MutL homologs cut
Several of these interactors were not previously directly associ-
the nascent strand of the DNA. Conserved endonuclease motifs
ated with CFTR. Knock-down of 12 out of the 19 genes selected
were identified in these MutL. In the alignment of 1792 sequences
for validation impacted significantly on the efficiency of CFTR
of bacterial MutL homologs we identified five endonuclease
trafficking to the PM. Further validation and characterization of
motifs comprising the catalytic site responsible for DNA cleavage
such CFTR interactors will clarify their mechanism of action and
in 1390 sequences. MutL proteins possessing the endonuclease
contribute to the identification of targets to specifically increase
motifs have been purified only from five bacterial species. Mecha-
CFTR PM stability. Work supported by PEst-OE/BIA/UI4046/
nism of searching of cleavage site in DNA by MutL is still
2011 – BioISI (from FCT – Portugal). JD Santos supported by:
unclear. We have purified the MutL protein from Rhodobacter
PD/BD/106084/2015 (from FCT – Portugal).
sphaeroides (rsMutL) for the first time. We demonstrated the
influence of metal ions on specificity and efficiency of DNA
hydrolysis by rsMutL. The protein showed the highest activity in P.18-018-Wed
the presence of Mn2+. The extent of plasmid DNA hydrolysis
Mapping of plasma membrane proteins
declined in the row Mn2+ > Co2+ > Mg2+ > Cd2+; Ni2+ and
Ca2+ did not activate rsMutL. Zn2+ inhibited rsMutL endonu- interacting with Arabidopsis flotillin-like
clease activity in the presence of Mn2+ excess. Apparently, Zn2+ protein 2
is not involved in DNA cleavage by rsMutL and could perform a P. Junkova1, M. Danek2, M. Janda1,2, J. Petrasek2,
structural function. Mn2+ and Mg2+ together induced double D. Kocourkova2, R. Hynek1, J. Martinec2, O. Valentova1
1
strand break followed by an increase in the amount of linear Department of Biochemistry and Microbiology, University of
DNA form that could be a result of rsMutL second subunit acti- Chemistry and Technology Prague, Technick a 3, 166 28, Prague 6,
vation. Analysis of amino acid sequences and biochemical prop- Prague, Czech Republic, 2Institute of experimental Botany AS CR,
erties of studied bacterial MutL homologs with endonuclease Prague, Czech Republic
activity revealed that rsMutL is similar the MutL proteins from Flotillins form a group of SPFH (stomatin/prohibitin/flotillin/
N. gonorrhoeae and P. aeruginosa. The work was funded by HflK/C) domain containing proteins localized in plasma mem-
RFBR grant 16-04-00575 and RSF grant 16-14-10319. brane microdomains, which act as signalling hub mediating the
cell response to stress. Such microdomains and flotillin homo-
logues were detected also in plants. Moreover, the similarity of
plant and animal flotillins, whose function in signalling pathways
most likely lies in the affection of other proteins via protein-pro-
tein interaction, suggests the similar role of plant flotillins. How-
ever, the mechanisms of plant response to various stress cues
differ from those in metazoans. Therefore, the determination of
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 393
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
plant flotillins interacting partners could contribute to the revela- biominerals involved in sensing of balance. Collagenous fibers
tion of their role in signalling pathways and deepen knowledge of and globular heads of otolin-1 may bind other proteins from
plants defence. To provide the reliable determination of plasma biomineral matrix, possibly enabling the spatiotemporal control
membrane proteins interacting with A. thaliana flotillin-like pro- of biomineralization. Despite that, little is known about the
tein 2, we used approach based on the immunoaffinity purifica- structure of otolin-1 and its globular C1q-like domain. We devel-
tion of enriched plasma membrane proteins with mass oped expression and purification procedures of C1q-like domain
spectrometry detection. Hereby, the proteins playing role in the of otolin-1 from human and zebrafish and examined their struc-
plant response to various biotic and abiotic stress factors were tures. The results show that the C1q-like domain forms trimers
identified as potential interacting partners of flotillin-like protein in solution in the presence of calcium ions. Without calcium ions,
2. Additionally, the employment of split-ubiquitin yeast two C1q-like domain exists in a monomer-trimer equilibrium with
hybrid system helped us to confirm that proteins such as plasma high association and dissociation rates. Calcium ions strongly
membrane-type ATPase 1 (AHA1), harpin-induced protein-like influenced the secondary structure and stability of the proteins.
(NHL3), hypersensitive-induced response protein 3 (HIR3) or The human variant was particularly sensitive to the calcium ions.
probable aquaporin PIP2-6 (PIP2-6) interacts directly with flotil- Fluorescent properties of the C1q-like domain indicate that the
lin-like protein 2. Those proteins have already been connected protein has hydrophobic regions exposed to the solvent, which
with the mediation of the response to bacteria, viruses, oomycetes may participate in binding of macromolecular ligands from
or nematodes as well as the response to water deprivation and biomineral matrix. These observations indicate that C1q-like
abscisic or salicylic acid signalling. domain may facilitate formation of otolin-1 trimers. Influence of
calcium ions on the structure and stability of C1q-like domain
indicates the importance of the C1q-like domain for the assembly
P.18-019-Mon of the organic matrix of otoliths and otoconia. Destabilisation of
Cancer metabolism and the human hexokinase the organic matrix induced by changes resulting from aging or
2 as an anticancer target disease may facilitate dissolution and release of otoconia and
J. Ferreira, M. H. Nawaz, W. Rabeh contribute to benign paroxysmal positional vertigo (BPPV). This
New York University Abu Dhabi, Abu Dhabi, United Arab project was funded by the National Science Centre (Poland)
Emirates grants UMO-2015/19/B/ST10/02148 and DEC-2012/06/M/ST4/
00036, and a statutory activity subsidy from the Polish Ministry
Cancer utilizes glucose at elevated levels to support its growth and of Science and High Education for the Faculty of Chemistry of
proliferation, historically known as “ Warburg effect”. Targeting Wroclaw University of Science and Technology.
glucose metabolism in cancer cells to limit its growth will enhance
patients’ survival rate. Hexokinase catalyzes glucose phosphoryla-
tion, and is a major step in regulation of glycolysis. One of four iso- P.18-021-Wed
zymes found in humans, hexokinase 2 (HK2), the first enzyme of Amyloid properties of the Escherichia coli
the glycolytic pathway, catalyzes the phosphorylation of glucose
YghJ protein
for its activation and further metabolism. Moreover, gene expres-
M. Belousov1,2, S. Bondarev1, A. Kosolapova1,2, K. Antonets1,2,
sion profiling experiments of different types of cancer showed high
M. Belousova2, A. Sulatskaya3, M. Sulatsky3, G. Zhouravleva1,
expression levels of HK2, and various biological studies high-
I. Kuznetsova3, K. Turoverov3,4, A. Nizhnikov1,2
lighted the importance of HK2 in tumor metastasis making it an 1
Saint Petersburg University, Saint Petersburg, Russia, 2All-Russia
ideal target to characterize cancer metabolism and for the develop-
Research Institute for Agricultural Microbiology, St. Petersburg,
ment of new class of cancer therapeutics. The crystal structure of
Pushkin, Russia, 3Institute of Cytology of the Russian Academy of
human HK2 was determined in complex with glucose and glucose-
Sciences, Saint Petersburg, Russia, 4Peter the Great St. Petersburg
6-phosphate (PDB code: 2NZT), where it is a homodimer with cat-
Polytechnic University, Saint Petersburg, Russia
alytically active N- and C-terminal domains linked by a seven-turn
helix. Through biochemical and biophysical characterization of Amyloids are protein fibrils with a characteristic spatial structure.
HK2, we found that the N-terminal domain not only catalyzes a In previous studies, using a method for the proteomic screening
reaction but it thermodynamically stabilizes the entire enzyme, and identification of amyloids, we identified 61 proteins of
where deletion of the N-terminal helix altered the stability and cat- Escherichia coli that formed detergent-resistant aggregates in vivo
alytic activity of the full-length enzyme. Also, conformation of the and without overproduction. Among these proteins, YghJ was
N-terminal active site but not C-terminal is important in stabilizing the most enriched with bioinformatically predicted amyloidogenic
the enzyme. Understanding the structural and molecular mecha- regions. YghJ is a lipoprotein with the zinc metalloprotease M60-
nisms of human HK2 in cancer metabolism and apoptosis will like domain (YghJM) that is involved in the mucin degradation
accelerate the design and development of new class of cancer thera- in the intestine as well as in proinflammatory responses. We
peutics. detected detergent-resistant aggregates of YghJM by SDS-PAGE
and SDD-AGE and confirmed that these aggregates are resistant
to a-chymotrypsin. Next, we demonstrated that the YghJM
P.18-020-Tue aggregates bind Thioflavin-T amyloid-specific dye and exhibit
Structural properties of a C1q-like domain of CD-spectra typical for protein aggregates rich in b-sheets. The
otolin-1 from human and zebrafish Congo red stained YghJM fibrils demonstrated bright apple-green
R. Hołubowicz1, M. Taube2, M. Kozak2, A. O_zyhar1, birefringence which is considered to be the “gold standard” for
P. Dobryszycki1 verification of the amyloid structure. Finally, we showed that
1
Department of Biochemistry, Faculty of Chemistry, Wroclaw YghJM forms amyloid fibrils on the surface of the Escherichia
University of Science and Technology, Wroclaw, Poland, 2Faculty coli cells using the curli-dependent amyloid generator system (C-
of Physics, Adam Mickiewicz University, Poznan, Poland DAG). Overall, we demonstrated that YghJM forms amyloid fib-
rils in vitro and in vivo. Our data on the amyloid properties of
Otolin-1 is a collagen-like protein expressed in the inner ear of the YghJ protein contribute to the knowledge of bacterial amy-
vertebrates. It provides an organic scaffold for otoliths of fish loids and is useful for investigations of the relationship between
and otoconia of land vertebrates, which are calcium carbonate amyloidogenesis and virulence of bacteria. This study was
394 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
supported by the Russian Foundation for Basic Research (16-34- same experimental design we have investigated the influence of
60153 to AAN, 17-54-150002 to GAZ), and the Grants of the Cr(VI), Mg, Ca ions on bacterial proteome changes by 2-D elec-
President of the Russian Federation (MK-3240.2017.4 to AAN, trophoresis. Differentially expressed bands were identified by
MK-512.2017.4 to MVB). Mass-Spectrometry. Differentially expressed proteins included:
Carboxy muconolactone decarboxylase, DNA-directed RNA
polymerase subunit beta, Capsular glucan synthase, Aldehyde
P.18-022-Mon dehydrogenase, ABC transporter, ABC transporter substrate-
Lipids inhibit aggregation of a-synuclein binding protein, FAD-linked oxidoreductase and many others.
A. Kurochka, V. Shvadchak, D. Yushchenko This work was supported by Shota Rustaveli National Science
Institute of Organic Chemistry and Biochemistry IOCB Research Foundation (SRNSF) FR/218 018/16 project title: “The influence
Centre & Gilead Sciences, Academy of Sciences of the Czech of different metal ions on the Cr(VI) reduction process ongoing
Republic, Flemingovo nam. 2, 166 10, Prague, Czech Republic, in bacteria such as Arthrobacter species”.
Prague, Czech Republic
a-Synuclein (aSyn) is a small neuronal protein that exists in equi- P.18-024-Wed
librium between unstructured cytosolic and helical membrane
Deepening into the modulation of human
bound forms. In neurons affected by Parkinson’s disease aSyn
can aggregate forming pathological amyloid fibrils composed of D-amino acid oxidase
thousands of monomers. aSyn fibril formation is an autocatalytic G. Murtas1, Z. Motta1, L. Pollegioni1,2, S. Sacchi1,2
1
multistage process. Slow spontaneous formation of first fibrils University of Insubria, Varese, Italy, 2The Protein Factory,
(lag phase) is followed by fast autocatalytic fibril elongation by Politecnico di Milano and University of Insubria, Milano, Italy
binding monomers to the ends (elongation phase). Interaction of Human D-amino acid oxidase (hDAAO, EC 1.4.3.3) is a peroxi-
aSyn with lipid membranes can inhibit fibril formation. We somal FAD-dependent enzyme that catalyzes the degradation of
aimed to investigate the role of lipids in aSyn fibril formation D-amino acids, taking part into different physiological processes:
studying separately the influence on lag phase and on elongation in the brain, the favorite substrate is D-serine (D-Ser), the main
phase. We measured aSyn fibrillization kinetics in the presence of endogenous co-agonist of the N-methyl-D-aspartate receptors
various concentration of model membranes (vesicles) quantifying (NMDAr). Converging lines of evidence suggest that a dysregula-
amount of formed fibrils using Thioflavin T fluorescence. To tion in processes tuning D-Ser concentration, and thus in
measure the fibril elongation rate selectively, we added to solu- NMDAr transmission, is involved in the mechanism of predispo-
tion some amount of pre-formed short aSyn fibrils serving as sition in various pathologies. The evidence of the important role
growth centers. We found that decrease in fibril elongation rate played by hDAAO in modulating D-Ser levels has resulted in an
in the presence of lipids correlates with the fraction of mem- increased interest for this flavoenzyme. Although its structural
brane-bound aSyn monomer. It indicates that lipids inhibit fibril and biochemical properties have been extensively investigated,
elongation by depletion aSyn monomer from solution. The influ- several aspects in the modulation of the enzymatic activity and
ence of lipids on the initial fibril formation (lag phase) was much stability remain elusive. hDAAO is known to be regulated by
stronger. It was observed at lipid concentrations not sufficient for small size ligands and cofactor binding as well as by the interac-
binding significant fraction of aSyn monomer. So we propose the tion with the proteins pLG72 and bassoon. Furthermore,
model where lipids can preferentially bind rare intermediate spe- hDAAO has been proposed to be regulated by nitrosylation and
cies (oligomers) in primary nucleation process. phosphorylation [NetPhos 2.0 server], but the consequences of
these modifications on the enzyme’s properties were poorly inves-
tigated. Moreover, it has been suggested that the flavoenzyme
P.18-023-Tue could be mistargeted (to cytosol and nuclei) or secreted. Here, we
Effect of chromate(VI), magnesium and investigated hDAAO post-translational modifications (PTMs) in
calcium on the proteome of Arthrobacter several tissue lysates by immunoprecipitation and Western blot
globiformis 151B analysis. Moreover, the enzyme modifications and their effects on
O. Rcheulishvili1,2, R. Solomonia1, L. Tsverava1, the structural and functional properties of hDAAO were studied
A. Rcheulishvili2, E. Gelagutashvili2, E. Ginturi2, N. Metreveli1, in vitro using the recombinant enzyme. Finally, hDAAO PTMs
M. Gurielidze2, L. Lomidze1, L. Tugushi2, H. Holman3 were also investigated by cellular studies. The identification and
1
Ilia State University, Tbilisi, Georgia, 2Ivane Javakhishvili Tbilisi the comprehension of the mechanisms of hDAAO modulation
State University Andronikashvili Institute of Physics, Tbilisi, might allow the identification of new targets for the treatment of
Georgia, 3Lawrence Berkley National Laboratory, Berkeley, CA, pathologies in which this flavoenzyme is involved.
United States of America
In our study, we investigated bacterial cell culture of Arthrobac- P.18-025-Mon
ter globiformis 151B isolated from a most contaminated site of b-sheet breaker NABi reduces Ab aggregates
Georgia (Kazreti). According to our previous research,
and prevents neuronal cell death
Arthrobacter species are characterised with chromium(VI) reduc-
J. Jang1, M. Nam2, E. Kim3, H. Rhim2, S. Kang3
tion capability from Cr(VI) to Cr(III). Fe and Mg are among the 1
Division of Life Sciences, College of Life Sciences and
most abundant elements in earth’s crust and play an important
Biotechnology, Korea University, Seoul, Korea, 2Department of
role in all living organisms. Natural habitats are often character-
Medical Life Sciences, College of Medicine, Catholic University of
ized by the coexistence of Cr, Mg and Fe. A. globiformis 151B
Korea, Seoul, South Korea, 3Division of Life Sciences, College of
cells were grown in a TS Broth medium, containing up to 50 ug/
Life Sciences and Biotechnology, Korea University, Seoul, South
ml Cr(VI), Mg and Ca. The exposure of bacterial cells in metal
Korea
containing growth medium continued up to 40 h (late stationary
phase). The metal accumulation ability of bacteria and the influ- Amyloid beta (Ab) aggregates are an important therapeutic tar-
ence of Mg2+, Ca2+ ions, on the reduction process of Cr6+ was get for Alzheimer’s disease (AD), a fatal neurodegenerative dis-
determined by atomic absorption spectroscopy. According to the ease. Previously, we found that intracellular Ab specifically
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 395
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
396 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
towards BJSP as compared with that of other known trypsin transduceosome together with other proteins (VDAC1, TSPO,
inhibitors. The distinct BJSP preparations were then analyzed by PKA regulatory subunits a (PKAR1a), ANT etc.) in the outer
SPR on a Biacore T200 equipment fitted with CM5 chip deriva- mitochondrial membrane. ACBD3 works as a scaffold protein of
tized with SFTI and PAB. Sensorgrams obtained with the BJSP TSPO and PKAR1a. Knockdown of ACBD3 suppresses hor-
purified by affinity chromatography on SFTI-agarose showed a mone-induced steroidogenesis (Li et al., 2003). In patient with
high affinity for SFTI inhibitors, reaching a steady state and dis- homozygous mutation in ACBD3 gene, we observed different
playing fast association (kon) and dissociation (koff) rates. Thus distribution of cholesterol in fibroblasts and increased and
far we were unable to get unambiguous SPR results regarding enlarged mitochondria in muscle together with decreased amount
interactions between all of the purified BJSP and PAB. Possible of OXPHOS complexes CI, CIII, CIV and ATP synthase. The
causes for these inconclusive results are being discussed. The aim of study was to characterize the impact of full absence of
overall results concerning the interactions between BJSP and ACBD3 in HeLa cells. We created knockout cell lines of ACBD3
SFTI provide useful insights for the development of new inhibi- by CRISPR/CAS9 system and studied overall impact of absence
tors of snake venom proteases. of the gene on mitochondrial metabolism, using proteins and
functional analysis of the OXPHOS system or by microscopy
analysis. Supported by research projects: GAUK 542217, GACR
P.18-029-Tue 14-36804G, RVO VFN64165/2012 and SVV 260367.
Role of internal dynamics in ligand binding of
the bile acid transporter protein gastrotropin
Z. Harmat1, A. L. Szabo1, O. T}
oke2, Z. Gaspari1
P.18-031-Mon
1
Pazmany Peter Catholic University, Faculty of Information Structural ensembles of 3rd PDZ domain of
Technology and Bionics, Budapest, Hungary, 2Research Centre for PSD-95 describing a dynamic ligand-binding
Natural Sciences, Hungarian Academy of Sciences, Budapest, mechanism
Hungary A. Hinsenkamp, Z. Gaspari
The protein gastrotropin, also known as intestinal ileal bile acid- Pazmany Peter Catholic University, Faculty of Information
binding protein (I-BABP) or ileal intestinal lipid-binding protein Technology and Bionics, Budapest, Hungary
(ILBP) or fatty acid binding protein 6 (FABP6), is a member of PDZ domain containing proteins in the postsynaptic density
intracellular lipid binging binding protein family. Gastrotropin (PSD) of excitatory synapses play a fundamental role in synaptic
binds bile acids in the cytoplasm of intestinal cells and takes part plasticity. The third PDZ domain (PDZ3) of PSD-95, the most
in the enterohepatic circulation of bile salts. The protein adopts a abundant scaffold protein in the PSD, has an additional alpha
beta clam structure, composed by two beta sheets having 10 beta helix on its carboxy terminus besides the common PDZ architec-
strands altogether and a helical cap encompassing a helix-loop- ture. Although this helix does not make direct contacts with the
helix motif. The protein binds glycocholate and glycochen- ligand, its phosphorylation/truncation is a potential regulator of
odeoxycholate in a cooperative way. NMR investigations suggest ligand binding. Atomic-level details of this intermolecular allos-
that gastrotropin in its apo form is in slow exchange with a low- teric process, involving conformational entropy, are not yet eluci-
populated conformation reminiscent to the doubly-liganded holo dated in detail. We performed a number of molecular dynamic
structure. In this work we performed ensemble-based molecular simulations of different lengths incorporating NMR-derived
dynamics simulations with and without NMR-derived experimen- backbone and side-chain mobility data as restraints using bound
tal data (NOE distances and backbone S2 order parameters) as and unbound, as well as full and truncated PDZ3 structures. The
restraints. The generated structural ensembles are in good agree- obtained ensembles correspond well to experimental data and
ment with NMR parameters not included in the simulations and show characteristic differences near their ligand-binding regions.
reveal two kinds of internal motions that can be relevant for Extensive comparative analysis, including PCA, were used to
ligand binding. Extensive docking calculations as well as addi- decipher the motions and intramolecular interactions of the
tional investigations in search for a structure similar to the low- PDZ3 domain in its different states. Estimates of changes in con-
populated form in exchange with the apo state allowed us to out- formational entropy were also calculated between the free and
line an initial atomic-level model on the role of internal dynamics bound forms. Our results suggest that dynamic structural ensem-
in ligand binding in gastrotropin. bles are capable of revealing atomic-level insights into the mecha-
nistic action of PDZ domains beyond what can be deciphered
based merely on the experimental observations.
P.18-030-Wed
The relevance of ACBD3 protein in energy
metabolism in various cell lines P.18-032-Tue
T. Danhelovska, H. Stufkova, M. Rodinova, J. Sladkova, Interactions of 17beta-hydroxysteroid
T. Honzik, H. Hansikova, J. Zeman, M. Tesarova dehydrogenase type 10 and cyclophilin D in
Laboratory for Study of Mitochondrial Disorders, Department of Alzheimer disease
Pediatrics and Adolescent Medicine, First Faculty of Medicine,
Z. Kristofikova1, A. Hofmannova1, L. Hromadkova1,
Charles University and General University Hospital in Prague, E. Gedeonova2, T. Springer2, M. Bockova2, J. Homola2
Prague, Czech Republic 1
National Institute of Mental Health, Klecany, Czech Republic,
2
Acyl-CoA biding domain contain 3 (ACBD3) is a protein of Institute of Photonics and Electronics, Prague, Czech Republic
Golgi apparatus which has many functions in the cell such as Nucleus-encoded mitochondrial matrix protein 17beta-hydroxys-
division of neuronal cells, in neurodegeneration, lipid homeosta- teroid dehydrogenase type 10 (17beta-HSD10) binds to amyloid
sis, in response of cell stress, apoptosis or in maintenance of
beta (Abeta). In people with Alzheimer disease (AD), 17beta-
Golgi. Moreover, ACBD3 has important role in replication of HSD10 brain overexpression was observed. It is assumed that
some plus RNA viruses. In steroidogenic cells ACBD3 is essen- cytosolic 17beta-HSD10 is imported into mitochondrial matrix
tial in transport of cholesterol into mitochondria. In this type of
where binds to cyclophilin D (CypD) and, by preventing its
cells, ACBD3 coexists in multiprotein complex named translocation to the inner mitochondrial membrane, regulates the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 397
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
opening of the mitochondrial permeability transition pore medi- work was supported by a grant from the Russian Foundation for
ated by CypD. Mitochondrial Abeta could influence the regula- Basic Research (# 17-04-00032).
tion of CypD by 17beta-HSD10. In this study, we proved the
interaction between 17beta-HSD10 and CypD by using the sur-
face plasmon resonance (SPR) biosensor method in which 17- P.18-034-Mon
beta-HSD10 immobilized on the surface of SPR sensor was Single-molecule FRET reveals Zn(II)-dependent
interacting with CypD in solution. The formation of 17beta- switch in S. cerevisiae Rad50 dimer’s
HSD10 – CypD complex was further confirmed by indirect quaternary structure
approach in which the 17beta-HSD10 – CypD complex previ-
M. Padjasek1, B. Krajnik2, A. Pomorski1, J. Tran1,
ously formed in solution was determined by second antibody on
A. Podhorodecki2, A. Kre_zel1
the surface of SPR sensor. Then we evaluated the ability of 17- 1
Faculty of Biotechnology, University of Wroclaw, Wroclaw,
beta-HSD10 to regulate CypD in brain mitochondria isolated
Poland, 2Wroclaw University of Science and Technology Faculty
from 11-month old transgenic McGill-R-Thy1-APP rats, one of
of Fundamental Problems of Technology, Wroclaw, Poland
animal models of AD with accumulated Abeta. We observed no
changes (levels of 17beta-HSD10, CypD, Abeta 1-42 and 17beta- Rad50 is an internal constituent of the MRN(X) complex, a com-
HSD10 – Abeta complexes), the significant increase (Abeta) and plex responsible for double-stranded DNA damage sensing and
the significant drop (17beta-HSD10 – CypD complexes) in a repair, as well as triggering the protein network of DNA damage
comparison with wild-type rats. We also estimated levels of 17- response pathway. Recent studies unveiling the structure of the
beta-HSD10 – Parkin complexes in cerebrospinal fluid of AD central part of human Rad50 shows two Rad50 protomers with
people but we did not find marked alterations compared to con- tetrathiolate Zn(II)-binding site in a rod-shaped assembly, with
trols. Our results suggest that up-regulation of 17beta-HSD10 in coiled-coil segments of both protomers arranged in a parallel
AD does not have to be followed by its increased transport into fashion. Our preliminary data indicate that yeast ortholog may
mitochondrial matrix via the PINK1-Parkin-TOM/TIM pathway. significantly differ from human one and presents itself as a
Moreover, the ability of 17beta-HSD10 to regulate CypD in dynamic entity able to adopt two distinct dimer assemblies –
mitochondrial matrix, under conditions of accumulated Abeta, is open and closed, depending on the Zn:Rad50 stoichiometric ratio
weakened. Supported by GACR (P304-12-G069) and AZV (16- of the complex, namely Zn(II)-mononuclear and Zn(II)-binuclear
27611A) projects. Rad50 dimers corresponding to 1:2 and 2:2 ratio, respectively.
Using tetracysteine tag and hetero-biarsenical labeling (FlAsH-
ReAsH) of yeast Rad50 zinc hook domain we were able to visu-
P.18-033-Wed alize resonance energy transfer between protomers inside Rad50
Yeast stress granule protein Pub1 is involved dimer with single-molecule resolution. Single-molecule FRET,
in translation termination and detoxification of anisotropy decay and cross-linking experiments indicate that
additional metal ion could initiate zinc-hook domain refolding
the [PSI+] prion
from open – P. furiosus-like to closed – H. sapiens-like, assembly.
O. Mitkevich, V. Urakov, A. Dergalev, M. Ter-Avanesyan
Such event would have to manifest itself in global conforma-
Bach Institute of Biochemistry RAS, Federal Research Centre
tional change of the entire MRN(X) complex and therefore
“Fundamentals of Biotechnology” of the Russian Academy of
change of its functional status as well, linking together three sep-
Sciences (Russia), Moscow, Russia
arate physiological pathways – DNA damage response, cell cycle
Termination of translation in eukaryotes is controlled by inter- and redox-intertwined Zn(II) distribution. Financial support by
acting polypeptide chain release factors, eRF1 and eRF3 (known National Science Center (grant no. 2016/23/N/NZ1/00040) is
as Sup45 and Sup35 in yeast, respectively) and the ABCE1 (Rli1 gratefully acknowledged.
in yeast) protein facilitating ribosome recycling. Here we describe
a novel participant involved in termination. We show that Pub1,
a yeast protein known to be involved in stress granule formation, P.18-035-Tue
regulation of gene expression, and organization of the tubulin Cracking the code of N-myristoylation at
cytoskeleton, also plays a role in translation termination. This structural and genome scales
protein was bound to ribosomes and was preferentially associated C. Dian1, B. Castrec1, S. Ciccone1, C. L. Ebert1,
with heavy polysomes enriched with terminating ribosomes. W. V. Bienvenut1, J. Le Caer2, J. Steyaert3, C. Giglione1,
Besides, it interacted with the N-terminal glutamine-/asparagine- T. Meinnel1
rich prion-forming domain of eRF3, though its binding to ribo- 1
Institute for Integrative Biology of the Cell (I2BC), Gif sur
somes did not depend on interaction with Sup35. Lack of Pub1 Yvette, France, 2Centre de Recherches de Gif, Institut de Chimie
decreased efficiency of nonsense readthrough at a majority but des Substances Naturelles, CNRS UPR 2301, Gif sur Yvette,
not all tetranucleotide stop signals. This distinguishes Pub1 from France, 3Ecole Polytechnique, Laboratory of biochemistry,
most other known binding partners of the release factors which Palaiseau, France
were shown to modulate readthrough of all nonsense codons uni-
formly. Besides its effect on translation termination, Pub1 influ- Comprehensive characterization of the entire repertoire of a pro-
enced properties of [PSI+], a prion form of Sup35. Deletion of tein modification of an organism is yet unachieved. N-Myristoy-
PUB1 (ΔPUB1) caused approximately a 2-fold increase of Sup35 lation (MYR) is a crucial eukaryotic N-terminal protein
prion polymer levels in cells containing [PSI+]. Importantly, modification consisting on the covalent addition of a C:14 lipid
combining ΔPUB1 with deletion of UPF1, a protein involved by the N-Myristoyl Transferases (NMTs) to the amino-termini of
both in nonsense mediated mRNA decay and translation termi- a subset of proteins displaying unmasked N-terminal glycine.
nation, but does not affect Sup35 prion polymerization, was This modification is involved in major biological processes influ-
lethal. Analysis of this effect showed that lethality was caused by encing membrane association of peripheral membrane proteins,
excessive sequestration of Sup45 into Sup35 prion aggregates. protein targeting to membrane subdomains, protein-protein inter-
The obtained data show that Pub1 can act as an accessory trans- action, and protein trafficking. We assembled complementary
lation factor involved in fine-tuning of translation termination strategies to tackle the complete MYR status in proteomes. A
and as an important factor of [PSI+] prion detoxification. The series of crystal structures of human NMT1 in complex with
398 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 399
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
evolution, revealing a latent internal N-degron that facilitated modern enzymes. This ancestral enzyme dubbed AncHLD-RLuc
PRC2 neo-functionalisation through coupling su(z)12 stability to exhibits both activities, with dehalogenase as its main activity
environmental signals. (10x lower than LinB), luciferase as secondary promiscuous activ-
ity (10000x lower than RLuc). Comparison between crystal struc-
tures of ancestral and extant enzymes uncovered high structural
P.18-039-Wed similarity between these proteins. The main difference lies in the
A major protein Rv0341 in the membrane of arrangement of secondary structure elements of the cap domain
dormant Mycobacterium tuberculosis binds and in the conformation of a loop connecting the main and the
cap domain. The flexibility of this region is believed to contribute
DNA and reduces the rate of RNA synthesis
to the binding of the luciferase substrate, coelenterazine. Random
M. Shleeva, K. Trutneva, M. Shumkov, G. Demina,
insertion and deletion mutagenesis was then performed on the
A. Kaprelyants
ancestral AncHLD-RLuc to investigate whether altering the pro-
A.N. Bach Institute of Biochemistry, Federal Research Centre
tein backbone would lead to a change in specificity. The gener-
‘Fundamentals of Biotechnology’ of the Russian Academy of
ated variant libraries were screened and the best hits were
Sciences, Moscow, Russia
characterized. Interestingly, variants exhibiting up to 60x increase
Formation of dormant Mycobacterium tuberculosis (Mtb) cells is in luciferase activity carried alterations in the specific regions
responsible for the phenomenon of latent tuberculosis. Molecular where the structure of the ancestral enzyme differs the most from
mechanisms of the transition of viable cells to dormant state are the structure of RLuc. High throughput molecular dynamics was
not fully elucidated. Recently we have shown that gradual acidifi- used to perform adaptive sampling of the Root mean square
cation of the external medium results in the formation of mor- deviation of the Ca atoms. Total simulation time was more than
phologically distinct dormant Mtb cells in vitro. Comparative 4000 ns and no less than 12 epochs. Molecular dynamic simula-
proteomic analysis between dormant and active Mtb cells resulted tions as well as experimental analysis based on hydrogen-deuter-
in accumulation of unknown Rv0341 as a major protein in mem- ium exchange connected with mass spectrometry are in progress
brane fraction of dormant forms. Using electrophoresis in native to analyze changes in the flexibility of the hot-spot regions.
conditions with low concentration of acrylamide and MALDI-
TOF analysis we found presence of large complexes (680 kDa and
greater) contained of Rv0341 protein in membrane fragment of P.18-041-Tue
dormant M. tuberculosis cells. In early stage of dormant cell reacti- In vivo incorporation of photoproteins into
vation, destruction of these complexes to monomer form of protein GroEL tetradecameric quaternary structure
begins and in logarithmic phase large complexes of Rv0341 disap- V. Marchenkov1, N. Marchenko1, T. Ivashina1,2, N. Ryabova1,
peared. M. tuberculosis strain with overexpression of Rv0341 A. Timchenko1, I. Kashparov1, V. Ksenzenko1, G. Semisotnov1
(ovRv0341) demonstrated reduction of growth rate on solid and 1
Institute of Protein Research, Russian Academy of Sciences,
liquid medium and the formation of large clumps during growth. Pushchino, Moscow Region, Russia, 2G.K. Skryabin Institute of
The cells of this strain were characterized by lower metabolic activ- Biochemistry and Physiology of Microorganisms, Russian
ity vs cells of control strain and by increased floating density show- Academy of Sciences, Pushchino, Moscow Region, Russia
ing significant changes in structure and metabolism of ovRv0341.
The recombinant Rv0341 protein bound to pDNA in a gel mobility To study protein location in a cell and protein-protein interac-
shift assay. This study for the first time demonstrates that Rv0341 tions in vivo, it is often necessary to introduce labels into protein
protein accumulates in significant amount in dormant mycobacte- structures. One of the promising approaches to mark proteins
ria, can bind to DNA and possibly participates in molecular events in vivo is expression of genes encoding fusion proteins consisting
resulted in transition of viable cells to dormancy and maintenance of a target protein and a photoprotein (such as various fluores-
of cell viability for long time in non-replicative conditions. This cent proteins and luciferases). However, when the target protein
work was financially supported by the Russian Science Foundation is a subunit of a complicated oligomeric protein, there arises the
grant 16-15-00245 (comparative proteomic analysis between dor- problem of packing the fused subunits into the native oligomeric
mant and active Mtb) and the Russian Foundation for Basic structure. Here, we report the insertion of the GroEL subunit
Research grant 18-015-00239a (experiments with Rv0341). fused with EGFP (M.w. 29 kDa) or with Gaussia princeps lucifer-
ase (M.w. 20 kDa) into the GroEL tetradecameric quaternary
structure. Expression of the prepared plasmids in E. coli cells
P.18-040-Mon resulted in folded GroEL subunits and folded photoproteins
Modification of protein dynamics by directed within the fusion proteins, but the fused subunits failed to form
evolution based on insertions and deletions the tetradecameric oligomeric structure and were purified as
monomeric forms. However, when these plasmids encoding
A. Schenkmayerova1,2, G. Pinto1,2, V. Liskova1,2, S. Emond3,
fusion proteins were co-expressed with plasmids encoding only
L. Hernychova4, D. Bednar1,2, R. Chaloupkova1,2,
GroEL (with an expression efficiency ratio of ~1:10), practically
F. Hollfelder3, J. Damborsky1,2
1 all subunits fused with photoproteins were incorporated into the
The International Clinical Research Center of St. Anne’s
tetradecameric quaternary structure. The purified GroEL with
University Hospital Brno (FNUSA-ICRC), Brno, Czech Republic,
2 incorporated fusion subunits was characterized by size-exclusion
Loschmidt Laboratories, Department of Experimental Biology,
chromatography, absorption and luminescence spectroscopy, as
Research Centre for Toxic Compounds in the Environment,
well as by urea transverse gradient electrophoresis, diffuse small-
Faculty of Science, Masaryk University, Brno, Czech Republic,
3 angle X-ray scattering, and electron transmission microscopy.
Department of Biochemistry, University of Cambridge,
The results show that only two subunits fused with photoproteins
Cambridge, United Kingdom, 4Regional Centre for Applied
are incorporated into the tetradecameric GroEL structure (possi-
Molecular Oncology, Masaryk Memorial Cancer Institute, Brno,
bly one fused subunit per one GroEL heptameric ring). Packing
Czech Republic
and stability of the GroEL quaternary structure with incorpo-
The common ancestral enzyme between haloalkane dehalogenase rated fusion subunits are similar to those of the wild-type ver-
(HLD, EC 3.8.1.5) and Renilla-luciferin 2-monooxygenase (RLuc, sion, and the photoproteins are apparently packed within the
EC 1.13.12.5) was resurrected in silico based on the sequences of
400 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
GroEL inner cavity. This work was supported by the Russian latter in vitro. The most pronounced inhibitory effect of YB-1
Science Foundation grant 14-24-00157. was observed at the initial stages of polymerization of the Ab-
peptide. Another AD symptom is the formation of intracellular
tangles consisting of hyperphosphorylated tau-protein that has
P.18-042-Wed lost its ability to bind to microtubules, thus provoking micro-
Characterization of Na-acetyltransferase 60 tubule destruction and disruption of mRNA transport along
(NAA60) isoforms axons. We have compared the effects of YB-1 and tau-protein on
S. Strømsøy1,2, H. Aksnes1, T. Arnesen1,3,4 tubulin polymerization in vitro and found that the stimulating
1
Department of Biomedicine, University of Bergen, Bergen, effect of YB-1 is even higher than that of tau-protein. We believe
Norway, 2Department of Clinical Science, University of Bergen, that YB-1 can functionally replace the inactivated hyperphospho-
Bergen, Norway, 3Department of Breast and Endocrine Surgery, rylated tau-protein, thus maintaining microtubule integrity and
Haukeland University Hospital, Bergen, Norway, 4Department of the active mRNA transport in AD patients. This study was sup-
biological sciences, University of Bergen, Bergen, Norway ported by RSF (# 14-14-00879) and RFBR (#18-04-00595).
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 401
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
402 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
A546p toxin variants on artificial lipid bilayers does not correlate P.18-050-Tue
with their hemolytic activities on erythrocytes. The molecular
Crystal structures of the complex of kallikrein
basis of this intriguing phenomenon is under investigation and
will be presented. inhibitor BbKI with trypsin and a comparison
of its inhibitory properties for various
kallikreins
P.18-049-Mon A. Gustchina1, M. Li1, J. Srp2, Z. Dauter3, M. Mares4,
A yeast model for genetic variants in human A. Wlodawer1
1
NAA15 associated with autism spectrum National Cancer Institute, Frederick, MD, United States of
disorder America, 2Institute of Organic Chemistry and Biochemistry AS
S. Varland1,2,3, H. Cheng4, A. V. D. Dharmadhikari4, CR, Flemingovo nam. 2, Prague, Czech Republic, Prague, Czech
A. Stray-Pedersen4,5,6, G. M. Mancini7, L. Meng4, G. J. Lyon8, Republic, 3National Cancer Institute, Argonne, IL, United States
T. Arnesen2,3,9 of America, 4Institute of Organic Chemistry and Biochemistry,
1
Donnelly Centre for Cellular and Biomolecular Research, Prague, Czech Republic
Toronto, Canada, 2Department of Biomedicine, University of Crystal structures of a recombinant Kunitz-type serine protease
Bergen, Bergen, Norway, 3Department of biological sciences, inhibitor from Bauhinia bauhinioides (BbKI) complexed with
University of Bergen, Bergen, Norway, 4Department of Molecular bovine trypsin were determined in two crystal forms. The crystal
and Human Genetics, Baylor College of Medicine, Houston/Texas, structure of a single-site mutant of BbKI (L55R) was determined
United States of America, 5Division of Pediatric and Adolescent in two space groups, P64 and in the monoclinic space group P21,
Medicine, Oslo University Hospital, Oslo, Norway, 6Institute of at resolution 1.94 A and 3.95 A, respectively. Additionally, the
Clinical Medicine, University of Oslo, Oslo, Norway, 7Department structure of the native BbKI complexed with trypsin was deter-
of Clinical Genetics, Erasmus MC University Medical Center, mined at 2.0 A resolution in the isomorphous P64 form. Since
Rotterdam, Netherlands, 8Stanley Institute of Cognitive Genomics, BbKI has been previously found to be a potent inhibitor of
Cold Spring Harbor, United States of America, 9Department of plasma kallikrein, it was tested against several tissue kallikreins
Surgery, Haukeland University Hospital, Bergen, Norway as well. We determined that BbKI is a potent inhibitor of the
According to WHO 1 in 160 children has an Autism Spectrum chymotrypsin-like human tissue kallikrein 7 (KLK-7). We have
Disorder (ASD), which is a neurodevelopmental disorder mani- built models of its complex with the catalytic domain of human
festing in a range of symptoms such as impaired social skills, plasma kallikrein, as well as KLK-7, and analyzed the interac-
repetitive behaviors, and restricted interests. There is a broad tions responsible for its potency. Inhibition properties of BbKI
consensus that ASD has a strong hereditary component and towards other tested tissue kallikreins were rationalized in struc-
extensive research efforts have been undertaken to identify tural terms.
genetic risk factors of ASD. We have identified and phenotypi-
cally characterized 37 individuals from 32 unrelated families with
P.18-051-Wed
25 different and likely gene disruption variants in NAA15, which
is emerging as a high impact ASD gene. All subjects have vari- The loss of the tertiary structure of fibrinogen
able degrees of neurodevelopmental disabilities, including intellec- induced by the external factors
tual disability, delayed speech and motor milestones, and ASD. D. Klinov, N. Barinov, E. Dubrovin
In most cases the mutation occurred de novo, while familial Federal Research and Clinical Center of Physical-Chemical
inheritance was observed in three families. The NAA15 gene Medicine, Federal Medical-Biological Agency of Russia, Moscow,
encodes the auxiliary subunit of a protein complex called NatA, Russia
which N-terminally acetylates proteins while they are being syn- Fibrinogen is a blood plasma protein responsible for blood coag-
thesized. To gain an insight into the genotype to phenotype rela- ulation. Its denaturation may be utilized in biomedical applica-
tionships in ASD we turned to the budding yeast Saccharomyces tions, e.g., for development of bioactive matrixes to favour
cerevisiae. Functional assays in yeast confirmed a deleterious hemostasis or to resist protein adsorption. In this work, using
effect for two of the truncating variants in NAA15. We propose high resolution atomic force microscopy (AFM) we have investi-
that defects in NatA-mediated protein N-terminal acetylation gated unfolding of single fibrinogen molecules induced by their
lead to variable levels of neurodevelopmental disorders in heating and adsorption on highly oriented pyrolytic graphite
humans, supporting the importance of NatA activity in normal (HOPG) surface modified with oligoglycine-hydrocarbon graphite
human development. This work gives new insight into the hered- modifier (GM). The morphology of heat denatured fibrinogen
ity of ASD and may lead to better treatment of patients with molecules was significantly different not only from their trinodu-
neurodegenerative disorders. lar nativelike structure but also from the morphology of fibrino-
gen molecules unfolded by extended contact with a GM-HOPG
surface. Thermal denaturation has led to formation of globular
structures surrounded by short fibrils. Surface induced denatura-
tion has resulted in appearance of fibrillar structures that resem-
bled organization of the nativelike protein molecule. Moreover, a
lot of fibrils have been formed after extended adsorption of
heated molecules onto a GM- HOPG surface. The obtained
results provide better understanding of fibrinogen denaturation
at a single molecule level that may be important in biotechnologi-
cal applications. Moreover, GM-HOPG surface may be used for
AFM investigation of fibrinogen unfolding in real time. The
authors acknowledge funding from the Russian Science Founda-
tion [17-75-30064 to D.V.K.].
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 403
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
P.18-053-Tue P.18-056-Tue
Nur77 phosphorylation by CK2a regulates its Extending the N-end rule concept for Gly-
stability and cytokine production starting proteins from protein half-life to
B. Huang1, H. Z. Pei1, H. Chang2, S. Baek1 subcellular compartmentalization
1
Yeungnam University, College of Medicine, Daegu, South Korea, T. Meinnel1, B. Castrec1, C. Dian1, S. Ciccone1,
2
Yeungnam University, College of Pharmacy, Daegu, South Korea W. V. Bienvenut1, J. Le Caer2, W. Majeran3, C. L. Ebert4,
J. Steyaert5, C. Giglione1
Nur77, also known as NGFI-B, TR3, or NR4A1, was the first 1
Institute for Integrative Biology of the Cell, Paris-Saclay
member of the NR4A family to be identified as a gene induced University, Gif sur Yvette, France, 2Institut de Chimie de
by NGF. Nur77 is an immediate early response gene and plays a Substances Naturelle, Paris-Saclay University, Gif-sur-Yvette,
critical role in cellular processes in response to diverse stimuli France, 3Institute of Plant Sciences, Gif sur Yvette, France, 4The
including cytokines, stress, and apoptotic signals. Nur77 has been Weizmann Institute of Science, Rehovot, Israel, 5Laboratoire
implicated in a many pathological processes, including cancer, d’informatique, Ecole Polytechnique, Palaiseau, France
immune alterations and metabolic or neurological diseases. Here
we show that CK2a binds to and phosphorylates a serine 154 The N-end relies the nature of the N-terminal amino acid to pro-
residue on Nur77. CK2a-mediated phosphorylation stimulates tein half-life. For the past three decades, a comprehensive num-
Nur77 degradation through ubiquitin-dependent proteasomal ber of achievements has allowed to reveal the various pathways
mechanism, thereby reducing Nur77 protein stability, which describing how 19 out of the 20 natural amino acids do con-
accelerates IL-6 production. Conversely, the inhibition of CK2a tribute to the N-end rule. Glycine is the last residue for which a
or introduction of CK2a inactive mutant into cells reduces related mechanism has not been yet identified. Although N-term-
Nur77 degradation and IL-6 production. Taken together, these inal acetylation (NTA) is now recognized as an important feature
404 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
related to the N-end rule, Gly is not found acetylated (NTAed) P.18-058-Mon
in yeast, remaining as a result with a free N-terminus (FNT). In
Effect of pH on DNA binding and catalysis of
addition, Gly appears to be unique among other amino acids as
it lacks a side-chain, and may undergo N-Myristoylation (MYR). human apurinic/apyrimidinic endonuclease 1
MYR corresponds to a C:14:0 lipid modification used by a num- I. Alekseeva1, A. Bakman2, Y. Vorobjev1,2, O. Fedorova1,2,
ber of proteins all starting with Gly. MYR acts as an anchor tar- N. Kuznetsov1,2
1
geting the modified protein to various cell compartments, Institute of Chemical Biology and Fundamental Medicine,
including plasma membrane, ER-Golgi and mitochondrion. We Siberian Branch of the Russian Academy of Sciences (ICBFM SB
have characterized the modification state of this N-terminal resi- RAS), Novosibirsk, Russia, 2Department of Natural Sciences,
due in both plants and human proteomes by focusing on all pro- Novosibirsk State University, Novosibirsk, Russia
tein isoforms starting with a Glycine. This set defines the Gly- Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key
ome. We show first that MYR involves as much as 20% of all participant in the cascade of base excision repair reactions. APE1
proteoforms starting with an N-terminal Gly while acetylation is initiates the search and repair of such DNA damage, as AP-sites,
found on 30% of the proteins belonging to the Gly-ome. Inter- which can lead not only to the cytotoxic effect, but also to muta-
estingly, 10–20% of the proteins of the Gly-ome is made of pro- genesis, if left unrepaired. It is known from X-ray diffraction
teoforms displaying mixed modifications at their respective data that enzyme amino acid residues interact preferentially with
amino termini (MYR/NTA, NTA/FNT, MYR/FNT or MYR/ one of the duplex strands to form usually hydrogen bonds and
FNT/NTA). Next we carried out quantitative analysis of plant electrostatic contacts. The enzyme active site is formed by a large
cell accumulation of the proteoforms of the Gly-ome in plant. number of polar amino acids, which provide extensive contact of
This analysis revealed that proteins with NTA accumulate signifi- the DNA binding site with the phosphate groups of the DNA
cantly more than those featuring MYR or FNT. Together our substrate. The phosphate residue located on the 50 -side of the
data suggest that the acylation state of an N-terminal Glycine AP-site is directly coordinated by amino acid residues Asn-174,
residue plays not only a role in cell compartmentalization of the Asn-212, and His-309. The catalytic reaction begins with an
protein targets but also in protein half-life, resulting in differen- attack of the water molecule, presumably coordinated by Asp-
tial accumulation. 210. Alternative mechanisms suggest that Tyr-171 in the pheno-
late form attacks the scissile phosphate or His-309 as the general
base, generating the attacking nucleophile. In this case, the car-
P.18-057-Wed boxyl group Asp-283 redistributes the electron density on the
The effect of chemical chaperones on imidazole ring of histidine to stabilize the positive charge. There-
aggregation of glutamate dehydrogenase and fore, the aim of the present work was to elucidate the influence
alpha-lactalbumin of pH on the efficiency of formation of the enzyme-substrate
V. Borzova, D. Kara, K. Markossian, B. Kurganov complex and catalytic reaction. Pre-steady-state kinetic analysis
FRC Fundamentals of Biotechnology RAS, Moscow, Russia of mutual conformational changes of the enzyme and DNA-sub-
strates in the course of their interactions was performed by the
Test systems based on thermal and dithiothreitol (DTT)-induced stopped-flow technique. It was shown that the activity of APE1
aggregation of proteins are widely used for the estimation of the increases with increasing pH due to acceleration of the rate of
antiaggregation activity of various low molecular compounds catalytic complex formation as well as the rate of catalytic
called chemical chaperones. In this work DTT-induced aggrega- reaction. This work was supported by grant from the Russian
tion of bovine alpha-lactalbumin (a-LA) and thermal aggregation Foundation for Basic Research (16-04-00037). The part of the
of bovine liver glutamate dehydrogenase (GDH) from bovine work with FRET detection combined with stopped-flow kinetics
liver were studied using the combination of dynamic light scatter- was specifically funded by Russian Science Foundation grant 16-
ing and asymmetric flow field-flow fractionation. The data on the 14-10038.
aggregation kinetics were analyzed using the quantitative
approaches and aggregation pathway graphs proposed in our
earlier works [1, 2]. It was shown that in the case of heat-induced P.18-059-Tue
aggregation of GDH (0.1 M Na-phosphate buffer, pH 7.6, 40°C Impact of Rad50 phosphorylation in zinc hook
and 50°C) arginine and its derivatives (argininamide and arginine
domain on its stability and global architecture
ethyl ester) acted as aggregation suppressors. The antiaggregation
J. Tran, M. Padjasek, A. Kre_zel
effect of these chemical chaperones was more prominent at 50°C,
Uniwersytet Wroclawski, Wroclaw, Poland
with argininamide and arginine ethyl ester having the higher anti-
aggregation activity than arginine at both temperatures. In the The Rad50 protein is a part of conserved Mre11-Rad50-Nbs1
case of DTT-induced a-LA aggregation (0.1 M Na-phosphate complex (MRN) – required e.g. in initial DNA double-stranded
buffer, pH 6.8, 20 mM DTT, 37°C) polyamines (putrescine and breaks response pathway or DNA recombination. The Rad50
spermidine) had a dual effect on the test system. Relatively low protein consists of two ATP-ase globular domains separated by
concentrations (up to 50 mM spermidine and 100 mM putres- coiled coils elements and zinc hook domain at the apex. Our
cine) caused the enhancement of aggregation by promoting the structural study of the central part of human Rad50 protein
sticking of aggregates. The increase in the polyamines concentra- shows two coiled-coil protomers in a rod-shaped arrangement
tion suppressed protein aggregation. This study was funded by with interprotein zinc hook. The role of the zinc hook is to con-
Russian Science Foundation (grant number 16-14-10055). nect two Rad50 monomers together, promoting the activation of
Reference ATM upon ATP binding in Rad50 catalytic domains. It has been
1. Borzova V., Markossian K., Kara D., Kurganov B. (2015) shown that phosphorylation of Rad50 at S635 by ATM in
Kinetic regime of dithiothreitol-induced aggregation of bovine response to DNA damage is essential in regulating downstream
serum albumin. Int. J. Biol. Macromol., 80, 130–138. signaling. High throughput analysis has identified another phos-
2. Borzova V., Markossian K., Kleymenov S., Kurganov B. (2017) phorylation site at T690 in human Rad50 protein. Our crystal
A change in the aggregation pathway of bovine serum albumin in structure of the fragment of human Rad50 showed that both
the presence of arginine and its derivatives. Sci. Rep., 7:3984. phosphorylated residues are located at major loops constituting
coiled-coil extrusions facing outwards hydrophobic core of the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 405
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
406 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
and ionization mass spectrometry (MALDI MS). For the pur- P.18-064-Mon
pose of determining exact binding sites, Tf was incubated with 10
Relationship of growth rate and muscle
fold molar excess of each complex for 24 h. Unbound portion of
the complexes was removed by ultrafiltration and the obtained protein turnover in Atlantic salmon Salmo
adducts were subjected to trypsin digestion. The resulting pep- salar L. under natural and artificial
tides were separated using LC, and Ru-containing fractions were photoperiods
collected for MALDI MS analysis. The obtained spectra revealed N. Kantserova, L. Lysenko, N. Nemova
presence of five ruthenated peptides. Binding amino acids have IB KarRC RAS, Petrozavodsk, Russia
been determined by MS/MS analysis of target sequences. Accord-
It is known that protein degradation in fish muscles depends on
ing to the obtained results, both Ru(II) complexes bind five his-
three proteolytic systems such as cathepsins, calcium-dependent
tidine residues, namely: His642, His300, His585, His289 and
proteases (or calpains), and the ubiquitin-proteasome system
His273. Only His585 is included in Fe3+ binding site, while other
(UPS). Calcium-dependent proteolysis is a major pathway regu-
His residues are mainly located on the protein surface. It can be
lating muscle turnover in fish, while cathepsins and ubiquitin-tar-
concluded that both complexes show high affinity towards His
geted protein digestion by the proteasome are primarily
residues and since the binding of the complexes does not cause
responsible for bulk protein degradation. Calpain activity is con-
changes in Tf structure, it can be suggested that these compounds
sidered as a marker of fish growth and health state at different
can use Tf cycle to be actively transported into tumor cells.
life stages along with myofibrillar protein expression levels, key
digestive and metabolic enzyme activities, lipid contents, etc. This
P.18-063-Wed study was conducted to evaluate the effects of natural and artifi-
cial (24:0 or 18:6 light:dark, L:D) photoperiods on the growth
The effect of arginine and ionic strength on
rate and calpain activity in the skeletal muscles in Atlantic sal-
aggregation of UV-irradiated glycogen mon. Maximal growth rate was attained at 24L:0D, followed by
phosphorylase b 18L:6D, and a minimal was inducted by a natural photoperiod.
V. Mikhaylova, T. Eronina, N. Chebotareva, B. Kurganov The results suggested positive correlation between length-weight
Bach Institute of Biochemistry, Research Center of Biotechnology growth dynamics in individuals and calcium-dependent proteoly-
of the Russian Academy of Sciences, Moscow, Russia sis level in their skeletal muscles as well as their orchestrated reg-
Arginine (Arg) is widely used not only because it stabilizes pro- ulation by photoperiod variations. Stimulating effect of a
teins and accelerates their folding, but also because of its ability continuous (24L:0D) light photoperiod on growth performance
to suppress protein aggregation. However, Arg is a charged and muscle protein turnover in salmon was concluded. The work
molecule, and therefore, when studying the action of Arg on pro- was supported by the Russian Science Foundation, project no.
tein aggregation, the effects of ionic strength should be taken 14-24-00102.
into account. In this work the effect of ionic strength and Arg on
aggregation of UV-irradiated glycogen phosphorylase b (UV-
P.18-065-Tue
Phb) was studied. With this test system we can obtain informa-
tion on the effects of different agents on aggregation of dena- Inhibition of the actin N-terminal
tured protein molecules. The process of UV-Phb aggregation acetyltransferase NAA80
includes the rate-limiting monomolecular stage of structural reor- M. Baumann1, L.M. Myklebust1, M. Goris1, R. Magin2,
ganization of the Phb molecule, containing concealed damages, H. Foyn1, S.I. Støve1, R. Marmorstein3, B. E. Haug1,
and a relatively fast aggregation stage. The process of structural T. Arnesen1
1
reorganization of UV-Phb is characterized by a first-order rate University of Bergen, Bergen, Norway, 2University of
constant (kI). The kinetics of UV-Phb aggregation was studied Pennsylvania, Philadelphia, United States of America, 3Perelman
using dynamic light scattering (DLS) at 37°C (0.03 M Hepes buf- School of Medicine, University of Pennsylvania, Philadelphia,
fer, pH 6.8) at various ionic strength values (0.02–0.7 M NaCl or United States of America
Arg). It was shown that an increase in NaCl concentration A common modification of proteins is the acetylation of their N-
caused a decrease in the kI value, suggesting a slowdown of the termini by N-terminal acetyltransferases (NATs). As a member
UV-Phb structural reorganization process. Circular dichroism of the NAT-family, NAA80 transfers an acetyl group from acetyl
data confirmed this conclusion. Analytical ultracentrifugation coenzyme (Ac-CoA) to the acidic N-termini of processed animal
and DLS data have shown that an increase in ionic strength actins. These actins are integral parts of the cytoskeleton that is
leads to the formation of smaller aggregates, testifying that the responsible for cell shape and motility, thus making NAA80 a
change in the aggregation pathway occurs. To evaluate the effect viable target for the regulation of cytoskeletal functions. In this
of Arg, we conducted experiments at fixed values of ionic work, peptidic bisubstrate inhibitors based on Ac-CoA and the
strength (0.15 M and 0.5 M NaCl or Arg). It was shown that at natural actin substrates of NAA80 were developed and for their
a low ionic strength Arg accelerated the process of protein aggre- synthesis we employed Fmoc-based solid phase peptide synthesis
gation and induced changes in the aggregation pathway. At high (SPPS) to prepare tetrapeptides, which were subsequently
concentrations Arg acts as a charged molecule, and its effect on attached to coenzyme A through an acetamide linker. Mass spec-
protein aggregation is due solely to a change in ionic strength of trometry (MS) and nuclear magnetic resonance (NMR) spec-
the solution. The study was funded by the Russian Science Foun- troscopy were used for structure confirmation of the inhibitors,
dation (grant 16-14-10055). whereas the potency and selectivity of the inhibitors was evalu-
ated through acetylation assays. Furthermore, in complex with
the most potent inhibitor, the crystal structure of NAA80 was
determined. This work marks the basis for development of more
potent and selective inhibitors of NAA80 thereby increasing the
number of tools available for regulation of actin and the
cytoskeleton.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 407
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
408 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 409
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
410 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
P.18-075-Wed program nucleoporin Nupl1 was one of the most probable candi-
dates. This protein is a part of a midplane ring of nuclear pore
Oxidative cellulose degradation orchestrated
complex, regulating nucleocytoplasmic traffic. Using advantages
by cellulose dehydrogenase and lytic of yeast model system we demonstrated that the full length pro-
polysaccharide monooxygenase studied by tein fused to EGFP forms fluorescent foci when overproduced.
structural mass spectrometry Nupl1 aggregates were resistant to cold SDS treatment according
F. Filandr1,2, D. Kracher3, L. Slavata1,2, R. Ludwig3, to the results of SDD-AGE analysis. Using C-DAG (curli-depen-
P. Halada1,2, P. Man1,2 dent amyloid generator) system we demonstrated that Nupl1
1
Faculty of Science, Charles University, Prague, Czech Republic, overproduction leads to staining of the bacterial cells with Congo
2
Institute of Microbiology of the CAS, Prague, Czech Republic, Red. Taken together these results allow to suggest that Nupl1 is
3
University of Natural Resources and Life Sciences, Vienna, a candidate for new human amyloid. The research was supported
Austria by the Russian Science Foundation (17-74-10159).
Cellobiose Dehydrogenase (CDH) and its redox partner Lytic
Polysaccharide Monooxygenase (LPMO) are essential parts of P.18-077-Tue
cellulolytic system in wood degrading fungi. Their combined
Dynamic structural ensembles of selected
activity allows for oxidative decomposition of recalcitrant cellu-
lose structures. The function of CDH depends on intramolecular folded and disordered postsynaptic proteins
electron transfer (IET) between CDH’s disaccharide oxidizing fla- B. Kovacs, M. Handbauer, B. Peterfia, Z. Gaspari
vin domain and electron transferring cytochrome domain, which Pazmany Peter Catholic University, Faculty of Information
then transfers electrons to copper-dependent cellulose oxidizing Technology and Bionics, Budapest, Hungary
LPMO by yet not clearly described mechanism. We used various Protein internal dynamics is a key determinant of protein func-
structural MS techniques namely H/D exchange and chemical tion and its regulation. Dynamic structural ensembles aim at pro-
cross-linking, to obtain more precise idea about the protein-pro- viding an atomic-level description of protein motions by
tein and protein-substrate interaction, product formation and enumerating a number of possible conformers characteristic of
protein stability under the oxidative conditions. Using HDX-MS, the conformational space explored by the molecules at a given
changes were observed on CDH induced by lactose oxidation. time scale. We have generated structural ensembles reflecting
Interestingly, no differences were observed on CDH when it experimentally determined internal dynamics of the tandem
interacted with LPMO either in its reduced or oxidized state. On PDZ1-2 domains of the postsynaptic density protein PSD95. In
the other hand, structural perturbance occurred on LPMO. In this domain pair ligand binding influences interdomain mobility.
the fully functional system, when CDH or small molecular reduc- Dynamic structural ensembles of free and ligand-bound forms of
tants are fueling LPMO with electrons, we saw increase in deu- the domain pair were generated with an in-house modified ver-
terium exchange around the active site and we attribute it to sion of GROMACS and analyzed to understand the atomic basis
oxidative damage and protein unfolding. Several oxidative modi- of this behavior. Our models made it possible to point out the
fications of amino acids in peptides surrounding the active site key determinants of interdomain mobility and the atomic-level
were indeed identified. These adverse effects can be significantly basis of its changes upon ligand binding. In addition, we will pre-
diminished in the presence of substrate (PASC or cellulose) where sent our efforts to generate structural ensembles of different
we observed strong stabilizing effect on LPMO. So far, our find- intrinsically disordered segments of the postsynaptic scaffold pro-
ings indicate that the interaction between CDH and LPMO may tein GKAP, along with biochemical studies on its partner-bind-
be in fact just direct cofactor-cofactor interaction between the ing properties. Our results highlight the usefulness of ensemble-
propionates of the heme group in cytochrome domain of CDH based structural models in understanding interactions within the
and a copper ion of LPMO. MALDI-MS product formation postsynaptic density.
analysis also demonstrated, that reductant is necessary for reac-
tion priming but the actual process requires nothing more than
just hydrogen peroxide. Financial support: CSF (16-34818L) P.18-078-Wed
Abscisic acid-induced re-localization of
P.18-076-Mon phytaspase
S. V. Trusova, P. I. Volik, N. V. Chichkova, A. B. Vartapetian
Human nucleoporin Nupl1 forms amyloid-like Belozersky Institute of Physico-Chemical Biology, Moscow State
aggregates University, Moscow 119991, Russia
S. Moskalenko1,2, L. Danilov2, D. Likholetova2, M. Belousov2,3,
A. Matveenko2, G. Zhouravleva2, S. Bondarev2 Phytaspase belongs to a family of plant subtilisin-like proteases
1
Vavilov Institute of General Genetics, St Petersburg Branch, St and possesses a rarely occurring aspartate specificity of hydroly-
Petersburg, Russia, 2St Petersburg State University, St Petersburg, sis. Phytaspase is a cell death-related protease. Upon autocat-
Russia, 3All-Russia Research Institute for Agricultural alytic proenzyme processing, active phytaspase is secreted out of
Microbiology, St Petersburg, Russia the plant cell into the apoplast. However, death-inducing stresses
cause retrograde transportation of phytaspase from the apoplast
Amyloids are a group of protein aggregates possessing a set of to inside the plant cell. Here we demonstrated that intracellular
unusual features including high resistance to detergent or pro- localization of phytaspase can be achieved in yet another, cell
tease treatment and their ability to induce the transition of some death-unrelated way. In Nicotiana benthamiana leaves transiently
proteins from soluble to aggregated form. Numerous investiga- producing Nicotiana tabacum phytaspase, leaf treatment with
tions of amyloids are in the top of interest due to increasing inci- phytohormone abscisic acid resulted in redistribution of the
dence of amyloid-associated disorders, for instance Alzheimer’s enzyme from the apoplast to the cell interior. Likewise, Arabidop-
disease, Parkinson’s disease, type II diabetes, etc. We analyzed a sis thaliana phytaspase transiently produced in A. thaliana leaves
set of proteins, which physically interact with Htt (Huntington’s became spread throughout the cell upon abscisic acid application.
disease) according to BioGrid database, for the ability to form Consistent with these findings, co-production of the A. thaliana
amyloid aggregates. According to the prediction of ArchCandy ABI3 transcription factor, a key regulator of abscisic acid
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 411
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
signaling pathway, together with the phytaspase, also resulted in (State University), Dolgoprudny, Moscow Region, Russia,
4
re-localization of the protease. It seems likely therefore that some Dmitry Rogachev National Medical Research Center of Pediatric
of the ABI3 target gene products may be responsible for phytas- Hematology, Oncology and Immunology, Moscow, Russia, 5Center
pase movement. Comparison of the intracellular phytaspase for Theoretical Problems of Physicochemical Pharmacology,
localization in response to cell death induction or to phytohor- Russian Academy of Sciences, Moscow, Russia, 6Skolkovo
mone treatment revealed marked difference between the two, sug- Institute of Science and Technology, Skolkovo, Russia
gesting that the underlying mechanisms of phytaspase re-import
Fibrinogen, which plays a key role in the physiological reaction
may be distinct. This work was supported by the Russian Science
providing plasma hemostasis, is the most vulnerable to reactive
Foundation grant # 16-14-10043.
oxygen species among all the blood plasma proteins. Oxidation
of fibrinogen damages its structure as well as affecting the pro-
P.18-079-Mon tein function. The induced oxidative modifications of the all three
polypeptide chains of the fibrinogen have been studied by the
N-terminal acetylation stabilizes alpha-
mass-spectrometry method. The alphaC-connector has convinc-
synuclein protein and modulates its toxicity ingly been shown to be most vulnerable to oxidation as com-
R. Vinueza1, I. I~
nigo-Marco1, L. Larrea1, M. Lasa1, B. Carte2, pared to other structural elements. The E region turned out to be
R. Aldabe2, M. Arrasate1 the most protected area of the protein. Oxidative modifications
1
Center for Applied Medical Research (CIMA), University of of Tyr338, Met367, Met373 in the area 330–375 of the beta-
Navarra, Pamplona, Spain, 2Department of Gene Therapy and nodules, oxidized Met476 and group modifications in the
Regulation of Gene Expression, CIMA, University of Navarra,, alphaC-region seem to contribute to the lateral aggregation dam-
Pamplona, Spain age. Detected modifications of Pro243 and Met264, Lys273,
Increased alpha-synuclein (aSyn) protein levels causes neurode- Tyr274 in gamma chain may influence the formation of interfa-
generative diseases like Parkinson’s disease or dementia with cial D:D contacts. The mutation Ala68 in beta chain results in
Lewy Bodies, grouped as synucleinopathies. Still, the mechanisms defective binding of thrombin to fibrin and consequent thrombo-
by which altered aSyn protein levels causes neuronal death are sis, so the His67 modification possibly affects this binding too.
unknown. In vivo, all aSyn molecules are posttranslationally Some structural elements of fibrinogen and a part of easily oxi-
modified by an acetyl group attached to the alpha-amino group dizable amino acid residues are supposed to be endowed with
of the N-terminal amino acid by the enzyme NatB. Some in vitro antioxidant properties. The gained data on the identified oxida-
evidences suggest that this modification stabilizes the helical tive modifications of fibrinogen appear to support the concept
structure of the aSyn N-terminal domain and affects the lipid previously proposed by us that the fibrinogen structure is
binding and aggregation capacity of the protein. However, the adapted to oxidation. The methods of confocal laser scanning
biological significance of aSyn N-terminal acetylation in neurons microscopy and elastic light scattering revealed the disturbances
has not been addressed. Recently, our laboratory has developed in the structure of fibrin clots assembled from oxidized, throm-
a neuronal model based on automated microscopy that enables bin-treated fibrinogen molecules. The study was supported by
longitudinal tracking of individual primary living neurons RFBR projects № 16-34-60244 mol_a_dk and № 18-04-01313a.
expressing aSyn and survival analysis. Using Cox proportional The mass spectrometry measurements was supported by the Rus-
regression models the specific contribution of different factors sian Science Foundation № 16-14-00181.
(e.g. mutations and protein expression levels) can be quantified
and dissected. To address the role of aSyn N-terminal acetylation
in neurons we generated three N-terminal aSyn mutants; D2A
P.18-081-Wed
and D2P that alter and block aSyn N-terminal acetylation Overproduction of Sch9 leads to its
respectively, and D2E, a conservative mutant. The toxicity of aggregation and cell elongation in
these mutants was evaluated by longitudinal survival analysis. Saccharomyces cerevisiae
Mutant D2P exhibited lower protein levels when expressed in pri- S. Bondarev1, P. Drozdova2, P. Lipaeva1, T. Rogoza1,3,
mary cortical neurons and consequently it decreased the risk of G. Zhouravleva1
neuronal death compared to normal aSyn forms. Using an opti- 1
Saint Petersburg State University, St. Petersburg, Russia,
cal pulse-chase methodology that allows measuring protein half- 2
Institute of Biology at Irkutsk State University, Irkutsk, Russia,
life in situ in primary neurons we found that D2P mutant 3
Vavilov Institute of General Genetics, St. Petersburg Branch,
decreased aSyn stability compared to wild-type aSyn. Our results Russian Academy of Sciences, St. Petersburg, Russia
strongly suggests that N-terminal acetylation stabilizes aSyn pro-
tein modulating its toxicity. We are currently evaluating N-term- The Sch9 kinase of Saccharomyces cerevisiae is one of the major
inal acetylation-dependent molecular mechanisms that lead to TOR pathway effectors. It regulates such important processes in
aSyn destabilization. the yeast cell as progression through cell cycle, aging and regula-
tion of response to nutrient. Sch9 belongs to the AGC kinase
family. Even though amplification of AGC kinase genes has been
P.18-080-Tue connected with cancer in human, not much is known about the
The effects of oxidation on the fibrinogen effects of Sch9 overproduction in yeast. We addressed this issue
by developing a model system to monitor subcellular location
molecule and its functional properties
and aggregation state of overproduced Sch9 or its regions fused
L. Yurina1, A. Vasilyeva1, M. Indeykina1,2,3, A. Bugrova1,
to a fluorescent protein. We found an earlier unknown feature of
A. Bychkova1, M. Biryukova1, N. Podoplelova4,5,
Sch9 protein, formation of specific intracellular structures, at
A. Kononikhin1,2,3, E. Nikolaev1,2,6, M. Panteleev4,5,
least some of which possess amyloid-like properties. In addition,
M. Rosenfeld1
1 we attempted to separately analyze functions of the different
N.M. Emanuel Institute of Biochemical Physics, Russian
Sch9 domains and revealed specific effect of the C-terminal
Academy of Sciences, Moscow, Russia, 2Institute for Energy
region overproduction on cell elongation and existing of several
Problems of Chemical Physics, Russian Academy of Sciences,
aggregation-prone regions in different parts of the protein. Even
Moscow, Russia, 3Moscow Institute of Physics and Technology
though this analysis may be further continued, we believe that
412 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
our results add to the prior knowledge about Sch9 functions and functional features of the proteins, suggest intrinsic disorder is
create a basis for further investigation. The constructs we present indeed an important feature of postsynaptic proteins and might
can be used to create superior yeast-based model systems to comprise one of the main factors contributing to the versatility
study processes behind kinase overproduction in cancers. These and dynamics of the postsynaptic network.
potential model systems could also be useful for testing novel
inhibitors of AGC kinases, for example p70S6K, inhibitors of
which are already being developed. The research was supported P.18-084-Wed
by the Russian Science Foundation (17-74-10159). Local structural factors governing the fold-
switching mechanism of RfaH
P. Galaz-Davison1, E. A. Roman2, D. U. Ferreiro3,
P.18-082-Mon I. Artsimovitch4, E. A. Komives5, C. A. Ramırez-Sarmiento1
Molecular insight in the interaction 1
Institute for Biological and Medical Engineering, Pontificia
mechanism of farnesylated hGBP 1 Universidad Catolica de Chile, Santiago, Chile, 2IQUIFIB,
L. Sistemich, C. Herrmann Universidad de Buenos Aires, Buenos Aires, Argentina, 3FCEyN,
Ruhr-University Bochum, Bochum, Germany Universidad de Buenos Aires, Buenos Aires, Argentina,
4
Department of Microbiology and The Center for RNA Biology,
Human guanylate-binding proteins (hGBPs) belong to the dyna-
The Ohio State University, Columbus, United States of America,
min superfamily of large GTPases and are upregulated in cells 5
Department of Chemistry & Biochemistry, University of
after stimulation with IFNc-II. Three of the seven isoforms con-
California San Diego, San Diego, United States of America
tain a C-terminal CaaX motif which can be isoprenylated. The
isoprenyl anchor allows the hGBPs to attach to membranes Metamorphic proteins challenge the paradigms of protein folding
within the cell and to artificial membranes in a nucleotide depen- by switching between distinctive functional and structural native
dent manner in vitro. In absence of membranes farnesylated states within biologically relevant timescales. The bacterial viru-
hGBP1 (hGBP-1fn) reversibly polymerizes by addition of GTP to lence factor RfaH constitutes a dramatic example of such behav-
a tube-like structure. hGBP fulfils its main function in the innate ior, with its C-terminal domain (CTD) refolding from a stable a-
immunity against bacteria and viruses by interacting with intra- helical hairpin bound to and occluding the RNA polymerase
cellular membranes. However, by now the molecular mechanism binding site of the N-terminal domain (NTD) into a dissociated
of interactions among the hGBPs when inserting into the mem- small b-barrel, enhancing transcription and enabling its coupling
brane is poorly understood. For a better understanding a range with translation. Here we explore in detail the metamorphic pro-
of in vitro methods, including fluorescence microscopy and spec- cess of RfaH using a combination of multiscale molecular
troscopy, turbidity assay and hydrolysis assays, have been per- dynamics and biophysical experiments. Coarse-grained simula-
formed to shed light on the missing knowledge. We showed that tions using dual-basin structure-based models reveal that interdo-
polymerization as well as membrane binding can be altered by main contacts control the fold-switching behavior of RfaH and
introducing different point mutations to hGBP1fn. These point highlight the role of specific NTD-CTD interactions in stabilizing
mutations lead to changes in the crucial properties such as GTP the a-folded state, with the CTD residue F130 having a dual role
hydrolysis, dimer formation or intramolecular rearrangements in stabilizing both folds. Moreover, simulations using the
that are needed for the function of the farnesylated protein. With AWSEM coarse-grained protein force-field correctly predict the
these findings, we gain a more detailed insight into the molecular NTD-CTD interface for the a-folded state of RfaH from
interactions among the hGBPs in the process of polymerization sequence and local structure information alone. Finally, compu-
and membrane binding. tationally predicted per-residue free energy differences using all-
atom confine-convert-release simulations of RfaH unveil local
structural preferences towards either native state, with the tip of
P.18-083-Tue the helical hairpin (CTD residues 129–146) having preferential
Protein intrinsic disorder in postsynaptic thermodynamic stability towards the a-folded state whereas the
density rest of this domain is ~2 kcal/mol more stable if folded as b-bar-
oth, A. Angy
A. Kiss-T an, B. Ligeti, B. Peterfia, G. Lukacs, rel. Experimental evidence of these heterogenous local stabilities
Z. G
aspari was obtained from deuterium incorporation kinetics using hydro-
Pazmany Peter Catholic University, Faculty of Information gen-deuterium exchange mass spectrometry analysis of RfaH in
Technology and Bionics, Budapest, Hungary both native states, showing remarkable spatial and magnitude
consistency. Our results reveal how the stabilities of the native
The synapse is a structure, which provides a way for neuronal states of RfaH are locally encoded within its sequence. Funding:
communication. The postsynaptic density, responsible for modu- FONDECYT 11140601.
lating signal transduction and orchestrating a number of pro-
cesses linked to learning and memory, is an elaborate protein
network composed of a many different proteins with a high num- P.18-085-Mon
ber of interaction sites. The molecular basis of its ability for Interaction of human GOLPH3 with RAB1A
dynamic rearrangement is still elusive. We investigated the possi-
and RAB1B: identification of a putative non-
ble role of intrinsically disordered segments of pre- and postsy-
naptic proteins and compared it to features of a number of other canonical effector
proteins associated with other well-characterized functions, such V. Cavieres1, C. Cerda2, A. Rivera-Dictter1, R. Castro1,
as the cytoskeleton, the immunome and signaling proteins. We P. Burgos1,2, G. Mardones1,2
1
took care to use consensus predictions as much as possible and Department of Physiology, Universidad Austral de Chile,
also to exclude fibrillar segments like coiled coils from predicted Valdivia, Chile, 2Center for Cell Biology and Biomedicine,
disordered regions to get unbiased statistics. We have calculated Universidad San Sebastian, Santiago, Chile
different structural attributes based on intrinsic disorder and also Golgi phosphoprotein 3 (GOLPH3) is a trans-Golgi network
on annotated protein-protein interactions from different sources. protein implicated in several aspects of membrane trafficking.
Our results, based on detailed analysis of the structural/ Intriguingly, GOLPH3 has been implicated also in tumorigenesis.
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 413
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
414 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
fluorescence signal, especially when being irradiated by UV-light. before the +3 nucleotide after transcription start site is presented
In contrast to UnaG, the addition of BSA leads to the fluores- as ssDNA for template or non-template strand, we revealed that
cence enhancement of the entire cluster indicating that its native phiKZ nvRNAP requires template strand for promoter interac-
structure remains unchanged upon penetration. In all cases, tion in single-strand form. It is the significant difference with bac-
MCCs don’t undergo significant changes of the shape or size dur- terial RNAP, which specifically binds non-template strand in
ing the interaction with proteins. Moreover, BSA and apoUnaG promoter consensus. Moreover, in binding with DFJ-templates,
may selectively interact with MCCs contained both bodipy and consensus nucleotides aren’t important for effective nvRNAP
BR simultaneously, leading to appropriate bright “turn-on” transcription start. To study the possible role of the fifth subunit
either of BR-UnaG or BSA-bodipy fluorescence responses. This in promoter recognition and nvRNAP maturation two variants
part of research was supported by Russian Science Foundation of co-expression systems were made: the first one encoded only 4
grant No 17-73-10408. b/b’-like subunit and the second contains all 5 nvRNAP subunits.
Both producing complexes from co-expression systems were
tested by size-exclusion chromatography and native PAGE. 4-
P.18-090-Wed subunit system a small amount of 4-subunit (4-s) complex with-
Mms19 modulates mitotic spindle dynamics out any polymerase activity and DNA-binding capacity. 5-subu-
by recruiting Xpd to microtubules nit system produced full recombinant nvRNAP, which
R. Chippalkatti1, B. Egger2, B. Suter1 transcribed late phage promoters with native nvRNAP specificity.
1
University of Bern, Institute of Cell Biology, Bern, Switzerland, Experiments with the addition of the fifth subunit to 4-s complex
2
University of Fribourg, Fribourg, Switzerland showed a significance of Gp68 to the formation of nvRNAP and
its DNA-binding capacity. The work was supported by the Rus-
Mms19 is a component of the Cytoplasmic Iron-sulfur cluster sian Science Foundation (grant № 17-74-10220) for M. Yakun-
assembly (CIA) machinery, which regulates the incorporation of ina.
iron-sulfur clusters in target proteins. Recent evidence, however
points towards cell cycle regulatory activity of Mms19 as down-
regulation of Mms19 in human cells leads to mitotic defects and P.18-092-Tue
mitotic tissues are absent or deformed in Mms19 loss-of-function Stopped-flow kinetic study of DNA
mutant Drosophila larvae. Apart from the CIA, Mms19 also
demethylation by Fe(II)/2-oxoglutarate-
forms a distinct complex, MMXD which consists of Mms19,
Xpd, and Mip18. The mitotic defects in the Drosophila larvae dependent dioxygenase AlkB
can probably be attributed to the inhibition of Cdk activity by L. Kanazhevskaya1, I. Alekseeva1, N. Kuznetsov1,2,
Xpd. But overexpression of the Cdk activating kinase complex in O. Fedorova1,2
1
Mms19 mutant background did not fully rescue the mutant lar- Institute of Chemical Biology and Fundamental Medicine of SB
val phenotype, indicating alternative pathways of mitotic regula- RAS, Novosibirsk, Russia, 2Novosibirsk State University,
tion by Mms19. We performed Mass-spectrometry screen to Novosibirsk, Russia
identify novel Mms19-binding proteins, in which, we found tubu- Proteins of AlkB family are involved in the direct oxidative
lin and microtubule associated proteins. We confirmed these repair of alkylated DNA bases. Bacterial AlkB protein is a Fe
observations by additional microtubule (MT) binding assays. We (II)/2-oxoglutarate dependent dioxygenase that removes methyl
were surprised to find that along with numerous mitotic spindle groups from N1-methyladenine, N3-methylcytosine and exocyclic
defects in Mms19 mutant larvae, the spindle microtubules poly- DNA adducts such as 1,N6-ethenoadenine (eA). The AlkB-
merize at a reduced rate. In further MT-assays, we observed that mediated catalysis is accompanied by oxidation of 2-oxoglutarate
the MMXD components Xpd and Mip18 also bind to MTs and to succinate resulting in formation of “oxyferryl” intermediate.
if MMXD complex formation is inhibited, MT polymerization The complexity of reaction mechanism requires multiple confor-
defects are seen in vitro and in vivo. Here, we establish that mational changes within the enzyme-substrate complex including
Mms19 recruits Xpd to spindle microtubules and the association flipping of the alkylated base. In the present work to gain deeper
of MMXD with MTs is crucial for regulating spindle structure insight into the damage recognition mechanism and catalytic step
and polymerization during mitosis. of reaction, we performed a pre-steady-state kinetic analysis of
conformational transitions of AlkB and DNA in the course of
their interaction. Using the stopped-flow fluorescence analysis
P.18-091-Mon together with a combination of biochemical assays, we have stud-
The unique mechanism of promoter binding ied conformational transitions and transient kinetics of AlkB
by phiKZ phage non-virion RNA-polymerase interactions with model ss and ds DNA containing damaged and
M. Orekhova1, T. Artamonova2, M. Yakunina2, undamaged nucleotides. An intrinsic fluorescence of the protein
M. Khodorkovskii2 Trp residues, the fluorescent base 2-aminopurine and dye–
1
Peter the Great St. Petersburg Polytechnic University, Saint- quencher pair FAM/BHQ1 introduced into 15 bp oligonu-
Petersburg, Russia, 2Peter the Great St. Petersburg Polytechnic cleotides were applied for registration of kinetic traces. The data
University, St. Petersburg, Russia obtained under single-turnover conditions have been described
quantitatively by equilibrium and rate constants. A comparison
Non-canonical multisubunit non-virion DNA-dependent RNA-
of kinetic data obtained by means of Trp and 2-aminopurine flu-
polymerase (nvRNAP) encoded by the bacteriophage phiKZ con-
orescence and F€ orster resonance energy transfer (FRET) detec-
tains 5 subunits, 4 of which are homologs of b and b’ bacterial
tion allowed us to elucidate the steps of specific and nonspecific
RNAP subunits, a function of the fifth subunit (Gp68) is
DNA binding and catalysis. This work was supported by grant
unknown. NvRNAP transcribes late phage promoters with ulti-
from the Russian Science Foundation (Grant № 16-14-10038).
mate short consensus TATG, where G is transcription start
point. In experiments with double-strand DNA-templates was
identified that for a specific transcription initiation every single
pair of consensus nucleotides is very important. Using down-
stream fork junction templates (DFJ-templates) where DNA
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 415
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
416 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 417
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
Hsp110 system is regulated by the AS of Apg2, one of the three neutralizing pdAbs is discontinuous and amino acid residues
representatives of the family in humans. located within two p35 regions, 15–19 aa and 232–237 aa, are
involved in binding with neutralizing anti-p35 antibodies. This
study was supported by the Russian Scientific Foundation (pro-
P.18-100-Mon ject #16-14-00083).
Intrinsic protein disorder in histone lysine
methylation
T. Lazar1, E. Schad2, B. Szabo2, T. Horvath2, A. Meszaros1,
P.18-102-Wed
P. Tompa1,2, A. Tantos2 The C-type lectin-like receptor Nkrp1b:
1
VIB-VUB Center for Structural Biology, Brussels, Belgium, structural features affecting protein
2
Research Centre for Natural Sciences, Budapest, Hungary conformation and interactions
Histone lysine methyltransferases (HKMTs), catalyze mono-, di- L. Hernychova1,2, M. Rosulek1, A. Kadek1, V. Mareska3,
and trimethylation of lysine residues, resulting in a regulatory J. Chmelik4, L. Adamkova1, V. Grobarova2, O. Sebesta2,
pattern that controls gene expression. Their involvement in many Z. Kukacka1, K. Skala1, V. Spiwok3, J. Cerny2, P. Novak1
1
different cellular processes and diseases makes HKMTs an inten- BIOCEV, Institute of Microbiology of the CAS, Vestec, Czech
sively studied protein group, but scientific interest so far has been Republic, 2Faculty of Science, Charles University, Prague, Czech
concentrated mostly on their catalytic domains. In this work we Republic, 3University of Chemistry and Technology, Prague, Czech
set out to analyze the structural heterogeneity of human HKMTs Republic, 4Institute of Microbiology of the CAS, Prague, Czech
and found that many contain long intrinsically disordered regions Republic
(IDRs) that are conserved through vertebrate species. Our predic- NK cells represent substantial part of the innate immunity as
tions show that these IDRs contain several linear motifs and con- they promptly recognize virally infected and tumor cells or regu-
served putative binding sites that harbor cancer-related SNPs. late adaptive immune responses. These functions are controlled
Although there are only limited data available in the literature, by the NK receptors. This project elucidates structure of the inhi-
some of the predicted binding regions overlap with interacting bitory C-type lectin-like receptor Nkrp1b and highlights its sev-
segments identified experimentally. The importance of a disor- eral structural features (the stalk, loop and oligomerization
dered binding site is illustrated through the example of the tern- state), which might affect whole protein conformation or interac-
ary complex between MLL1, menin and LEDGF/p75. Our tions. To study these structural aspects, two Nkrp1b protein
suggestion is that intrinsic protein disorder plays an as yet unrec- forms were recombinantly prepared: entire ectodomain and
ognized role in epigenetic regulation, which needs to be further ligand-binding domain lacking the stalk. Both protein forms fold
elucidated through structural and functional studies aimed specif- as monomers and homodimers, therefore, their functionality was
ically at the disordered regions of HKMTs. tested. Bone-marrow derived cells expressing Nkrp1b ligand were
incubated with fluorescently labeled recombinant proteins and
their interaction was analyzed by scanning fluorescence micro-
P.18-101-Tue scopy. Thereafter, homology models of the protein forms were
Phage display antibodies (selected to proposed using distance restrains obtained by chemical cross-
Ectromelia virus) capable of Variola virus linking and disulfide bonds characterization. Distance constrains
in vitro neutralization: implementation for p35 related to dimerization interface were taken in account as well.
neutralize epitope mapping Finally, homology models were validated by ion mobility-mass
spectrometry, when their collision cross sections were compared
Y. Khlusevich1, A. Matveev1, I. Baykov1, L. Bulychev2,
to the experimentally obtained values of native proteins. Taking
I. Ilyichev1, G. Shevelev1, V. Morozova1, D. Pyshnyi1,
our results together, we suggest the stalk region does not affect
N. Tikunova1
1 protein fold, nor Nkrp1b dimerization as it was thought for dec-
Institute of Chemical Biology and Fundamental Medicine, SB
ades, or even its ability to interact with the ligand. Moreover, we
RAS, Novosibirsk, Russia, 2State Research Center of Virology and
show that only monomeric Nkrp1b forms are able to interact,
Biotechnology, Novosibirsk, Russia
nevertheless, homodimers occur. This might point out the regula-
Five phage display antibodies (pdAbs) against ectromelia virus tory function of the stalk region. This work was supported by
(ECTV) were selected from vaccinia virus (VACV)-immune the projects no. 200816, LH15010, LD15089, “NPU II” project
phage-display library of human scFv antibodies. ELISA demon- LQ1604, LM2015043 CIISB for CMS BIOCEV; LTC17065,
strated that selected pdAbs could recognize ECTV, VACV, and CZ.1.05/1.1.00/02.0109 BIOCEV, CZ.1.05/4.1.00/16.0347 and
cowpox virus (CPXV). Atomic force microscopy visualized bind- CZ.2.16/3.1.00/21515.
ing of the pdAbs to VACV. Three of the selected pdAbs neutral-
ized variola virus (VARV) in the plaque reduction neutralization
test. Western blot analysis of ECTV, VARV, VACV, and CPXV P.18-103-Mon
proteins indicated that neutralizing pdAbs bound orthopoxvirus Isolation of mycobacterial monooxygenase
35 kDa proteins, which are encoded by the open reading frames EthA
orthologous to the ORF H3L in VACV. The fully human anti- M. Zahorszka, J. Madacki, D. Mikulasova, J. Kordulakova
body fh1A was constructed on the base of the VH and VL Department of Biochemistry, Faculty of Natural Sciences,
domains of pdAb, which demonstrated a dose-dependent inhibi- Comenius University in Bratislava, Bratislava, Slovakia
tion of plaque formation after infection with VARV, VACV, and
CPXV. To determine the p35 region responsible for binding to Protein EthA is NADPH dependent, FAD-containing mycobac-
neutralizing pdAbs, a panel of truncated p35 proteins was terial monooxygenase that catalyses activation of thioamide and
designed and expressed in Escherichia coli cells, and a minimal thiourea drugs, like ethionamide, isoxyl or thiacetazone. These
p35 fragment recognized by selected neutralizing pdAbs was antitubercular drugs inhibit the biosynthesis of mycolic acids,
identified. In addition, peptide phage-display combinatorial which are the hallmark of the cell envelope of mycobacteria,
libraries were applied to localize the epitope. The obtained data including the important human pathogens Mycobacterium tuber-
indicated that the epitope responsible for recognition by the culosis and Mycobacterium leprae. EthA encoded by the ethA
418 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 419
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
protein and its two potential DNA binding domains, using interference in bacterial cells. RsAgo was proposed to use guide
pGEX/E. coli expression system and performed interaction stud- RNAs to recognize complementary target DNA, leading to inhi-
ies with respect to the conformation of target DNA: electromo- bition of transcription and also its nucleolytic cleavage by acces-
bility shift assay, as well as, filter binding assay (DOT-blot) sory nucleases. However, the mechanisms of specific DNA
showed, that the N-terminal domain does not bind to the region, targeting by RsAgo and, in particular, the details of its interac-
irrespectively of its conformation. N-domain may loss its binding tions with double-stranded DNA molecules are unknown. In the
activity due to bulkiness of N-terminal tag. In contrast, the C- present study, we analyzed the interactions of guide-loaded
terminal domain does bind, but without any DNA-structural RsAgo with dsDNA targets in vitro. Using the gel shift assay, we
selectivity. The full protein SAF-A preferably binds to G4 form showed that successful loading of RsAgo onto dsDNA requires
of oct4 promoter region. To characterize this interaction in more prior DNA melting. The boundaries of the assembled ternary
detail we performed the ITC calorimetry experiments, followed complex of RsAgo with guide RNA and dsDNA were revealed
by CD spectroscopy measurements, which indicate changes of by footprinting methods. Possible interactions of RsAgo with
the secondary structure of interacting partners. This work was RNA polymerases of Escherichia coli and R. sphaeroides were
supported by the CSF (17-19170Y)and by the project SYMBIT tested using the bacterial two-hybrid system, and the domains of
reg. nr. CZ.02.1.01/0.0/0.0/15 003/0000477 financed by the the b- and b՛-subunits of RNA polymerase that are likely
ERDF. involved in interactions with RsAgo were identified. The results
suggest that recognition of dsDNA targets by RsAgo in vivo may
be facilitated by DNA replication and/or transcription, a hypoth-
P.18-107-Tue esis that is now under investigation. This work was supported in
Metabolism of different types of polyubiquitin part by the Grant of the Ministry of Education and Science of
chains Russian Federation 14.W03.31.0007.
A. Kudriaeva1, A. Tupikin2, M. Kabilov2, A. Belogurov Jr1
1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the
Russian Academy of Sciences, Moscow, Russia, 2Institute of
P.18-109-Mon
Chemical Biology and Fundamental Medicine, Siberian Branch of Intrinsically disordered proteins – how the
the Russian Academy of Sciences, Novosibirsk, Russia solvent influences the interaction between the
Ubiquitination is one of the major post-translational modifica- intrinsically disordered transcription factor
tions of proteins in eukaryotic cells. In addition to the well- c-Myb and its interaction partner CBP
known functions such as the targeting of substrates for proteaso- J. Schnatwinkel, C. Herrmann
mal degradation and DNA repair, polyubiquitin chains are also Faculty of Chemistry and Biochemistry, Ruhr-Universit€
at Bochum,
involved in other major cellular processes, for example, they par- Bochum, Germany
ticipate in the regulation of the cell cycle and functioning of the Intrinsically disordered proteins (IDPs) are characterized by a
immune system. The incredible diversity of polyubiquitin signals
partial or global lack of secondary structure elements under
derives from the ability of ubiquitin to form chains of various physiological conditions, which allows them to bind to their
branched types via isopeptide bonds between the e-NH2 group of interaction partner with low affinity and high specificity. A com-
any of the seven lysines (K6, K11, K27, K29, K33, K48, K63) of mon feature among IDPs is a folding upon binding process, dur-
one ubiquitin with the C-terminus of the other ubiquitin. Abun-
ing which the IDP samples through its funneled energy landscape
dance of these chains in the cell is different. It has been supposed in search of favorable intra- and/or intermolecular interactions,
that each of these different linkages imparts the unique structural stabilizing the final complex. This structural plasticity enables
and dynamic properties of the polyubiquitin chain. The function
IDPs to play key roles in cellular signaling and regulatory path-
of the polyubiquitinated chains in proteasome degradation and
ways. However, thermodynamics, kinetics and binding mecha-
DNA repair consisting of K48 and K63 bonds, respectively, are nisms of IDPs are poorly understood. In this work we present
well studied. The cellular functions of other, so-called non-cano- the impact of solvent and co-solutes on the interaction of the
nical chains of ubiquitin, are substantially less defined. This study
intrinsically disordered transcription factor c-Myb and its interac-
examined the intracellular metabolism of polyubiquitinated con- tion partner CREB-binding protein (CBP). We use different tech-
jugates and the substrate specificity of the proteasome for differ- niques, including temperature jump relaxation and microscale
ent ubiquitin linkage types, using a combination of flow thermophoresis to study both kinetics and thermodynamics of
cytometry and high throughput sequencing. The reported study
the interaction. By applying a variety of co-solutes, such as
was supported by Russian Scientific Foundation project #14-14- osmolytes, electrolytes, artificial polymer crowders, as well as
00585. protein crowders like BSA and Lysozyme we are able to quantify
the effect of each co-solute on the interaction of c-Myb and
P.18-108-Wed CBP. Further work will include a mutagenic study, revealing the
impact of the solvent at nearly residue level.
Interactions of a bacterial Argonaute protein
with DNA targets in vitro
L. Lisitskaya1, I. Petushkov1, D. Esyunina1, A. Aravin2, P.18-110-Tue
A. Kulbachinskiy1 In vivo study of copper transporter 1
1
Institute of Molecular Genetics, Moscow, Russia, 2Division of ectodomain (NdCTR1) chelating properties
Biology and Biological Engineering, California Institute of
I. Orlov1, T. Sankova1,2, A. Saveliev2, L. Puchkova2,3
Technology, Pasadena, CA, United States of America 1
ITMO University, Saint-Petersburg, Russia, 2Peter the Great St.
Argonaute proteins are central components of RNA interference Petersburg Polytechnic University, Saint Petersburg, Russia,
3
in eukaryotes but the functions of homologous proteins in Institute of Experimental Medicine, Saint-Petersburg, Russia
prokaryotes remain largely unknown. Rhodobacter sphaeroides Copper (Cu) is an essential trace element because it is a cofactor
Argonaute protein (RsAgo) was shown to preferentially recognize
for variety of vital enzymes and it also acts as a modulator of tran-
foreign genetic elements in vivo suggesting its role in RNA scription factors, cell cycle, apoptosis and signalling. In opposite
420 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
to plenty Cu pathways inside the cell, there are only two Cu Delaunay tessellation. Degree of conservation of residues in beta-
importers into the cell: highly specific CTR1 and nonspecific structural part and in beta-bend separately was determined by
DMT1. NdCTR1 contains three metal-binding motives and plays multiple-alignments of homologous structures and sequences of
crucial role in import by catching Cu(II) from extracellular donors corresponded protein domains using various matrices of amino
and guiding them as Cu(I) to the membrane pore. Besides Cu acid similarities. Possible biological implementations are discussed.
NdCTR1 can also bind silver (Ag) ions, which are isoelectronic to This work was supported by RFBR (project No 17-04-02105) and
Cu(I), and cisplatin. Mechanism of NdCTR1 action during Cu the Program of fundamental research for state academies for 2013–
import by CTR1 is not fully understood yet, also remains unclear 2020 years (No 01201363825 and No AAAA-A16-11611610173-5).
whether it participates in Cu(II) reduction prior to transport. In
present work hNdCTR1 fused with GST was cloned in E. coli. As
was shown by AAS bacteria synthetizing GST-NdCTR1 accumu- P.18-112-Mon
lated significantly more Ag and Cu compared to GST-producing Design of lipoate ligase library with following
strain. NdCTR1 increased resistance of bacteria to Ag and Cu selection of variants for intracellular site-
ions as well as to silver nanoparticles. Gel filtration and immuno- specific protein labeling by derivates of lipoic
precipitation experiments with subsequent measurement of metal
acids
concentration in fractions revealed that major amount of it is bind
K. Gaynova, A. Kudriaeva, A. Belogurov
to NdCTR1. As GST-NdCTR1 formed inclusion bodies, however
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
GST alone did not, we performed bioinformatical analysis of its
Moscow, Russia
structure and showed that last 13 a.r. of NdCTR1 demonstrate
high hydrophobicity. Due to presence of 8 (13%) histidine resi- Nowadays there is a wide diversity of methods for protein visual-
dues in NdCTR1, GST-NdCTR1 was isolated on Ni-charged ization in a living cell. However, existent approaches have major
IDA-Sepharose column in a buffer with 6M guanidine chloride. deficiencies and limitations such as cell toxicity or substrate imper-
Now we are obtaining NdCTR1 without mentioned hydrophobic meability, large size and low substrate specificity, therefore alterna-
cluster to purify NdCTR1 for further experiments. In present tive methods are intensively developed. Currently, there are several
work we’ve shown that NdCTR1 chelates Ag and Cu ions in vivo variants of lipoate ligase (LplA), an enzyme of 338 amino acids
and increases E. coli resistance to heavy metals. It’s important that and 38 kDa known to be involved in bacterial oxidative metabo-
as NdCTR1 binds Cu and cisplatin, potentially, it can be used in lism, which are capable for attaching fluorophores with red and
treatment of Cu-related disorders, characterised by presence of blue fluorescence. Here we report design of the lipoate ligase
unbound copper, and therapy of malignancies. library with a subsequent selection of variants for intracellular site-
specific protein labeling by derivates of the lipoic acid. We selected
seven amino acid residues in the wild-type lipoate-ligase sequence
P.18-111-Wed that form the substrate-binding pocket. These amino acid residues
Conformationally predetermined segments of are replaced by each of 20 amino acid residues, thus creating all
polypeptide chain in proteins: their sorts of combinations, each of which will form a unique configura-
tion of the active center. The library is constructed from a set of
characteristics and role of context
overlapping primers containing random nucleotides at positions
A. A. Anashkina1, L. A. Uroshlev2, I. Y. Torshin3,
encoding the corresponding amino acid residues. The reported
N. G. Esipova1, V. G. Tumanyan1
1 study was supported by Russian Scientific Foundation project #14-
Engelhardt Institute of Molecular Biology of the Russian
14-00585.
Academy of Sciences, 119991 Moscow, Russia, Moscow, Russia,
2
Vavilov Institute of General Genetics of the Russian Academy of
Sciences, 119991 Moscow, Russia, Moscow, Russia, 3Department P.18-113-Tue
of Chemistry, Lomonosov Moscow State University, Leninskie
Gory 1/3, 119991 Moscow, Russia, Moscow, Russia
Mechanistic insight into HsNMT1-mediated
acylation
A beta-hairpin included beta-bend can be treated as an example of F. Riviere1, C. Dian1, I. Perez-Dorado2, M. Ritzefeld3, J. Shen4,
composite local structure which components (beta-bend and frag- E. Cota2, T. Meinnel1, E. W. Tate3, C. Giglione1
ment of beta-structure) play different structural, energetic and geo- 1
CNRS-I2BC, Gif-sur-Yvette, France, 2Department of Life
metric role. In accord to our conception, the fragment of Sciences, Imperial College London, London, United Kingdom,
antiparallel beta-structure characterized by increased stability 3
Department of Chemistry, Imperial College London, London, United
imposes to the linker the definite set of conformations. Wherein, Kingdom, 4Imperial College London, London, United Kingdom
the number of independence conformational variables reduces due
to pseudo-cycle formation across hydrogen bonds. As a result the N-myristoylation is an essential and irreversible lipid modifica-
overall number of conformations in this system does not exceed tion in eukaryotes, corresponding to the addition of a myristate
two for main types of beta-bends. Thus the conformation of beta- group (C14:0) on the N-terminal of a glycine. This modification
bend is determined by context, namely assigning beta-structural is carried out co-translationally after excision of the initiating
part of the whole structure. It must be underline that definite con- methionine directly on the nascent protein chain by the N-myris-
formation is enforced to beta-bend independently to the primary toyltransferase enzyme (NMT) or by post-translational manner
structure of beta-bend itself even if this conformation encounters after proteolytic cleavage of certain proteins. This modification
sterically hinders (so called ‘disallowed’ conformations). In current has the role of targeting a protein to specific membranes where it
study the primary and three-dimensional structures of both parts can interact with other partners triggering signaling pathways
of the composite structure have been studied. On the basis of repre- essential for many cellular processes. The NMTs are monomeric
sentative sample it can be found that the sequence and the struc- enzymes composed of two GCN5-related N-acetyltransferase
ture provide enlarge stability of the context part. In spite to (GNAT) fusing domains, a large superfamily of enzymes that use
predetermined character of the beta-bend part of the whole struc- as substrates acyl-CoA derivatives to acylate their substrates.
ture some peculiarities in amino acid composition rather than in Nevertheless, previous studies suggested that the catalytic mecha-
sequence take place. Contacts within beta-hairpin and its part as nism of NMTs is different from that of other GNAT proteins.
well as long-distance contacts were estimated by Voronoi- We present several 3D structures of hsNMT1 in complex with its
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 421
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Protein – folding, dynamics, interaction and degradation POSTERS
two substrates and its two products (Myr-CoA/Octapeptide and characteristics of ThT incorporated into mature alpha-synuclein
Coa/Myristoyl-peptide), recapitulating snapshots of the different fibrils and ThT-amyloid fibrils binding parameters a special
steps of catalysis and providing unprecedented details at the approach based on spectroscopic investigation of solutions pre-
active site pocket. Here, we propose a brand-new portrait to both pared by equilibrium microdialysis was used. One mode (type) of
protein catalysis and substrate specificity. ThT binding to alpha-synuclein fibrils with binding constant
about 104 M1 and binding stoichiometry of ThT per protein
molecule about 1:4 was observed. The comparative analysis of
P.18-114-Wed the results obtained by a wide range of physical-chemical
Knockdown and inhibition of the N-terminal approaches for amyloid fibrils formed from different amyloido-
acetyltransferase Naa10/NatA in zebrafish genic proteins, allowed to assume the possibility of the existence
R. Ree1,2,3, L. M. Myklebust3, H. Foyn1, S. I. Støve1,3, of one more ThT binding mode to alpha-synuclein fibrils with
B. E. Haug4, T. Arnesen1,3,5 higher affinity and fluorescence quantum yield of the bound dye,
1
Department of biological sciences, University of Bergen, Bergen, which is difficult to detect because of the very small amount of
Norway, 2Department of Surgery, Haukeland University Hospital, these binding sites. This work was supported by grants of the
Bergen, Norway, 3Department of Biomedicine, University of RFBR (16-04-01614, 18-54-00022_Bel, 18-34-00366_mol_a and
Bergen, Bergen, Norway, 4Department of Chemistry, University of 18-34-00965_mol_a) and Fellowship of President of the Russian
Bergen, Bergen, Norway, 5Department of Surgery, University of Federation (SP – 841.2018.4).
Bergen, Bergen, Norway
N-terminal acetyltransferases (NATs) catalyze the addition of an P.18-116-Tue
acetyl group to the N-terminal amine moiety of a protein. Protein-nucleic acid complexes: insight into
Although this modification is extremely common, occurring on
their gas-phase stability
80–90% of human proteins, little is currently known about the
L. Slavata1,2, A. Kadek1, J. Chmelik2,3, R. Liskova1,2,
physiological role of N-terminal acetylation. The major eukary-
P. Novak1,2
otic NAT, NatA, of which Naa10 is the catalytic subunit, acety- 1
BioCeV – Institute of Microbiology, Czech Academy of Sciences,
lates distinct subpopulations of N-termini on the ribosome. Both
Prumyslova 595, 25250, Vestec, Czech Republic, 2Charles
NatA and Naa10 are important for cancer cell survival and pro-
University in Prague, Faculty of Science, Prague, Czech Republic,
liferation and may be therapeutic targets. Recently, mutations in 3
Institute of Microbiology, CAS, Prague 4, Czech Republic
the NAA10 gene were implicated in several developmental disor-
ders, suggesting that this protein may have an important role in Interactions between proteins and nucleic acids play a crucial role
development. We employ knockdown techniques, in vitro enzyme in all kinds of cell regulations. Understanding of this part of reg-
assays, in vivo NAT inhibition and label-free quantitative shot- ulation pathways depends on the knowledge of the protein-
gun proteomics to investigate the functional role of the NatA nucleic acid complex structure and its dynamics. Field of struc-
complex in developing zebrafish. We identified the zebrafish tural mass spectrometry has undergone a remarkable develop-
orthologue of the human Naa10, determined its expression pat- ment in recent years. This include a huge increase of commercial
tern, and assessed its knockdown phenotype. Furthermore, we availability of instruments coupling ion-mobility to mass-spectro-
developed selective NatA-inhibitors and found overlapping devel- metry. Native mass spectrometry is capable of gently transferring
opmental phenotypes when targeting NatA and Naa10. Label- macromolecules into the gas phase and so preserving their ter-
free quantitative shotgun proteomics on inhibitor-treated tiary and quaternary structure. Ion mobility can then separate
embryos identifies potential downstream targets of NatA which these macromolecules or macromolecular assemblies according to
may contribute to the observed phenotypes. their size and shape. Stability of macromolecules can thus be
studied under different conditions by observing the shifts of a
drift-time caused by changes of the shape during the unfolding
P.18-115-Mon event. We demonstrate here the power of this technique to
Alfa-synuclein amyloid fibrils structure and observe the increase in stability of the protein when interacting
formation probed by the fluorescent dye with its specifically recognized ligand counterpart – nucleic acid.
thioflavin T Further we support these results by high-resolution mass spec-
trometry measurements, that reveals an involvement of low
A. Sulatskaya1, N. Rodina1,2, M. Sulatsky1, I. Gagarskaia1,
molecular weight adducts in protein-nucleic acid interaction sta-
O. Povarova1, I. Kuznetsova1, K. Turoverov1,2
1 bility in gas-phase. Well characterized complex of the DNA-bind-
Institute of Cytology of the Russian Academy of Sciences, Saint-
ing domain of Forkhead-box transcription factor (FOXO4), with
Petersburg, Russia, 2Peter the Great St. Petersburg Polytechnic
its target DNA sequence – DAF-16 DNA native response ele-
University, Saint-Petersburg, Russia
ment was used as an object of this study. Acknowledgement: This
Alpha-synuclein amyloid fibrils formation accompanies the devel- work was supported by the Czech Science Foundation (grant
opment of the Parkinson’s disease, one of the most common neu- numbers 16-24309S), the Ministry of Education of the Czech
rodegenerative diseases. Although, alpha-synuclein amyloid fibrils Republic (project LH15010; programs “NPU II” – LQ1604 and
attracted the researchers attention for a long time, not all the fea- LM2015043 CIISB for CMS BIOCEV – LTC17065), and the
tures of their structure and formation are revealed. In this work European Regional Development Funds (BIOCEV – CZ.1.05/
alfa-synuclein amyloid fibrils structure and fibrillogenesis were 1.1.00/02.0109).
investigated using the fluorescent dye thioflavin T (ThT). Alfa-
synuclein amyloid fibrils formation was visualized by electron
microscopy and the kinetics of this process was detected by
absorption, fluorescence and CD spectroscopy (absorption, fluo-
rescence, fluorescence excitation spectra and fluorescence lifetime
of ThT and CD spectra of fibrils were determined). Formation of
amyloid fibrils was also proved by confocal microscopy in the
presence of ThT. For correct determination of photophysical
422 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Protein – folding, dynamics, interaction and degradation
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 423
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural biology POSTERS
424 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural biology
the C-terminus has also been studied in complex with Clp1. accumulation of the pathogenic forms of the amyloid-b peptide.
Other known domains, although less characterized, include a This work was supported by RFBR 17-04-02045-a.
region involved in association with Rna14/Rna15, a region rich
in glutamine and two zinc-finger domains. We have identified a
novel domain adjacent to CID and have completely determined P.19-003-Wed
the backbone and side chain 1H, 13C and 15N chemical shift Bacterial synthesis, purification and structural
assignments for the bacterially produced construct. Relaxation study of the Toll-like receptor 1 cytoplasmic
analysis indicates that the domain is independently folded and domain
composed of three well-defined alpha-helix. The final high resolu-
V. Lushpa, M. Goncharuk, S. Goncharuk, K. Mineev,
tion structure confirms the helical fold. The proximity of this pre-
A. Arseniev
viously uncharacterized domain close to the CID prompts
Institute of Bioorganic Chemistry, Moscow, Russia
speculation for a putative role in modulating CTD and RNA
binding, or intermolecular contacts within CF IA. We have used Today special attention is paid to transmembrane proteins,
NMR spectroscopy to characterize the RNA binding by this widely represented by ion channels and membrane receptors. The
domain and to reveal its role in enhancing RNA affinity by family of Toll-Like Receptor (TLR) refers to the first type of
cooperating with the adjacent CID. Despite this cooperativity, MPs. TLRs play a critical role in the innate immunity as the first
we have found motional independence between CID and this line of host defense. Despite the facts that general scheme of
domain by using paramagnetic relaxation enhancement (PRE) TLRs functioning is known and that the structures of some indi-
and 15N relaxation studies. Deletion and site-directed mutagene- vidual domains of several TLRs are solved, the detailed mecha-
sis indicate preliminary effects of cell growth when the domain is nism of signal transduction into the cell remains unclear.
absent or disrupted. Further tests will aim to pinpoint the pro- Obtaining the full-length receptor structures, or at least junctions
cesses affected by these mutants. of individual domains, would provide an answer to many ques-
tions of this problem. To date, structural problems are solved
mainly employing methods of cryoelectron microscopy, X-ray
P.19-002-Tue diffraction analysis and NMR spectroscopy. However, main suc-
Pathogenesis mechanism of familial cess in the field of first type MPs investigation was achieved by
“Australian” L723P mutation of Amyloid-b NMR spectroscopy. NMR study of the MPs in membrane-
mimicking environment allows not only to obtain an information
precursor protein revealed by structural and
about the structure of the protein but also about its mobility,
dynamical NMR data and to follow the conformational changes directly in the process
A. Urban1,2, E. Bocharov1,2, K. Nadezshdin1,2, A. Arseniev1,2, of interaction with other proteins or ligands. Such data makes it
O. Bocahrova1,2 possible to make an accurate model of MP functioning and shed
1
Moscow Institute of Physics and technology, Dolgoprudny, light on the process of signal transduction into the cell. The
Moscow Region, Russia, 2Institute of Bioorganic Chemistry results of bacterial expression, purification and NMR spectra
Russian Academy of Sciences, Moscow, Russia assignment of the intracellular domain of TLR1 are shown. The
Despite some progress in study of the molecular mechanisms of interactions of this domain with the model membrane were stud-
initial steps of Alzheimer’s disease pathogenesis are still ied. The obtained data allow us to clarify previous results about
unknown. Amyloidogenic Ab-peptides forming plaques in the the mechanism of the Toll-Like Receptors functioning, serving
brain are products of intramembrane cleavage of Amyloid-b Pre- the starting point to study the mechanism of signal transduction
cursor Protein (APP). At the same time cholesterol interactions into the cell when TLR is activated. This work was supported by
with APP have been rather extensively explored, and Alzheimer’s Russian Science Foundation (project No 14-14-00573).
disease pathogenesis is known to be closely correlated with the
membrane concentrations of various lipid species, the dependen-
cies on cholesterol content being especially frequently discussed. P.19-004-Mon
“Australian” (L723P-APP) mutation is identified to be associated Construction of recombinant fragments of
with developing early onset Alzheimer’s disease. Using of cell-free tick-borne encephalitis virus E glycoprotein in
expression we produced the 13C/15N-isotope labeled, wild-type order to determine an epitope recognized by
and mutant APP686-726 fragments. We performed high-resolu- protective chimeric antibody Encemab
tion NMR in membrane mimetics followed by Molecular
I. Baykov, E. Karelina, L. Emelianova, A. Matveev,
Dynamics simulations. We found L723P-APP mutation to cause
N. Tikunova
local unfolding of the last C-terminal turn of the APP transmem-
Institute of Chemical Biology and Fundamental Medicine, Siberian
brane domain helix and increased accessibility to water. More-
Branch of the Russian Academy of Sciences, Novosibirsk, Russia
over, we found that unlike the wild-type fragment, the mutant
fragment APP686-726 gradually converts from the a-helical to An antibody-based therapeutic drug for tick-borne encephalitis
the b-sheet conformation. With the aid of a spin-labeled choles- post-exposure prophylaxis and treatment is under development in
terol analogue we also characterized the sterol interactions with Novosibirsk, Russia. This drug is based on high affinity protec-
the APP transmembrane domain in two different membrane- tive chimeric antibody Encemab derived from murine 14D5 anti-
mimicking systems we described novel site near central hinge body against tick-borne encephalitis virus (TBEV) E
region of the APP transmembrane helix. The observed diversity glycoprotein. When developing a therapeutic drug based on anti-
of sterol interactions with APP implies an important role of over- body Encemab, it is necessary to determine its antiviral activity
all state of lipid environment in the Alzheimer’s disease develop- mechanism as well as localize an epitope recognized by this anti-
ment. These findings suggest a straightforward mechanism of the body on the surface of E glycoprotein. The major goal of this
pathogenesis associated with “Australian” mutation, which is study was to find the epitope by constructing various fragments
able to shift the distribution between the alternative APP trans- of E glycoprotein and examining the binding of the antibody to
membrane domain cleavage cascades, resulting in the these fragments. At the first step, location of the epitope was esti-
mated by creating three recombinant fragments of E
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 425
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural biology POSTERS
glycoprotein: rED3_294 and rED3_301 proteins, being two vari- in H/D exchange rate upon complexation with both human and
ants of domain D3 and differed by N-terminus, as well as rED12 phage dUTPases. This peptide therefore will be subjected to fur-
protein representing domain D1 together with domain D2 of E ther studies as a potential candidate for development of a new,
glycoprotein. Western blot analysis showed that the antibody peptide-type inhibitor against this enzyme.
Encemab binds to both variants of the D3 domain and does not
bind to the rED12 protein. Surface plasmon resonance-based
affinity measurements revealed that both D3 variants have a P.19-006-Wed
comparable affinity for the antibody. This suggests that amino Lectin RS20L from plant pathogen Ralstonia
acid residues 294–300, which are only present in rED3_294 pro- solanacearum – stability studies and
tein, do not appear to be part of the epitope region. Next, several crystallization
recombinant fragments of domain D3 were generated. Western
P. Kysel’1,2, N. Kostlanova1,2, J. Houser1,2, E. Dubska1,2,
blot analysis revealed that the antibody Encemab can bind only ak1,3, M. Wimmerova1,2,4
O. Sul
to the N-terminal fragment of the D3 domain of the glycoprotein 1
National Centre for Biomolecular Research, Faculty of Science,
E, which includes amino acid residues 301 to 339. These results
Masaryk University, Kotl arsk a 2, 611-37, Brno, Czech Republic,
were also complemented by in silico docking of predicted 3D 2
Central European Institute of Technology, Masaryk University,
structure of the antibody to the D3 domain of E glycoprotein.
Kamenice 5, 625 00, Brno, Czech Republic, 3Molecular
The research was supported by the Russian Science Foundation
Glycobiology, CERMAV-CNRS, BP53, 38041, Grenoble Cedex 9,
(project # 17-74-10146).
France, 4Department of Biochemistry, Faculty of Science,
Masaryk University, Kotl arsk a 2, 611-37, Brno, Czech Republic
P.19-005-Tue Ralstonia solanacearum is a Gram-negative b-proteobacterium,
Exploring a proteinaceous inhibitor of the inhabiting water and soil and causing wilt in more than 200
human dUTPase: pioneering structural model plants. Plant pathogens use protein-carbohydrate interactions for
host invasion. Lectins are non-enzymatic carbohydrate binding
of the inhibitory complex
proteins of non-immunoglobulin nature. Lectin RS20L (the Ral-
K. Nyıri1,2, H. D. T. Mertens3, B. Tihanyi4, G. N. Nagy4,
stonia solanacearum 20 kDa lectin) has no sequence similarity to
B. K} ohegyi4, J. Matejka4, M. J. Harris5, J. E. Szab
o4,
any known lectin amino acid sequence. Multiple sugars were
O. Ozohanics6, K. Vekey6, D. I. Svergun3, A. J. Borysik5,
tested for RS20L binding by isothermal titration calorimetry.
B. G. Vertessy4
1 Despite negative testing of monosaccharides, addition of manno-
Institute of Enzymology, Research Centre for Natural Sciences,
sylated oligodendrimers or mannosylated BSA provided positive
Hungarian Academy of Sciences, Budapest, Hungary, 2Budapest
results. RS20L without a sugar ligand showed low solubility.
University of Technology and Economics, Faculty of Applied
Additives can suppress precipitation. A selected panel of addi-
Biotechnology and Food Science, Budapest, Hungary, 3European
tives was tested using the nano differential scanning fluorimetry
Molecular Biology Laboratory, Hamburg Unit, c/o DESY,
(nanoDSF). This method is based on intrinsic fluorescence of
Hamburg, Germany, 4Institute of Enzymology, RCNS, HAS,
aromatic amino acids in proteins. Fluorescence intensity and the
Budapest, Hungary, 5King’s College London, London, United
fluorescence maximum heavily depend on the surroundings of the
Kingdom, 6Institute of Organic Chemistry, Research Centre for
tryptophan and can indicate protein unfolding. MnCl2 with basic
Natural Sciences, Hungarian Academy of Sciences, Budapest,
pH 8.1 dramatically increased the stability of RS20L. The result-
Hungary
ing protein solution was used for crystallization screening. Crys-
As a part of preventive repair, dUTPase enzyme prohibits uracil tals emerged in 0.1 M Hepes pH 7.5 with 10% (v/v) PEG3350.
misincorporation into DNA by hydrolysis of dUTP. This impor- Selenomethionine substitution is currently chosen to solve the
tant role of dUTPase entitles it as an essential enzyme in most phase problem by multi-wavelength anomalous dispersion
organisms. It has been shown that Stl, a Staphylococcus aureus (MAD). Beside the expected 20 kDa monomer, a higher molecu-
pathogenicity island repressor protein interacts with several tri- lar form of this protein appears on the SDS-PAGE. Analytical
meric dUTPases. Upon dUTPase-Stl complex formation dUT- ultracentrifugation confirmed the trimeric form of RS20L in solu-
Pase enzymatic activity is inhibited while the repressor function tion at room temperature. Similar properties have been described
of Stl protein is also perturbed. Besides the possible perspective for the model porin protein OmpF from Escherichia coli. Despite
of designing new antibacterial drugs after the detailed mechanism the different architecture of porins from Gram-positive and
is revealed, our group has shown that this system has also a Gram-negative bacteria, antiparallel ß-sheets in secondary struc-
potential to design human dUTPase inhibitors for chemotherapy, ture represent a common denominator. This work was supported
which are recently in the scope of medicinal interest. We investi- by the Ministry of Education, Youth and Sports of the Czech
gated the structure of human dUTPase-Stl protein-protein com- Republic under the project CEITEC 2020 (LQ1601).
plex by applying an integrated structural biology approach, while
combining the results of in vitro laboratory techniques (native gel
electrophoresis, steady-state and transient kinetics, isothermal P.19-007-Mon
titration calorimetry, native and hydrogen-deuterium exchange Structure-based design of human carbonic
mass spectrometry (HDX-MS)) with synchrotron radiation struc- anhydrase XII inhibitors
ture determination techniques as circular dichroism spectroscopy, acova1,2, M. Fabry2, V. Kral2,
M. Kugler1,2, J. Brynda1,2, P. Rez
small-angle X-ray scattering (SAXS) and X-ray crystallography. ıcha3, J. Nekvinda3, B. Gr€
K. Pospısilova1, J. Holub3, V. S uner3
The obtained hitherto unprecedented structural models revealed 1
Institute of Organic Chemistry and Biochemistry of the CAS,
that upon dUTPase-Stl complex formation the functional Prague, Czech Republic, 2Institute of Molecular Genetics, Prague,
homodimer of Stl repressor dissociates, which abolishes the DNA Czech Republic, 3Institute of Inorganic Chemistry of the CAS,
binding ability of the protein. Active site forming dUTPase seg- Re z, Czech Republic
ments were directly identified to be involved in the dUTPase-Stl
interaction by HDX-MS, explaining the loss of dUTPase activity Carbonic anhydrase XII (CA XII), one of the isoform of car-
upon complexation. Strikingly a relatively short, 10–15 residues- bonic anhydrase overexpressed in solid hypoxic tumours, was
long peptide segment of Stl showed the most significant decrease recently identified target for cancer therapy and diagnostics.
426 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 427
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural biology POSTERS
Here, we show the cryo-EM structures of initiation complexes SAXS experiment. Interestingly, we show that u-ParR is not able
with a constituent small ribosomal subunit (30S), three special- to bind its DNA sequence target. SAXS result also shows folded
ized translation initiation factors: IF1, IF2 and IF3, spa mRNA protein containing flexible region. This was confirmed by limited
and fMet-tRNA. Our structures could be used for new therapeu- proteolysis, which leads to the C-terminal domain (CTD) isola-
tic developments in the design of new antibiotics against this bac- tion. In addition, the N-terminal domains carrying (NTDw2) or
teria. not (NTDwt) the mutation M59I was cloned and purified. Struc-
tural investigation in solution showed that NTDwt is a well-
folded and globular domain. Crystallization screens on NTDwt
P.19-011-Tue gave diffracting crystals that were optimized and X-ray data were
The function of N-terminus in human 4- then collected at ESRF and SOLEIL synchrotrons. Molecular
hydroxyphenylpyruvate dioxygenase replacement solution and structure refinement of this domain is
H. Lee1, A. Feng2, C. Lin1 now in progress. These structures will shed light on the role of
1
National Defense Medical Center, Taipei, Taiwan, 2Cheng Hsin the particular mutation M59I of ParR and will offer a new target
General Hospital, Taipei, Taiwan for in silico drug-design in order to restore AG activity.
428 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural biology
P.19-014-Tue survival of Met null mutants. The functions of Met and Gce are
not fully redundant and proteins exhibit tissue specific distribu-
Prevotella intermedia produces two
tion. Bioinformatic analyses assign Met and Gce to the bHLH-
homologous proteins to Porphyromonas PAS transcription factors family. Their sequence homology is
gingivalis HmuY with different heme-binding limited to defined domains while C-termini (MetC and GceC)
properties exhibit significant differences. These fragments are predicted as
M. Bielecki1, S. Antonyuk2, R. W. Strange3, J. W. Smalley4, intrinsically disordered regions (IDRs). The differences between
M. Smiga 1
n1, P. Mackiewicz1, P. Noce
, P. Stepie n1, M. Olczak1, C-termini of Met and Gce can be crucial for proteins distinction.
T. Olczak1 In this study, we present purification protocol and initial struc-
1
Faculty of Biotechnology, University of Wroclaw, Wroclaw, tural characterization of GceC. The size exclusion chromatogra-
Poland, 2Institute of Integrative Biology, University of Liverpool, phy (SEC) was used to estimate apparent molecular weight
Liverpool, United Kingdom, 3School of Biological Sciences, (MW) and Stokes radius (Rs). Other analyses were performed
University of Essex, Essex, United Kingdom, 4School of Dentistry, using analytical ultracentrifugation (AUC). The Circular Dichro-
University of Liverpool, Liverpool, United Kingdom ism (CD) was used for the secondary structures content determi-
nation. All results demonstrate the disordered character of the
Periodontitis is a group of multifactorial, infectious diseases, ini-
GceC with residual secondary structure and propensity for fold-
tiated by an ecological shift in the composition of the subgingival
ing. We hypothesize that high flexibility and a range of confor-
biofilm, resulting in inflammation and destruction of the tooth-
mations adopted by the GceC are essential for its activity. Some
supporting tissues, eventually leading to tooth loss. Among the
subtle structural differences between GceC and MetC, in example
putative periodontopathogens, Porphyromonas gingivalis is con-
localization of short structured fragments, could be crucial for
sidered as the main etiologic agent and key pathogen responsible
their distinction during D. melanogaster development.
for initiation and progression of chronic periodontitis. P. gingi-
valis is a heme auxotroph and uptake of this compound is central
for survival and efficient infection establishment by this bac- P.19-016-Mon
terium. Our hypothesis was that Prevotella intermedia might uti-
Deep-structure of the genetic code and the
lize similar mechanisms to the P. gingivalis hmu heme uptake
system, with leading role played by hemophore-like HmuY pro- origin of replication: path invariants as pre-
tein, to acquire heme and increase its virulence. Comparative and LUCA attachment sites
phylogenetic analyses suggested differentiation of HmuY homo- B. K. Davis
logs in a variety of bacteria and low conservation across phyla of Research Foundation of Southern California, La Jolla, United
heme-coordinating histidine residues of HmuY. The analyses also States of America
showed that the homologs were subjected to duplications before The genetic code conserves an imprint of its evolution before the
divergence of Bacteroidetes lineages, which could facilitate evolu- Last Universal Common Ancestor (LUCA), over 3.5x109 years
tion of the residues binding heme, and functional diversification. ago, as a model with the time-order of codon assignments based
In agreement with these findings, we found that P. intermedia on amino acid synthesis path-length unifies its diverse structural
produces two HmuY homologs, termed PinO and PinA. Both features. Consistent with evidence for pre-LUCA attachment to a
proteins bound heme, but with lower affinity, preferentially under cofactor/adaptor tRNA, intermediates of all amino acids (except
reducing conditions, and in a manner different to that of HmuY. histidine) retain a free a-carboxyl. Reductive pentose-phosphate
Analysis of the three-dimensional structures confirmed differences cycle (RPC) intermediates, likewise, retain an invariant, a free
between PinO and HmuY, mainly in the fold forming the heme- PO42-, and attributing it to a pre-LUCA polyphosphate scaffold
binding pocket and the lack of two histidine residues which coor- led to evidence of the origin of replication. Method: Model-build-
dinate heme in HmuY, being replaced by two methionine resi- ing software, optimized by restricted Hartree-Fock fragment
dues in PinO. We conclude that specific accommodation of molecular orbital calculations, was used to construct the poly(-
heme-binding pocket could be generated exclusively in P. gingi- sugar-phosphate) replicators responsible for the RPC invariant.
valis and other bacteria maintain hmu genes allowing transport Result: D-ribulose-1,5-bisphosphate (RuBP) molecules, linked by
of heme or other nutrients, depending upon redox conditions. terminal PO42-, form a ladder-like polymer with parallel
polyphosphate scaffolds, crosslinked by the RPC pentose. Hydro-
lytic cleavage at the C2-C3 bond within carboxylated-poly
P.19-015-Wed
(RuBP), in an RPC-like manner, yields two 3-phospho-glycerate
Purification and structural analyses of the C- (PG) bearing strands. The modified glyceraldehyde monomer
terminal part of germ-cell expressed protein obtained on strand separation links the origin of replication to
from D. melanogaster the spontaneous, autocatalytic formose cycle. Anti-parallel
M. Kolonko, B. Greb-Markiewicz, A. O_zyhar strands of the 2-ketopentol additionally form a H-bonded duplex,
Wroclaw University of Technology, Faculty of Chemistry, equipped to bind by charge-attraction to a cationic mineral sur-
Department of Biochemistry, Wroclaw, Poland face. Incorporation of a second monomer – a H-bonding 3-keto-
RuBP (anoxic conditions), or non-bonding ribose (spacer) –
Drosophila melanogaster is a model organism in the study of
yields a hetero-poly(pentotide) duplex with anti-parallel strands
molecular basis of organism development. The growth and matu-
as an antecedent of an RNA double helix. Conclusion: Invariant
ration of insects are controlled by the coordinated action of two
groups in the ancient pathways of central metabolism and amino
hormones: 20-hydroxyecdysone (20E) and juvenile hormone (JH).
acid synthesis were attributed to pre-LUCA attachment to a scaf-
Although the 20E receptor has been studied extensively, the iden-
fold, or cofactor. Evidence resulted that ladder-like polypen-
tity and function of the JH receptor has been long unknown.
totides bridged the interval between pre-sugar autocatalysis and
Recently, Wilson and Fabian identified the Methoprene tolerant
RNA replication in the origin of life.
protein (Met), acting as JH receptor. Met binds JH and mediates
JH action preventing the precocious differentiation. The deletion
of Met is lethal for insects. However in D. melanogaster exists
Met paralog: Germ cell-expressed protein (Gce), ensuring
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 429
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural biology POSTERS
P.19-017-Tue ribosomes are converted into hibernating 100S even in rich media.
Finding the way to prevent dimerization may significantly decrease
Crystallographic studies of the Fab based on
survival ability of S. aureus. Our recent cryo-EM studies demon-
high potency VHH specific for IL-17A strated that integrity of giant 100S disomes (5 MDa) is maintained
O. Kostareva1, A. Gabdulkhakov1, S. Evdokimov2, V. Ekimova2, primarily by interactions between C-terminal domains (CTD) of
U. Putintseva2, M. Garber1, S. Tishchenko1 SaHPF with each other. Based on NMR and crystal structures of
1
Institute of Protein Research, Russian Academy of Sciences, SaHFP-CTD (see Fatkhullin et al., this issue) we predicted the key
Pushchino, Russia, 2BIOCAD, St.-Petersburg, Russia amino acids and point mutations which may distort SaHPF
Cytokine interleukin-17A (IL-17A) is a target for therapy of dimers. Predicted mutants were purified and analyzed using several
immuno-inflammatory rheumatic diseases and psoriasis. Recently biophysical methods (i.e. AUC, DLS, gel filtration coupled MALS,
the company BIOCAD (Russia) has developed a novel human native PAGE). According to our data, hydrophobic residues F160,
IgG1/j based on VHH to IL-17A as a promising potential drug Ile173, Tyr175 are the key players in SaHPF dimerization. Selected
candidate for autoimmune disease therapy. First, antibodies from mutations will be next introduced into S. aureus cells in order to
Lama glama specific for IL-17A were obtained by immunization analyze their survival rate as well as their infectivity in mice. Con-
and phage display methods. Then, based on the obtained mon- cluding, our work paves the path for exploring of complementary
odomain antibody, the classical antibody (BCD-085) was con- approaches to combat this severe pathogen. This work was sup-
structed. BCD-085 has very high (KD = 2 pM) affinity to human ported by the Russian Science Foundation (grant 16-14-10014),
IL-17A and neutralizes the interaction of IL-17A with the recep- Agence Nationale de la Recherche ANR-10-LABX-
tor. Currently, BCD-085 is put on the stage of clinical trials. At 0036_NETRNA.
present time was known two crystal structures of IL-17A in com-
plex with Fab fragments of different specific antibodies (PDB ID:
2VXS, 4QHU). Each of the complexes contained one functional P.19-019-Mon
homodimer of IL-17A sandwiched between two Fab fragments. Studies of inhibition mechanisms,
We have shown by using the gel filtration method that the com- dimerization and DNA binding of the
plex of the homodimer of IL-17A with Fab BCD-085 is formed transcription regulator Fur
in the ratio 1:1. We speculate that in this case could be another S. Nader, G. Veronesi, I. Michaud-Soret, J. Perard, S. Crouzy
type of interaction cytokine with antibody like interaction the IL- Univ. Grenoble Alpes, CNRS, CEA, BIG-Laboratoire de Chimie et
17A with the receptor IL-17RA (PDB ID: 4HSA). We present Biologie des M
etaux, Grenoble, France
preliminary structural studies of the Fab BCD-085. The crystals
belonged to the space group P212121, with unit-cell parameters Facing the public health threat of bacterial resistance to antibi-
a = 90.56, b = 111.92, c = 165.43, a = b = c = 90. Diffraction otics, the development of new strategies to fight pathogens
data were collected to 1.9 A resolution using a synchrotron- should be a priority. The new ideal therapeutic targets should
radiation source ESRF (Grenoble, France). The crystal structure exert weak evolutionary pressure, be able to disarm or weaken
of Fab BCD-085 playing an important role in the initial phase the pathogen and be unique to the microorganism. An interest-
structural investigations of Fab BCD-085/IL-17A and can help ing strategy is to interfere with the “Battle for Iron” that takes
to provide new insights into the molecular details antibody-anti- place between hosts and pathogens as iron is vital for all forms
gen interactions in this complex. This work was supported by the of life. To do so, we must target iron dependent sensors like
Russian Science Foundation (grant № 17-74-10156). We the Ferric Uptake Regulator (Fur) protein, a transcription regu-
acknowledge the Structural Biology Group of the European Syn- lator with a large regulatory network implicated in iron home-
chrotron Radiation Facility for granting access to the syn- ostasis and bacterial virulence. Fur proteins from different
chrotron beamlines. bacterial species have a relatively similar tertiary structure but
their quaternary structures vary: some are homo-dimers and
others are homo-tetramers. Our main objective is to study the
P.19-018-Wed inhibition mechanisms of Fur proteins and design new inhibi-
Hibernating ribosomes of Staphylococcus tors. To reach this goal, structure-function studies of the rela-
tionship between Fur proteins from different species and their
aureus as a potential target for new
inhibitors have been performed and in silico models of the inter-
therapeutics actions (Docking, Free Energy Profiles) have been proposed
I. Khusainov1,2, S. Validov2, R. Ayupov2, K. Usachev2, providing us with mechanistic insight into Fur complexes. These
M. Yusupov1,2 models will guide the experiments answering the same problem-
1
IGBMC, University of Strasbourg, Strasbourg, France, 2Kazan atic using techniques like X-ray absorption spectroscopy and
Federal University, Institute of Fundamental Medicine and crystallography. The structural information gathered from this
Biology, Kazan, Russia work will be used to better understand the inhibition mecha-
Bacteria and especially pathogenic species often cope with stress nism of Fur proteins and the development of new anti-virulence
and nutrients limitations, and their survival strongly depends on molecules as an alternative to antibiotics to which pathogens
the ability to preserve energy. One of the most beneficial mecha- are becoming resistant.
nism to save energy for bacterium is deactivation of its own trans-
lation machinery, namely hibernation of ribosomes. In S. aureus
this process is prompted by binding of stress protein SaHPF
(S. aureus hibernation promoting factor) to 70S ribosomes, which
triggers their association into inactive 100S ribosome dimers (dis-
omes). In such stand-by mode ribosomes are preserved from degra-
dation and from binding of several antibiotics, at the same time,
they can rapidly (within 1–2 min) be transformed back into active
ribosomes upon energy recovery. Unlike other bacteria, in S. au-
reus SaHPF is constitutively expressed in the cell, and part of
430 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural biology
P.19-020-Tue investigate cell signaling events and many other cellular processes
in general.
CW-domain of ASHH2 methyltransferase:
structural basis of ligand binding and
specificity P.19-022-Mon
M. Brilkov1, O. Dobrovolska1, R. Aasland2, Ø. Halskau1 X-ray structure of Arthrobacter globiformis
1
University of Bergen, Bergen, Norway, 2University of Oslo, Oslo, M30 ketose 3-epimerase which is useful for
Norway
D-allulose production
The CW-domain is a histone-tail modification reader which is H. Yoshida1,2, A. Yoshihara2,3, P. K. Gullapalli4, K. Ohtani4,
found in many different protein families involved in epigenetic K. Akimitsu2,3, K. Izumori2,3, S. Kamitori1,2
1
regulation and chromatin remodeling, and functioning by recruit- Life Science Research Center and Faculty of Medicine, Kagawa
ing and directing the proteins to the chromatin. Studies have University, Kagawa, Japan, 2International institute of Rare Sugar
shown that CW is specific towards methylated H3K4 modifica- Research and Education, Kagawa University, Kagawa, Japan,
tion, however specificity is different for different protein families, 3
Faculty of Agriculture, Kagawa University, Kagawa, Japan,
4
thus, CW from ASHH2 was shown to be more specific to Matsutani Chemical Industry Co., Ltd, Hyogo, Japan
H3K4me1, while MORC3 and ZCWPW1 are more specific to
D-Allulose (or D-psicose) is one of rare sugars of which physio-
H3K4me3. It is not well understood what determines the speci-
logical functions are focused for human healthcare, such as mod-
ficity of the domains. The aim of the study is to understand the
erating a blood glucose level and fat accumulation. In recent
structural requirements underlying the specificity of ASHH2 CW-
years, the effects of a non-caloric sweetener, a growth regulator
domain. The objectives were approached by structural and calori-
to plants and an immune enhancer against pathogenic bacteria
metric analysis of interaction of wild type, followed up with
have been also reported as biological functions. Several enzymes
mutation studies. Results show that specificity of ASHH2 CW-
for the production of D-allulose have been reported so far, how-
domain towards H3K4me1 is conditioned by energetically favor-
ever the structural information of those enzymes is limited. The
able enthalpy driven interaction. Study of mutants showed that
available structures of the potential enzymes for D-allulose pro-
C-terminal a-helix is an important determinant in stabilization of
duction from abundant sugar D-fructose, are Pseudomonas
the structure and binding, functioning via intradomain interac-
cichorii D-tagatose 3-epimerase (homo dimer, PDBID, 2QUL),
tion of L915 and I915 residues with the tryptophan side chains in
Clostridium cellulolyticum D-psicose 3-epimerase (homo tetramer,
the binding pocket. Changes in chemical environment around
3VNK), and Agrobacterium tumefaciens D-psicose 3-epimerase
tryptophan side chains can affect the structure of the binding
(homo tetramer, 2HK1). Here we determined the X-ray structure
pocket and structure of the whole domain.
of Arthrobacter globiformis M30 ketose 3-epimerase which was
previously found as D-allulose 3-epimerase (AgD-AE) due to its
P.19-021-Wed similar substrate specificity to the known D-psicose 3-epimerases.
There are two molecules of AgD-AE in asymmetric unit, and
Cryo-high voltage electron microscopic
AgD-AE forms homo tetramer by two-fold symmetry operation.
tomography (Cryo-HVEMT) of whole cells of The overall structure is similar to that of the known structures of
the immortalized murine microglial cell line, C. cellulolyticum D-psicose 3-epimerase and A. tumefaciens
BV-2 D-psicose 3-epimerase, but rather similar to the structure of
S. Jun1,2,3, H. Ro1,2,3, H. Kim1,2,3, S. Yoo4, J. Kim4 Mesorhizobium loti L-ribulose 3-epimerase (homo tetramer,
1
Korea Research Institute of Chemical Technology, Daejeon, South 3VYL). In addition, we found that AgD-AE shows the highest
Korea, 2Department of Bio-Analytical Science, University of enzymatic activity for L-ribulose. Therefore, AgD-AE could be
Science and Technology, Daejeon, South Korea, 3Korea Basic categorized to be L-ribulose 3-epimerase. Nevertheless, AgD-AE
Science Institute, Cheongju, South Korea, 4Korea Basic Science shows the activity for D-allulose with 65% of L-ribulose that is
Institute, Daejeon, South Korea different from the substrate specificity of M. loti L-ribulose 3-epi-
merase which has almost no activity for D-allulose.
Cryo-ET allows three-dimensional (3D) visualization of cellular
structural details at nanometer resolution in a near-native state.
Acquiring high resolution 2D projection images is a key point of P.19-023-Tue
high quality 3D reconstruction. Thick sample suggests abundant
Structural basis of interactions between 14-3-3
structural information with augmented 3D sample volume. How-
ever, low penetration power of conventional cryo-EM only can proteins and the 14-3-3 binding motifs of
resolve samples embedded in thin (200–500 nm) layer of ice. Sec- calcium/calmodulin-dependent protein kinase
tioning or thinning thick vitrified samples has been suggested as kinase
a solution, but it mitigates given specimen volume and comes D. Lentini Santo1, V. Obsilova2, T. Obsil1,2
with difficulties in handling delicate vitrified sample. Here, we use 1
Faculty of Science, Charles University, Prague, Czech Republic,
high voltage electron microscopy (HVEM) at accelerating voltage 2
Biocev – Institute of Physiology, The Czech Academy of Science,
of 1250 kV to reconstruct 3D structure of large mammalian cell, Prague, Czech Republic
BV-2 without sectioning. Despite the fact that our cryo-HVEMT
Calcium/calmodulin-dependent protein kinase kinases (CaMKK1
has been performed manually between 20 and 60 as asymmet-
and CaMKK2) are members of the Ca2+/calmodulin-dependent
ric tilting angles and the lack of high tilt angle projections in one
kinase (CaMK) family involved in adiposity regulation, glucose
side acts as the missing wedge, this study shows the first visual-
homeostasis and apoptosis. These upstream activators of
ization of thick part (~1000 nm) of the whole cells in 3D using
CaMKI, CaMKIV and AMP-activated protein kinase are nega-
an advanced technique combining HVEM and cryo-ET. We
tively regulated by phosphorylation, which also triggers an asso-
anticipate that the methodology established here will not only
ciation with the scaffolding protein 14-3-3. Studies have shown
constitute a useful tool for studying virus-host cell interactions at
that CaMKKs bind to various 14-3-3 isoforms and that the con-
various stages during infection, but will also open new ways to
served N-terminal motif containing phosphorylated Ser74 in
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 431
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural biology POSTERS
432 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural biology
subsequent crystallographic studies. The complex and the full deaminase (GSDA), a plant-specific enzyme, was characterized to
length hCCS have been regarded as recalcitrant to crystallization be responsible for converting guanosine into xanthosine. Product
due to the highly flexible structure of hCCS. Previous crystallo- xanthosine is then catalyzed into xanthine for further conversion
graphic work on this interaction focused on the yeast CCS ortho- into urate, a substrate for the ureide pathway. Presence of plant
logue in combination with copper site SOD1 mutants. However, specific GSDA is very unique, because in most organisms gua-
comparison between hCCS and yCCS reveals significant differ- nine serves as a substrate of guanine deaminase (GDA) to pro-
ences such as absence of Zinc loop and binding sites in yeast. duce xanthine. In order to understand substrate specificity of
The objective of this work was to understand the structural basis GSDA from Arabidopsis thaliana (AtGSDA) and its mechanistic
on hSOD1 recognition and maturation catalysed by hCCS. Com- features, we are undergoing structural studies of AtGSDA. In
plex stability experiments on combinations of hCCS and SOD1 particular, AtGSDA is related in sequences to GDA but differs
mutants confirmed that disulphide formation seems to be both in its function. Specifically, they contain catalytic residues com-
the last step in the SOD1 maturation and the regulator of com- mon for the cytidine/deoxycytidylate deaminase superfamily but
plex formation with hCCS. Based on these observation, we chose their substrate specificities are unclear. Our analyses will provide
a set of complexes suitable for crystallographic assays. Heterodi- structural basis for substrate specificity of AtGSDA and charac-
mer crystals were obtained using a variety of crystallisation terize enzyme mechanism in the early events of purine degrada-
screens and optimized using different techniques e.g. microbatch, tion in plants. This work was supported by Next Generation
protein/precipitant proportion, micro, macro and streak seeding. BioGreen 21 program of Rural Development Administration
Data collections were performed at Soleil and Diamond syn- (Plant Molecular Breeding Center) of Republic of KOREA.
chrotrons and structure was solved using Molecular replacement.
The results showed a novel conformation of the SOD1 disulphide
sub-loop communicating the presence of hCCS to the SOD1 P.19-029-Tue
active site, and orchestrate the timing of copper and disulphide Staphylococcus aureus hibernation promoting
transfer, complexation and complex dissociation. factor interaction with 70S ribosome
determined by high resolution NMR
P.19-027-Wed spectroscopy
Glutamate transporters in archaea and K. Usachev1, S. Validov1, I. Khusainov1,2, R. Ayupov1,
V. Klochkov1, A. Aganov1, B. Kieffer2, M. Yusupov1,2
eukaryotes 1
Kazan Federal University, Kazan, Russia, 2IGBMC, University of
A. Guskov Strasbourg, Strasbourg, France
University of Groningen, Groningen, Netherlands
Recent structural cryoelectron microscopy studies have shown
Glutamate transporters catalyse the thermodynamically unfa- that in human pathogen Staphylococcus aureus, 100S ribosome
vourable transport of anionic amino acids across the cell mem- formation occurs in the presence of long two domain protein
brane by coupling it to the downhill transport of cations. This
SaHPF and its N-terminal domain (NTD) binds to the 30S sub-
coupling mechanism is still poorly understood, in part because unit at the P-site and A-site similarly to its homologs from
the available crystal structures of these transporters are of rela- E. coli, EcHPF, EcYfiA and a plastid specific YfiA and may inhi-
tively low resolution. In this contribution I will present the crys- bit protein synthesis by preventing mRNA and tRNAs from
tal structures of the archaeal transporter GltTk in the presence
accessing the 70S ribosome. Additionally, it occludes the binding
and absence of aspartate resolved to the highest up to date reso- of several antibiotics such as hygromycin B, tetracycline, edeine,
lution, and will report results of molecular dynamics simulations pactamycin, kasugamycin. C-terminal domain (CTD), connected
and binding assays to show how strict coupling between the
by a linker of 35 amino acids, protrudes out of each ribosome
binding of three sodium ions and aspartate takes place. Further-
through the E-site and forms a main contact in 100S dimer
more I will present the latest results, including Cryo-EM struc- through homodimerization with SaHPF CTD from the second
ture of a mammalian homologue, which has revealed a few very ribosome. In order to improve interpretation of recently obtained
interesting peculiarities when compared to archaeal homologues.
cryoEM data and provide more detailed characterization of the
interaction between SaHPF NTD and the functional sites of
P.19-028-Mon S. aureus ribosome we performed HETSOFAST NMR experi-
ments and observed an intensity changes of SaHPF NTD signals
Characterizing substrate specificity of in the presence of 70S ribosomes in solution during imino-pro-
guanosine deaminase from Arabidopsis tons saturation. For free SaHPF NTD sample we did not
thaliana observe any influence on the amide signals intensity, while after
S. Y. Yang, S. K. Rhee the addition S. aureus 70S ribosome a strong effect (up to ~25%)
Seoul National University, Seoul, South Korea was observed, which means that we observe a chemical exchange
between protein and ribosome protons. Obtained intensity
Nitrogen is an essential element for growth and reproduction of
changes allow us to determine that several amino acid residues of
plant. In the past decade, it has been characterized in plants and
SaHPF, located in close proximity to the 16S rRNA, may be
many microbes that purine, a nitrogen-rich compound, is subject
involved in contacts formation. Information on SaHPF protein-
to enzyme-dependent degradation and the resulting products are
ribosome contacts will provide crucial knowledge for further
ammonia and/or urea. In brief, the ureide pathway in plants is
antibiotic modification strategy. This work was supported by the
capable of utilizing urate, an early product from purine degrada-
Russian Science Foundation (grant 161410014).
tion, as substrate and undergoes sequential enzyme reactions to
produce ammonia, glyoxylate, and/or urea. This ureide pathway
has been suggested as one of the possible metabolic pathway for
recycling nitrogen in plants. Unlike the downstream of purine
catabolism by ureide pathway, the early biological events for pur-
ine degradation have been studied recently. In Arabidopsis thali-
ana, among those early events in purine degradation, guanosine
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 433
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural biology POSTERS
434 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural biology
identified the dephosphorylated form of HPr as a regulator deter- repetitive region is unknown. We hypothesise that the repetitive
mining the glucose preference over mannitol by interacting with region forms an elongated structure with the necessary rigidity to
and augmenting the repressor activity of MtlR in E. coli. Here, project the functional N-terminus away from the cell wall sur-
we solved the structure of the MtlR-HPr complex to understand face. We redefined the domain boundaries of the structural units
how phosphorylation of HPr impedes its interaction with MtlR. in the repetitive region, Domains of Unknown Function
The phosphorylation site His15 of HPr is recognized by Glu108 (DUF1542), using bioinformatics approaches. The structure was
and Glu140 of MtlR and phosphorylation at His15 induces elec- determined by X-ray crystallography and showed elongated heli-
trostatic as well as steric repulsion between the two proteins. cal bundles, arranged in tandem. Importantly, the new domain
Based on this structural insight and comparative sequence analy- boundaries for DUF1542 were verified. Biophysical characterisa-
ses, we show that determination of the glucose preference over tion confirmed that the interface between tandem domains was
mannitol solely by the MtlR-HPr interaction is unique among essential for both intra- and inter-domain structural stability. We
members of the Enterobacteriaceae family. assessed by Quasi-Elastic Light Scattering (QELS) that the shape
of DUF1542 domains in solution was elongated rather than glob-
ular. The approximate size in solution measured by Small Angle
P.19-034-Mon X-ray Scattering (SAXS) for 4 DUF1542 domains showed elon-
Towards characterizing substrate specificity of gation to the maximum length predicted from the crystal struc-
DAO1 from Arabidopsis thaliana ture, indicating rigidity. Taken together, the structural insight
S. Jin, S. Rhee gained for the repetitive region of SasC suggests that the biofilm-
College of Agriculture and Life Sciences, Seoul National mediating domain is displayed at the tip of an elongated and
University, Seoul, South Korea putatively rigid stalk, projecting it away from the cell wall sur-
face. Knowledge of the structure and function of domains in
Auxin is a central plant hormone, regulating many aspects of CWA proteins contributes to a better understanding of the mech-
plant growth and development. Therefore, maintaining its con- anism underlying protein-mediated biofilm formation.
centration at a critical level is a key biological event for plant.
Local concentrations of auxin have been known to be diverse
among various types of plant cells, suggesting that its regulation P.19-036-Wed
is crucial for plant development. Auxin homeostasis requires a Hydrogen bond network near OH group of 6-
metabolic balance of its biosynthesis and degradation. In the past
(p-hydroxyphenyl) substituent of
years, there have been intensive studies for IAA (indole-3-acetic
acid) biosynthesis, a major form of auxin, whereas its modifica- coelenterazine determines the
tion or degradation has not been investigated. It is only recently bioluminescence spectra differences among
identified that DIOXYGENASE FOR AUXIN OXIDATION 1 hydromedusan calcium-regulated
from Arabidopsis thaliana (AtDAO1) catalyzes a conversion of photoproteins
IAA into oxIAA (2-oxoindole-3-acetic acid), an oxidized form of E. Vysotski1, S. Markova1, P. Natashin1,2, G. Stepanyuk1,
IAA, which is a major IAA catabolite and subsequently subjected J. Lee3, N. Malikova1, Z. Liu2
to further degradation. Sequence analysis indicates that AtDAO1 1
Photobiology Laboratory, Institute of Biophysics SB RAS,
is a member of the Fe(II)/a-ketoglutarate-dependent dioxygenase, Federal Research Center “Krasnoyarsk Science Center SB RAS”,
and further suggests that AtDAO1 also follows an enzyme mech- Krasnoyarsk, Russia, 2iHuman Institute, ShanghaiTech University,
anism for IAA oxidation essentially identical for other dioxyge- Shanghai, China, 3Department of Biochemistry and Molecular
nases. In plant kingdom, AtDAO1-like gene is well conserved Biology, University of Georgia, Athens GA, United States of
including corn, rice, potato etc. From sequence perspective, America
AtDAO1 is one of the a-ketoglutarate-dependent dioxygenase
family, but its substrate specificity for IAA remains unclear. In Ca2+-regulated photoproteins are responsible for the light emis-
order to decipher a substrate specificity of AtDAO1, we initiated sion of many marine coelenterates. All hydromedusan photopro-
structural study of AtDAO1 using X-ray crystallography. Our teins are single-chain polypeptides to which the oxygen-activated
structural analysis will provide a platform for understanding sub- substrate, 2-hydroperoxycoelenterazine, is tightly but non-cova-
strate specificity and mechanistic features of AtDAO1. This work lently bound. Bioluminescence results from oxidative decarboxy-
was supported by Next Generation BioGreen 21 program of lation of 2-hydroperoxycoelenterazine, generating a protein-
Rural Development Administration (Plant Molecular Breeding bound product, coelenteramide, in an S1 excited state. The biolu-
Center) of Republic of KOREA. minescence spectral maxima of photoproteins from different
organisms vary in the range 462–495 nm, despite a high degree
of identity of amino acid sequences and spatial structures of these
P.19-035-Tue photoproteins. Based on the studies of obelin and aequorin
Tandem domains go a long way: the repetitive mutants with substitution of the residues located near oxygen of
region in a biofilm-forming protein forms an 6-(p-hydroxyphenyl) substituent of 2-hydroperoxycoelenterazine
by amino acids with different donor-acceptor properties of side
elongated stalk chains we found that the hydrogen bond network surrounding
L. van Beek1, A. Bateman2, C. G. Baumann1, J. R. Potts1 the oxygen atom influences a light emission spectrum of the pho-
1
University of York, York, United Kingdom, 2EMBL-EBI, toprotein. For instance, the replacement of Phe to Tyr and Tyr
Hinxton, United Kingdom to Phe in obelin and aequorin, respectively, makes biolumines-
Staphylococcus aureus is an opportunistic Gram-positive bac- cence spectrum of obelin similar to that of aequorin and the bio-
terium. It can colonise the surface of in-dwelling medical devices, luminescence spectrum of aequorin – to that of obelin.
forming a biofilm. Cell wall-anchored (CWA) proteins such as Determination of the crystal structures of some photoprotein
S. aureus surface protein C (SasC) mediate biofilm formation. mutants before and after bioluminescent reaction clearly supports
These multi-domain proteins feature a similar organisation: the the suggestion that different hydrogen bond patterns near the
N-terminal domain mediates adhesion and the C-terminus is hydroxyl group of 6-(p-hydroxyphenyl) substituent of the sub-
attached to the cell wall, while the function of the central strate molecule account for spectral differences among
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 435
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural biology POSTERS
hydromedusan photoproteins. This work was supported by structure becomes disorganized, and vesicle traffic is impaired;
RFBR grant 17-04-00764 and a China-Russia international col- however, the details of Hof1-Bnr1 interactions have remained
laboration grant from the Chinese Academy of Sciences and the unclear. Here, using single particle electron microscopy, we reveal
Natural Science Foundation of China. the structures of the FH2 domains of Bnr1 in complex with full-
length Hof1 or with its F-BAR domain. The Hof1-Bnr1 complex
is a dumbbell-shaped structure, in which the tips of an elongated
P.19-037-Mon F-BAR dimer hold two FH2 dimers apart, and these interactions
X-ray structural study of a novel S-type appear to obstruct the actin-binding surfaces on the FH2. In sup-
cardiotoxin (CTX) from N. naja port of this view, bulk actin assembly assays and TIRF micro-
V. R. Samygina1,2, K. M. Dubova1,2, G. Bourenkov3, scopy assays show that the F-BAR domain of Hof1 directly
P. V. Dubovskii4, Y. N. Utkin4 inhibits Bnr1-mediated actin nucleation. Taken together, our
1
FSRC Crystallography and Photonics, RAS, Moscow, Russia, results allow us to describe a mechanism for how Hof1 controls
2
NRC “Kurchatov Institute”, Moscow, Russia, 3European Bnr1 activity. The authors acknowledge funding from the Rus-
Molecular Biology Laboratory, Hamburg Unit, c/o DESY, sian Science Foundation (grant #14-14-00234 to O.S.), the grant
Hamburg, Germany, 4Shemyakin-Ovchinnikov Institute of of the President of the Russian Federation (grant MK-
Bioorganic Chemistry, Moscow, Russia 2614.2018.4 to T.S.-K.), and the National Institutes of Health
(RO1 GM083137 to B.G.).
Cobra cardiotoxins (CTXs) are amphiphilic three-finger (loops
L1-L3) basic cytolytic polypeptides (59–61 residue-long) that bind
to cell membranes, causing a wide range of effects. These include P.19-039-Wed
depolarization and necrosis of heart and skeletal muscles, lysis of Novel coiled coils observed in the C-terminal
blood and epithelial cells, induction of toxicity in cortical neu-
domains of human septins may aid in filament
rons and various types of cancer cells. Based on binding activities
of CTXs to zwitterionic membranes, two distinct types of CTXs, bundling
P- (Pro-30-containing) and S- (Ser-28-containing), have been R. Garratt1, D. Leonardo1, I. Uson2, N. Soler2, N. Valadares3
1
identified, of which the P-type CTX interacts more strongly than Instituto de Fısica de S~ ao Carlos, Brazil, 2Structural
ao Carlos, S~
the S-type with the membranes. We have isolated several CTXs Biology Unit, CSIC,, Barcelona, Spain, 3Department of Cellular
from Naja naja cobra venom using gel-filtration, ion exchange Biology, UNIV. Brasılia, Brasılia, Brazil
and reversed-phase HPLC and analyzed the amino acid Classical dimeric coiled coils are composed of two a-helices
sequences of isolated toxins by mass-spectrometry. The most which may be orientated either parallel or anti-parallel with
abundant was an S-type CTX 16-1, a L48V49 analogue of CX3 respect to one another. They are characterized by heptad repeats
(P24780) N. naja. In the present work, its 3D structure was (abcdefg) in which hydrophobic residues occupying the a and d
established by X-ray crystallography. Crystals of CTX 16-1 positions stabilize the helical interface. Characteristic residue pat-
belonging to C2221 and P6422 space groups were grown by the terns can often be used to predict their relative orientation, how-
hanging drop or counter diffusion method. Structures of hexago- ever, as far as we are aware, the predominance of hydrophilic
nal (with three molecules in the asymmetric unit) and orthorhom- interactions at the interface has yet to be described. Here we
bic (with six molecules in asymmetric unit) crystal form were observe this phenomenon for the first time in the crystal struc-
solved at 2.3 and 2.5 A respectively. X-ray analysis reveals unu- tures of the short coiled-coils present in the C-terminal domains
sual conformation of L2 functional loop in all subunits of hexag- of human group III septins. Septins are filament forming GTP-
onal structure and most subunits of orthorhombic form, while binding proteins involved in a wide variety of intracellular events
the remaining models reveal a structure reported earlier. Dis- most of which involve membrane remodelling or barrier forma-
tinctly different is the tip of the L2 in the structures of both crys- tion. Crystal structures of peptides derived from human septins
tal forms. This exhibits striking similarity to that found by NMR 1, 4 and 5 at <2 A resolution show examples of both the parallel
in dodecylphosphocholine-bound cytotoxin I from N. oxiana. and anti-parallel arrangement. In the latter the a position is
Thus, for the first time, via crystallization we obtained a mem- exclusively occupied by hydrophilic residues (including Asp and
brane-bound form of an S-type cardiotoxin. This work was Glu) which form a continuous chain of hydrogen bonds down
partly supported by the Federal Agency of Scientific Organiza- one strip of the interface. However, the amino-acid sequence and
tions and in the frame of Federal Space programm 2016–2025 the anti-parallelism guarantee that the charged residues are never
(ISS “Science”). consecutive along this chain. On changing to the parallel orienta-
tion those residues which previously occupied the a position
move out of the interface. Simultaneously those occupying the d
P.19-038-Tue position move into a and a new group of hydrophobic resides
Structural study of interactions between the assume the d position. The sequences appear therefore to be cha-
F-BAR domain protein Hof1 and the formin meleon in nature, possessing two partially-overlapping alternative
Bnr1 interfaces. Neither of the two orientations appear to provide
T. B. Stanishneva-Konovalova1, M. V. Garabedian2, great stability and we speculate that dynamic interchange may be
D. I. Begrova1, A. S. Chemeris1, B. L. Goode2, O. S. Sokolova1 physiologically relevant to the formation of individual filaments
1
Department of Bioengineering, Faculty of Biology, Lomonosov when parallel and cross-bridges between them when anti-parallel.
Moscow State University, Moscow, Russia, 2Department of This work was supported by FAPESP.
Biology, Rosenstiel Basic Medical Science Research Center,
Brandeis University, Waltham, United States of America
The formin Bnr1 is recruited to the bud neck in S. cerevisiae to
assemble actin cables and enable cargo transport between the
mother cell and the growing daughter cell. This process is regu-
lated by Hof1, which directly binds to Bnr1 and inhibits its actin
nucleation activity. In the absence of Hof1 the actin network
436 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 437
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural biology POSTERS
438 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural biology
P.19-046-Mon P.19-048-Wed
Structural studies of plant omega amidase Unravelling dimerization of the epigenetic
(M. truncatula) reader LEDGF/p75
B. Imiolczyk1, J. Barciszewski1, K. Szpotkowski1, M. Jaskolski1,2
V. Lux1, K. Cerm akova1, T. Brouns2, M. Fabry3,
1
Center for Biocrystallographic Research, Institute of Bioorganic M. Madlıkova1, M. Horejsı3, F. Christ2, J. Demeulemeester2,
Chemistry PAS, Poznan, Poland, 2Department of Crystallography, acova1,3, Z. Debyser2, V. Veverka1
J. De Rijck2, P. Rez
1
Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland Institute of Organic Chemistry and Biochemistry CAS, Prague,
Czech Republic, 2KU Leuven, Molecular Virology and Drug
Omega amidase (systematic name: omega-amidodicarboxylateami-
Discovery, Leuven, Belgium, 3Institute of Molecular Genetics,
dohydrolase; synonym: Nit 2, Nitrilase-like protein 2) is an enzyme
CAS, Prague, Czech Republic
metabolically linked to asparagine and glutamine transamination.
The role of omega amidase is to remove potentially toxic The Lens Epithelium Derived Growth Factor/p75 (LEDGF/p75)
intermediates by converting alpha-ketoglutaramate and alpha- is a chromatin reader recognizing H3K36me2/me3 marks through
ketosuccinamate to biologically useful alpha-ketoglutarate and the N-terminal PWWP domain. Versatile integrase binding domain
oxaloacetate, respectively. Subsequently, alpha-ketoglutarate and (IBD) of LEDGF/p75 serves for interaction with several other
oxaloacetate can enter the TCA cycle as an energy source. We partners, e.g. Mixed-lineage leukemia (MLL) protein, the Myc
overexpressed, purified and crystallized recombinant omega ami- interacting factor JPO2, Zinc-finger protein PogZ, transcription
dase from Medicago truncatula (MtOA). Synchrotron X-ray elongation factor IWS1, HIV-1 integrase, and tethers them to
diffraction data were collected and two MtOA structures were actively transcribed genes. Our major aim is detailed structural
determined, with the catalytic loop in an open and closed confor- characterization of LEDGF/p75 and a thorough understanding of
mation. The protein exists as a dimer with alpha-beta-beta-alpha its physiological functions in order to advance in drug discovery
fold in both structures. The quaternary arrangement of the sub- efforts. LEDGF/p75 was found to dimerize and using NMR, we
units is also observed in solution using small-angle X-ray scattering identified the minimal dimerization domain to comprise amino
(SAXS). acid residues 345–467. Surprisingly, the structured part of the
dimerization interface partially overlaps with the binding site rec-
ognized by all known LEDGF/p75 cellular binding partners. We
P.19-047-Tue will therefore follow the effects of LEDGF/p75 dimerization on
Structural and functional study of translation binding to various cellular partners and try to identify dimerization
initiation factor e/aIF2 defective LEDGF/p75 variants to decipher physiological relevance
E. Stolboushkina1, U. Dzhus1, M. Bukhtoyarova1, of LEDGF/p75 dimerization. Besides NMR spectroscopy, size
V. Arkhipova1,2, A. Anisimova3, S. Dmitriev3, M. Garber1 exclusion chromatography, small angle X-ray crystallography
1
Institute of Protein Research, Russian Academy of Sciences, (SAXS), analytical ultracentrifugation, cross-links and native elec-
Pushchino, Moscow Region, Russia, 2University of Groningen, trophoresis are employed to investigate LEDGF/p75 dimerization.
Groningen, Netherlands, 3Belozersky Institute of Physico-Chemical
Biology, Lomonosov Moscow State University, Moscow, Russia
P.19-049-Mon
In Eukarya and Archaea, translation initiation factor 2 (e/aIF2a,b, 3-D structure of L254N carboxypeptidase T
c) plays a key role in delivering initiator Met-tRNA to the small
mutant and its complexes with transition state
ribosomal subunit. Structures of the eukaryotic 43S and 48S com-
plexes bound to the translation initiation factors have been deter- analogues
mined (Hashem et al., 2013; Husain et al., 2014). However, there is V. Timofeev1,2, V. Akparov3, I. Kuranova1,2, T. Rakitina1,4
1
no interpretable density for eIF2b and c. Therefore, no model National Research Centre “Kurchatov Institute”, Moscow, Russia,
2
building or refinement was done for eIF2b and c, and its placement Shubnikov Institute of Crystallography of Federal Scientific
was based on the structure of the archaeal aIF2 (Schmitt et al., Research Centre “Crystallography and Photonics”, Russian
2012; Stolboushkina et al., 2013). aIF2 and eIF2 have a similar Academy of Sciences, Moscow, Russia, 3State Research Institute
structural organization, but the eukaryotic polypeptides have addi- for Genetics and Selection of Industrial Microorganisms
tional terminal extensions, most strongly pronounced in the case of (GosNIIgenetika), Moscow, Russia, 4Shemyakin-Ovchinnikov
the b subunit. Structure of the eukaryotic eIF2 is a great interest- Institute of Bioorganic Chemistry of the Russian Academy of
ing. Before all attempts to produce and isolate recombinant Sciences, Moscow, Russia
eukaryotic eIF2c were unsuccessful. Here, we report about the sol- Metallocarboxypeptidase T is a zinc-dependent exopeptidase.
uble isolated recombinant human and yeast eIF2c. In this study, Like other proteins of this family, carboxypeptidase T cleaves C-
we have used a strong ionic detergent SDS for solubilization of terminal amino acid residues from proteins and peptides, but,
inclusion body proteins and refolded proteins by removing SDS at unlike other enzymes of this class, it has broad substrate speci-
lower temperature with KCl. At present, we are testing this eIF2c ficity, cleaving, albeit at different rates, peptide bonds formed by
for binding with other subunits eIF2 and tRNA. After that we plan C-terminal hydrophobic, as well as positively and negatively
to use the reconstructed heterotrimeric eIF2 for crystallization. charged amino acid residues. Carboxypeptidase T from Ther-
Moreover, we have reconstructed chimeric e/aIF2 as an attractive moactinomyces vulgaris, for which a spatial structure is known,
tool for studying the role of the each subunit of archaeal and is a convenient object for constructing mutant forms with altered
eukaryotic e/aIF2 in translation. Using the toeprinting and yeast substrate specificity. One such form is L254N carboxypeptidase T
cell-free translation system, we have received first results. Chimeric mutant. It was shown that L254N carboxypeptidase T mutant
e/aIF2 can support ribosomal scanning during eukaryotic transla- has a specificity in comparison with the wild type with respect to
tion initiation but archaeal c in complex with eukaryotic a and b the charged substrate. In present study 3-D structures L254N
inhibits translation. This research requires continuation and was carboxypeptidase T mutant and its complexes with transition
supported by the Program for Basic Researches on Molecular and state analogues (N-sulfamoyl-L-glutamic acid, N-Sulfamoyl L-
Cellular Biology and Post-Genomic technologies of the Presidium Arginine, N-Sulfamoyl L-Leucine) have been determined by X-
of RAS to E.A.S. ray analysis method. The analysis of the obtained spatial
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 439
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural biology POSTERS
structures allowed to determine how the replacement of leucine dynamic membrane-protein system. The plant-specific REMOR-
with asparagine affects the binding of substrates. The work was INs (REMs) are proteins regulating hormonal crosstalk and host
supported by the Russian Science Foundation (Project 17-14- invasion. Located in the plasma membrane, they spatially segre-
01256) in part of macromolecule production and X-ray analysis gate into nanodomains and represent the best-characterized nan-
and by the Central Research Institute for Mechanical Engineer- odomain markers in plants. In order to establish a mechanistic
ing, ROSKOSMOS in part of microgravity crystallization. description of the interplay between REMs and membrane nan-
odomains we use magic-angle-spinning solid-state NMR
(ssNMR), a powerful emerging technique to reveal protein struc-
P.19-050-Tue ture and flexibility in the native membrane environment, in con-
Structure-function studies of formate junction with static ssNMR to assess membrane order, thickness,
dehydrohenase from pathogenic bacterium phase transition and curvature. Our studies of a short N-terminal
Staphylococcus aureus peptide, conferring membrane anchoring and nanodomain orga-
nization of REMs, reveal an original mechanism regulating the
V. I. Tishkov1,2,3, K. M. Boiko2, A. A. Pometun2,3,
dynamic nanodomain targeting of REMs, mediated by the inter-
A. Y. Nikolaeva2, D. A. Korgenevskiy2, I. S. Kargov2,3,
action of the anchor peptide with phosphoinositolphosphate
S. S. Savin1,3
1 PI4P. MD simulations and solid-state NMR propose unconven-
Department of Chemistry, M.V. Lomonosov Moscow State
tional binding motif of the anchoring peptide. While the mem-
University, Moscow, Russia, 2Federal Research Centre
brane anchor revealed essential for nanodomain organization, the
“Fundamentals of Biotechnology” of RAS, Moscow, Russia,
3 role of REM trimerization via a coiled-coil domain remained
Innovations and High Technologies MSU Ltd, Moscow, Russia
obscure; we therefore invested in a detailed dissection of the
Bacterium Staphylococcus aureus is one of the most dangerous structural mechanisms driving trimerization, and their implica-
hospital infection due to high multiple antibiotics resistance. The tions for native REM organization. We show that the coiled-coil
resistance increases more when S. aureus exists as biofilms. Gen- domain controls targeting of REMs into the PM and, combining
ome analysis of S. aureus showed presence in chromosomal solid-state NMR with molecular modeling, we propose a model
DNA of gene of formate dehydrogenase (FDH) which showed of the REMs when associated to the PM.
very low homology with FDH from other pathogenic and methy-
lotrophic bacteria as well as with ones from yeasts, fungi and
plants. It was shown that at biofilm conditions level of FDH P.19-053-Tue
mRNA is in the third place among all mRNAs. We have cloned Characterization of L-serine
and expressed in E. coli gene of S. aureus FDH (SayFDH). O-succinyltransferases involved in L-cysteine
Recombinant enzyme was purified and characterized. SauFDH
biosynthesis in prokaryotes
showed one of the highest chemical and thermal stability among
T. Bessonnet
all described formate dehydrogenase. The enzyme has high speci-
Genoscope, Institut de biologie Francois-Jacob, Cea, Evry, France
fic activity and very high Km values with formate and NAD+.
Multiple alignment of FDH amino acid sequences showed that in Until very recently, acetylation was the only known way for acti-
some conservative regions of active site the SauFDH has differ- vating L-serine in the penultimate step of L-cysteine biosynthesis,
ent amino acids residues. We carried out site-directed mutagene- in bacteria and fungi. This reaction is catalyzed by L-serine O-
sis experiments to check the role of differences in stability and acetyl transferases (SAT), encoded by cysE and srpH. These
catalytic activity. Some amino acid changes resulted in improve- enzymes belong to the transferase hexapeptide repeat family.
ment of Km values. To determine structure of SauFDH the crys- Nevertheless, we demonstrated that L-serine is succinylated in
tallization experiments (totally 1728 variants) were performed. Schizosaccharomyces pombe by an enzyme unrelated to the classi-
Crystals of apo- and holo- forms were obtained but its quality cal SAT (Bastard and Perret et al., Nature Chemical Biology,
was not enough for data collection. Optimization of crystalliza- 2017). This unique class of L-serine succinyl-CoA transferases
tion conditions resulted in crystals which were used for data col- (SST), called Cys2, is encoded by metX homologs whereas these
lection. Preliminary structures of apo- and holo- forms of latter are known to only be involved in L-methionine biosynthe-
SauFDH were solved with resolution 2.3 and 2.7 angstrom, sis. The conservation of Cys2 in fungi suggests that the biosyn-
respectively. Parts of this work were supported by Russian thesis of L-cysteine via the novel metabolite O-succinyl L-serine
Science Foundation (grant 16-14-00043) and Russian Foundation is a common treat in this kingdom. In contrast, few Cys2 homo-
for Basic Research (grant 17-04-001662a). logs have been identified in bacteria, particularly in Xanthomon-
adales species. The phylogenetic proximity of these enzymes,
along with the structural similarity of their active site suggest
P.19-051-Wed they share the same function. To support the in vivo role for O-
Insights into the structural mechanisms succinyl-L-serine in Xanthomonadales, we kinetically character-
controlling the dynamic nanodomain ized Cys2 from both Xanthomonas campestris and Frateuria
organization of REMORINs aurantia. Their catalytic efficiency (i.e. kcat/Km) is in favor of the
formation of O-succinyl-L-serine. We also report that their L-
D. Martinez1, A. Legrand2, J. Gronnier3, M. Decossas1,
cysteine synthase (the next enzyme in L-cysteine biosynthesis
P. Gouguet3, M. Berbon1, V. Germain3, A. Grelard1,
pathway) are actually O-succinyl-L-serine sulfhydrylases. This
O. Lambert1, S. Mongrand3, A. Loquet1, B. Habenstein1
1 new metabolite was detected in the metabolome of X. campestris.
CBMN CNRS UMR 5248, Pessac, France, 2LBM CNRS UMR
Together, these results demonstrate the role for this novel
5200, Pessac, France, 3LBM CNRS UMR 5200, Villenave
metabolite in L-cysteine biosynthesis in bacteria, too.
d’Ornon, France
Membrane proteins and lipids are dynamically organized in
domains or compartments, orchestrating important cellular event
such as trafficking, signalling and pathogen response. Structural
and mechanistic principles governing membrane organization are
often uncharacterized because of the challenging nature of the
440 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 441
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Systems biology POSTERS
hosts as harmless commensals, being kept under the control by 2. Hercik, K., Brynda, J., Nencka, R. & Boura, E. (2017) Struc-
the host immune system. One of the key survival strategies of tural basis of Zika virus methyltransferase inhibition by sine-
fungal pathogens is the ability to proliferate in different carbon fungin, Archives of virology. 162, 2091–2096. The work was
dioxide concentrations. CO2 is among the most important gases supported from European Regional Development Fund; OP
for living organisms. In nature, the concentration of CO2 is bal- RDE; Project: “Chemical biology for drugging undruggable tar-
anced by interconversion to hydrogen carbonate however, its gets (ChemBioDrug)” (No. CZ.02.1.01/0.0/0.0/16_019/0000729).
average amount required by organism is much greater than the
amount produced spontaneously from CO2. Therefore, bicarbon-
ate production requires a fine tuned regulation. For this reason, P.19-059-Tue
a highly diverse family of enzymes has evolved that are able to Structural basis of STING activation by its
accelerate the interconversion reaction up to 10 000-fold. The fluorinated agonists
members of this family, carbonic anhydrases (CAs) are able to M. Smola, E. Boura
catalyze reversible hydration of CO2 to bicarbonate. CAs evolved Institute of Organic Chemistry and Biochemistry of the CAS,
in all three domains of life, and are divided into five, evolutionar- Prague, Czech Republic
ily unrelated classes (a, b, c, d, and f) that independently arose
from different precursors during convergent evolution. ß-CAs are STING (stimulator of interferon genes) is a protein with four
present in many pathogenic microorganisms but not in the mam- transmembrane domains localized on the endoplasmatic reticu-
malian hosts and therefore represent possible target for drug lum membrane that is involved in innate immune signaling. After
development. We determined the crystal structure of CA form triggering by cyclo dinucleotides (CDNs) of bacterial or host ori-
Candida albicans (CaNce103) at 2.2 A resolution. CaNce103 gin, it is capable to activate both NF-jB and IRF3 transcription
assembles as a tetramer, with the active site localized at the inter- pathways to induce type I interferons (INF-a and INF-b), which
face between two monomers. At the bottom of substrate pocket, leads to potent anti-bacterial and anti-viral state of organism.
the zinc ion is coordinated by three highly conserved residues Therefore, STING is being investigated as a potential pharma-
Cys78, His133 and Cys136 in addition to water molecule. Activ- cophore for the treatment of viral diseases. A natural endogenous
ity assays of full length and truncated versions of CaNce103 indi- agonist of STING is 20 30 -cyclo-GMP-AMP (20 30 -cGAMP). This
cated that the N-terminal region is indispensable for enzymatic CDN is produced by cytosolic microbial DNA and RNA sensor
activity. This work was supported by grant GA17-08343S from cyclic-GMP-AMP synthase (cGAS). On the basis of 20 30 -cGAMP
the Czech Science Foundation, by project NPU LO1302. and also cyclo-dinucleotides of bacterial origin (c-di-AMP, c-di-
GMP or c-di-IMP), novel STING agonists were designed.
Among the very interesting ones are fluorinated analogs of
P.19-058-Mon CDNs, which exhibit higher potency in induction of type I inter-
Flavivirus methyltransferases as a target in ferons than their non-fluorinated analogs such as fluorinated 30 30 -
cGAMP. Mechanism how the fluorine atoms influence CDNs
antiviral treatment
ability to activate STING is unclear. Aim of our work is to com-
P. Krafcikova, E. Boura
pare structures of human STING in complex with fluorinated
Institute of Organic Chemistry and Biochemistry AS CR,
and non-fluorinated agonist using protein crystallography. To
Flemingovo nam. 2, Prague, Czech Republic, Prague, Czech
this date we have successfully co-crystallized human wild type
Republic
STING with fluorinated 30 30 -cGAMP and 30 30 -c-di-AMP. The
Viruses of the Flaviviridae family are widespread vector-borne protein:ligand structures were solved at 2.8 A and 3 A resolution
pathogens causing large epidemics. Members of the Flaviviridae in space group P 41212. The Project was supported by Project
family consist of a large group of enveloped viruses with a InterBioMed LO1302 from Ministry of Education of the Czech
+RNA genome. Dengue virus (DENV), yellow fever virus Republic, the support of the Academy of Sciences of the Czech
(YFV), tick-borne encephalitis virus (TBEV) and Zika virus Republic (RVO: 61388963) is also acknowledged.
(ZIKV) are all emerging or reemerging pathogens. Their RNA-
dependent RNA polymerase (RdRp) consists of RdRp subunit
Systems biology
and methyltransferase (MTase) subunit that is responsible for N-7
and 20 -O methylation of the viral RNA cap which protects the
RNA from being recognized by host sensors. We have already P.20-001-Mon
shown that the RdRp can be targeted by nucleotide analogs [1] and Regulatory network of Bacillus subtilis
we performed structural analysis of the Zika MTase [2]. Now, we alternative r factors during spore germination
explore the possibility to target different (ZIKV, TBEV, DENV
and outgrowth: kinetic modelling, meta-
and YFV) MTase domains by the same compound. We produced
recombinant MTase domains in E. coli and assayed them enzymat- analysis and experimental verification
ically. A key step was the removal of S-adenosyl-L-methionine O. Ramaniuk, M. Modrak, O. Ruiz Larrabeiti, J. Vohradsky,
(SAM) – the methyl donor that co-purifies with the MTases. We L. Krasny
have already designed several analogs of SAM. Our next aim is the Institute of Microbiology, The Czech Academy of Sciences,
crystallization of MTases with our compound and also with small Prague, Czech Republic
molecule fragments to obtain starting hits for inhibitor design. We r factor is an essential transcription initiation factor in bacteria.
believe that our approach can help the development of efficient A fundamental task is identification of target genes of various r
antivirals against Flaviviruses. factors. Each bacterial species has one primary and several alter-
References native r factors. Primary r factor drives gene expression in a
1. Hercik, K., Kozak, J., Sala, M., Dejmek, M., Hrebabecky, H., bacterial cell under favorable conditions, while alternative r fac-
Zbornikova, E., Smola, M., Ruzek, D., Nencka, R. & Boura, E. tors take control when diverse stresses come. In past years, there
(2017) Adenosine triphosphate analogs can efficiently inhibit the was a burst of sequencing data accumulation based on ChIP-seq
Zika virus RNA-dependent RNA polymerase, Antiviral research. and RNA-seq data. These approaches provide information on
137, 131–133. singular interactions between r factor and its potential targets.
442 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Systems biology
Nevertheless, this information is fragmentary and does not pro- transcriptional response and absolute transcriptional changes are
vide comprehensive view of the gene regulatory network in the relatively small and protein changes at the same time are negligi-
studied organism. A highly useful tool for discovering gene regu- ble. We used TSS’s map and cross-species conservation analysis to
latory networks that complements sequencing approaches is reconstruct transcription control network based on known regula-
kinetic modelling of gene expression. Bacillus subtilis is a widely tors. We concluded that conserved transcriptional regulators are
used Gram-positive model organism containing 18 r factors. In mostly involved in control of metabolic pathways, which are
our previous study we used data of gene expression profiles from reduced in mycoplasmas compared to other Mollicutes. To reveal
14 time points to model gene regulatory network of the primary both adaptation strategy and underlying regulatory network in
r factor – rA – in B. subtilis during spore germination and out- mycoplasmas we performed a combination of –omics studies with
growth, subsequently verifying selected predictions experimen- computational methods. We constructed a quantitative model of
tally. In this study we focused on the regulatory network of gene expression in M. gallisepticum. We identified that M. gallisep-
alternative B. subtilis r factors during spore germination and ticum features unexpectedly high amount of genes that undergo
outgrowth. We used kinetic modelling and meta-analysis to cre- regulation, a substantial amount of housekeeping genes including
ate regulatory network between alternative r factors and their components of translation machinery and glycolytic enzymes
targets. For selected r factors rB, rD, rH, r1, rM we set up require regulation as well. We used sub-diffraction microscopy to
in vitro transcription systems to verify predicted interactions directly quantitate translation efficiency and protein distribution in
experimentally. The outcome is a comprehensive overview of population under different combinations of promoters and transla-
alternative r factors regulatory network in B. subtilis. tion determinants. We identified that M. gallisepticum may feature
high heterogeneity and the protein distribution pattern close to all-
or-nothing. M. gallisepticum demonstrated the ability to retain the
P.20-002-Tue altered proteome state during long-term cultivation on artificial
Measuring interdependence of bacterial medium after the infection. Using genetic tools we identified sev-
optogenetic circuit and microbial growth eral key players (SpxA, YebC) that are involved in support of this
Y. T. Yang, Z. Dou transition of phenotype. We conclude that under physiological
National Tsing Hua University, Hsinchu, Taiwan conditions mycoplasma demonstrate strong ability of regulation
and adaptation and is capable of transition to stable phenotypic
Light can replace the chemical effector for programming on- states (attractors), while the exact mechanism is yet to be discov-
demand gene expression in bacteria and optogenetic bacterial cir- ered. This work was funded by RSF grant 14-24-00159.
cuits with wavelengths ranging from near infrared to UV have been
developed over the past decade. While system biology has always
aimed to decipher complex interactions of biological systems and P.20-004-Mon
identify mathematical models that best depict their behavior. Effect of repeated stress on gene expression in
Recent study reveals that these synthetic genetic circuits can be
the basal conditions in human skeletal muscle
strongly affected by the physiological states of the organism, and
D. Popov1,2, P. Makhnovskii1,2, E. Lysenko1,2, O. Vinogradova1,2
often the growth rate of cell and the level of gene expression are 1
Institute of Biomedical Problems of the RAS, Moscow, Russia,
often intimately intertwined. Characterization of this dependence 2
Lomonosov Moscow State University, Moscow, Russia
is also of practical importance because these synthetic circuits must
be placed under different growth conditions. In this work, we Repeated cellular stress leads to expression of specific proteins
report the measurement of the coupling of optogenetic circuit and which improve physiological function of cell and tissue. This adap-
microbial growth using our recently developed bioreactor array tation is related to transitory changes in gene expression after each
that enables automated, in vivo parallelizable simultaneous moni- acute stress. However, repeated stress may affect the expression of
toring of gene expression and growth. The CcaS-CcaR light sens- many genes during the basal conditions (several days after the last
ing system in Escherichia coli is chosen as a model system for such stress stimulus). Importantly, acute and repeated stress-related
study. The CcaS-CcaR light sensing system consists of membrane changes in gene expression usually are evaluated in cell culture and
bound histidine kinase CcaS and a response regulator CcaR. The in animal models. The goal of our study was to investigate the
interdependence between microbial growth and optogenetic gene effect of repeated stress on changes in the transcriptome in the
expression was confirmed in a growth experiment, varying light basal conditions in human skeletal muscle. We used regular physi-
intensity and three effectors of microbial growth (carbon source, cal exercise (exercise training) as a model of repeated stress. The
oxygenation, and antibiotic drug concentration). Specifically, the transcriptome in the basal conditions was evaluated prior to and
demand of optogenetic circuit for resource usage from the host is after exercise training (8 wk cycling training, 5/wk, 1 h/day) in
measured using light intensity as a tuning parameter. Growth biopsy samples from m. vastus lateralis of 7 males. RNA-seq was
under different carbon sources and oxygenation levels shows the performed by NextSeq 500. As expected 8 wk training increased
gene expression decreases as the growth rate increases. These stud- endurance performance, ADP-stimulated mitochondrial respira-
ies again confirm resource allocation trade off picture, recently tion in muscle fibers, and content of proteins related to mitochon-
proposed by Terence Hwa’ group. drial complexes I-V. Previous studies showed that acute exercise
affects the expression of several hundred genes. Surprisingly, here,
repeated stress changed the expression of 2000 genes in the basal
P.20-003-Wed conditions (48 h after the last exercise) and a half of these genes
The language of the genome – semantics of were down-regulated. Many genes, encoding mitochondrial pro-
the minimal cell organization teins, protein kinases, and transcriptional regulators, were both
V. Govorun up- and down-regulated, while genes, encoding cytokines and
Federal Research and Clinical Center of Physical-Chemical growth factors, were mainly up-regulated. These up- and down-
Medicine of Federal Medical Biological Agency, Moscow, Russia regulated genes were associated with different biological process.
In conclusion, the adaptation of human muscles to repeated stress
Mycoplasmas as the most reduced group of Mollicutes are believed associates not only with the transitory changes in gene expression
to have extremely reduced gene expression control systems along after each acute stress, but also with the marked changes in gene
with the general genome reduction. They demonstrate broad
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 443
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Systems biology POSTERS
expression in the basal conditions. This work was supported by the sequence contains a unique label for each of the TFs. Analysis is
Russian Science Foundation (14-15-00768). performed by qPCR with a pair of primers, common for all the
reporters, and a probe, specific for each TF and complement to the
TF label. It is possible to analyze four TFs with quadruplex qPCR
P.20-005-Tue using probes with different fluorophores. Normalization for the
Identification of novel mechanisms of number of the reporter integrations to the genome could be per-
transcriptional regulation in reduced bacteria formed with gDNA, a pair of primers, common for all the TFs,
using machine learning and high-throughput and the same probe as is used for the mRNA analysis. The system
could be easily modified for the analysis of the activity of the TF
identification of promoters of interest via one-step cloning of the corresponding response ele-
G. Fisunov1, D. Evsyutina2, I. Garanina2, V. Govorun2 ment. It could be scaled up and used for RNA-seq analysis instead
1
Federal Research and Clinical Centre of Physical-Chemical of qPCR. Therefore here we present a useful tool for the routine
Medicine FMBA Russia, Moscow, Russia, 2Federal Research and monitoring of the transcription factor activity in mammalian cells
Clinical Centre of Physical-Chemical Medicine, Moscow, Russia that could be easily modified to meet the needs of a particular
Bacteria of class Mollicutes and in particular mycoplasmas repre- research.
sent good approximation of minimal cell due to the severe reduc-
tion of genome. Previous studies revealed that mycoplasmas
feature very limited repertoire of transcriptional regulators and P.20-007-Mon
poorly react to different stimuli in perturbation models. These Stress causes the formation of a morphologically
findings led to conclusion that the systems of gene expression reg- new type of Mycoplasma hominis
ulation degraded in mycoplasmas along with the general reduc- G. Levina1, O. Barkhatova1, I. Rakovskaya1, T. Gribova2,
tion of genome. We applied a high-throughput method of the I. Butenko2, O. Pobeguts2, M. Levites2, V. Ladigina2,
identification of transcription start sites and the quantitation of G. Fisunov2
their activity with machine learning to construct the quantitative 1
Federal State Budgetary Institution «National Research Centre
model of bacterial promoter on the model of Mycoplasma gal- for Epidemiology and Microbiology named after the honorary
lisepticum. The model was able to predict promoter strength from academician N.F. Gamaleya» of the Ministry of Health of the
its sequence. Further we identified promoters, which predicted Russian Federation, Moscow, Russia, 2Federal Research and
strength deviated from their measured activity. Thus we identified Clinical Center of Physical-Chemical Medicine of Federal Medical
candidate promoters, which activity was putatively regulated. Biological Agency, Moscow, Russia
This group included the targets of few known regulators as well.
Surprisingly we identified numerous regulated promoters. Further Mycoplasma hominis (M. hominis) is the human pathogen belong-
using genetic engineering we identified novel DNA sites and ing to Mollicute class. M. hominis’ genome is extremely reduced
RNA structures, responsible for the activator or repressor effect and contains only 559 protein-encoding genes. In this work we
extracted unique M. hominis (“miniM”) isolates from blood of
on the promoters. We conclude that the gene expression regula-
tion in mycoplasmas is complex. The regulated genes code for patients with bronchial asthma, urogenital infection, from syn-
both stress-response proteins and housekeeping proteins. The lat- ovial fluid of patients with juvenile rheumatoid arthritis and from
ter is of particular interest for synthetic biology since the control the cell culture with gentamicin. In contrast with classic “fried-
egg” colonies they produce very small colonies, exhibit low
of housekeeping genes may be an essential function of cell. This
work was funded by RSF grant 14-24-00159. growth rate and are capable of growing without arginine and glu-
cose. The miniM’s cells have elongated shape and this morphol-
ogy is permanent – we did not observe reverse transition from
P.20-006-Wed miniM to the classic cells (“CC”). We suppose that human
Versatile reporter for multiplex analysis of the organism has a resistant form of M. hominis, which escapes the
immune system. We’ve developed a method to produce the
transcription factor activity in mammalian miniM cells from laboratory strain H34 using stress factors (UV,
cells blood plasma treatment, antibiotics). We identified 346 proteins
A. Khan1,2, A. Zolotarenko2, E. Piruzian2, E. Chekalin2,3, for CC and 291 proteins for miniM with shotgun proteomics
S. Bruskin2 approach and found several proteins (transcription factor yebC,
1
Lomonosov MSU, Moscow, Russia, 2VIGG RAS, Moscow, ABC-transporter, lipoproteins, metabolic proteins and others)
Russia, 3MIPT, Moscow, Russia unique to miniM cells. Quantitative analysis of data by Max-
Transcription factor (TF) activity plays pivotal roles in the regula- Quant has shown that the amount of proteins, which participate
tion of broad spectrum of signaling pathways in cells. Considering in the glycolysis, translation, transcription and replication,
cross-regulation loops between different TFs, it is an important decreases, but levels of lipoproteins (lmp1, p120), elongation fac-
task to perform simultaneous evaluation of the activity of several tors (fusA, IF-Tu), deoA protein and some proteins without
TFs for the analysis of a given signaling pathway. Not only it is a assigned function, increases more than 2-fold. This work was
topical issue for fundamental scientific research, but it also has funded by RSF grant 14-24-00159.
direct applications in screening for drug discovery. Here we present
a multi-reporter system based on qPCR evaluation of the activity
of 4 TFs (NFkB, AP1, STAT, IRF) for monitoring of the inflam-
mation-related signaling. It could be used both for transient trans-
fection of cells or for the lentiviral delivery and contains GFP gene
for the evaluation of the transfection efficiency as well as for the
selection of the reporter-containing cells via FACS-sorting. Each
of four reporters contains a TF response element (3–8 consensus
sites of the TF) followed with a minimal promoter and two consen-
sus sequences separated with a chimeric intron. Second consensus
444 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Synthetic biology
P.20-008-Tue levels”, and “Actin filament-based process” were found only after
the first exposure to acute stress. Consequently, as a result of
Small open reading frames of plants: trends in
adaptation to repeated stress, transcriptome changes in response
evolution and peptide coding capacity to acute stress become much less pronounced, but more specific.
I. Fesenko1, I. Kirov1, A. Knyazev1, A. Mamaeva1, V. Ivanov1, This work was supported by the Russian Science Foundation
V. Govorun1,2 (14-15-00768).
1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Russian Academy of Sciences, Moscow, Russia, 2Federal Research
and Clinical Center of Physical-Chemical Medicine of Federal P.20-011-Tue
Medical Biological Agency, Moscow, Russia Transcriptomic and lipidomic profiling reveals
Genomes of nearly all organisms contain hundreds of thousands a functional interplay between sex steroids
of small open reading frames (sORFs; < 100 codons). However, and growth hormone in the liver
gene annotation algorithms are generally not suited for dealing L. Fernandez-Perez1, B. Guerra1, R. Santana-Farre1, M. Mirecki-
with sORFs due to high false discovery rate and low conserva- Garrido1, I. Garcıa2, J. C. Dıaz-Chico1, A. Flores-Morales3,
tion. Nevertheless, recent reports suggest that sORFs may regu- D. Iglesias-Gato3, M. Dıaz2
late translation of proteins or produce bioactive peptides. 1
University of Las Palmas de Gran Canaria (ULPGC), Las
Although sORFs have been found in some plant species our Palmas de Gran Canaria, Spain, 2University of La Laguna, La
knowledge about sORF function and evolution in plants is Laguna, Spain, 3University of Copenhagen, Copenhagen, Denmark
scarce. Here we used moss, Physcomitrella patens, to address
questions about trends of sORF evolution between evolutionary Gonadal steroids (GS) and GH are critical regulators of body
diverged plant species and peptide coding capacity of sORF. We growth and intermediate metabolism in mammals. The metabolic
identified about 600 000 sORFs with high coding potential in influences of GS and GH deficiency have been well documented
moss genome, 6763 of which were potentially secreted. About 70 in adult men by the developing of chronic illness (e.g., fatty liver,
000 transcribed sORFs were located on mRNAs and lncRNAs. insulin resistance), a phenotype that can be ameliorated by E2/
Using mass spectrometry analysis, we identified hundreds trans- testosterone and GH replacement. The effects of GS on liver
latable sORFs in moss cell and secretome. Intensive evolutionary might be direct through their respective nuclear receptors. Indi-
analysis demonstrated that peptide coding capacity of sORFs rect mechanisms, related to the influence of sexual steroids on
and conservation rate are weakly correlated. We hypothesized the pituitary GH secretion and/or their influence on GHR signal-
that sORFs can be a source of biological novelties used by plant ing pathway in the target tissue, might also play a relevant role
cell for global regulation of proteome and adaptation. This work to regulate liver physiology. The molecular characterization of
was supported by grant № 17-14-01189 from Russian Scientific the hepatic changes induced by GS and how they influence the
Foundation. liver response to GH deserves to be explored. Hypothyroidism-
hypogonadism is accompanied by systemic and hepatic metabolic
disturbances with features that mimic deficiencies in GH and GS.
P.20-010-Mon In this study, the analyses of lipid and transcriptional profiles in
Specificity of transcriptome response to acute hypothyroid-orchidectomized rats were combined to obtain com-
prehensive information on the GS and GH crosstalk in liver.
stress in human skeletal muscle depends on
Testosterone (T) activated, among others, a metabolic transcrip-
adaptation to repeated stress tional program linked to glucose and fatty acids and lipid class
P. Makhnovskii1,2, D. Popov1,2, E. Lysenko1,2, O. Vinogradova1,2 metabolism. The overall impact of T on hepatic lipid content and
1
Institute of Biomedical Problems of the RAS, Moscow, Russia, transcriptome differed from the effects of E2. The combined
2
Lomonosov Moscow State University, Moscow, Russia administration of T and GH revealed biological processes related
Acute stress acting on the cell changes the expression of a specific to lipid biosynthesis, oxidation-reduction, unsaturated and long-
set of genes. However, the number of stress-dependent genes may chain fatty acid metabolism. GH showed permissive, additive, or
significantly differ after the first and subsequent stress actions. antagonistic effects on T actions on hepatic lipid content. Pro-
The aim of this work was to investigate the specificity of tran- tein-protein interactions analysis revealed a close interplay among
scriptome response to acute stress in human skeletal muscle proteins which are central in the metabolism of steroid and fatty
depending on the level of adaptation to repeated stress. As acute acids. These findings highlight the impact of GS-GH interplay on
stress, we used contractile activity: one-legged knee extension for liver metabolism which is relevant for physiological and thera-
1 h. This test was performed before and after adaptation to peutic roles of these hormones in human.
repeated stress (8 wk cycling training, 5/wk, 1 h/day). Transcrip-
tome was investigated in biopsy samples from m. vastus lateralis Synthetic biology
of 7 males before and at 1 h and 4 h after acute stress (one-
legged exercise). RNA-seq was performed using NextSeq 500
(Illumina). In nonadapted muscle the expression of a large num- P.21-001-Mon
ber of genes was changed at 1 h and 4 h after acute stress (about Peculiarities of the stereospecific interactions
400 and 900 genes, respectively). After adaptation to repeated of catalytic antibodies A5 and A21 with aryl-
stress, the acute stress induced significantly less number of DEG phosphonate X
(differentially expressed genes): about 100 genes for 1 h and 4 h. S. Pipiya1, Y. Mokrushina1, A. Golovin2, I. Smirnov1,
In both cases the increase in expression of the genes regulating A. Gabibov1
mitochondrial biogenesis, angiogenesis, carbohydrate and lipid 1
Shemyakin&Ovchinnikov Institute of Bioorganic chemistry RAS,
metabolism, i.e. biological processes specific for this stress, was Moscow, Russia, 2Lomonosov Moscow State University, Faculty of
found. Reduced amount of DEG in the adapted muscle was Bioengineering and Bioinformatics, Moscow, Russia
associated with a decrease in nonspecific response to stress: the
GO-terms, such as “Cellular response to biotic stimulus”, “Cellu- Specificity of biochemical reactions is the cutting-edge field of the
lar response to the external stimulus”, “Response to oxygen modern biochemistry and biotechnology. Many biochemical
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 445
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Computational biology POSTERS
446 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Computational biology
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 447
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Computational biology POSTERS
448 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Computational biology
modelling of the binding mode of HSP90 and AhR guided by of biological systems and large number of variables usually cou-
experimental data, umbrella sampling of the binding modes and pled with the relatively low number of observations can make
attempting AhR PAS-B expression in insect cells. Dealing with analyses quite challenging. What are some useful techniques that
the large system also led to the development of a tool to aid in help deal with high-dimensional data effectively or what statisti-
umbrella sampling preparation. By being able to know how cal or modeling methods perform well on high-dimensional data
ligands bind, how the complex translocates and when the binding of complex biological samples? Specifically, we will present a
with ARNT occurs, we would be better prepared for thoughtfully great potential of Raman spectroscopy for detection, identifica-
designing compounds. This work explores the questions – What tion and subsequent analysis of traces of body fluids like blood,
actually causes the translocation of the complex? What role do saliva, semen etc. found at a crime scene using some novel
agonists and antagonists play in the translocation? approaches. In addition, Raman spectroscopy is capable to pro-
vide information about the changes in biochemical composition
and molecular structure of the body fluids caused by a disease.
P.22-008-Tue The application of Raman spectroscopy of blood for Alzheimer’s
Development of suitable computational Disease diagnostics will be shown.
methods for intrinsically disordered proteins
(IDPs) as medicinal chemistry targets P.22-010-Mon
J. V. de Souza Cunha1, F. de Paula Sabanes Zariquiey2,
A. K. Bronowska1
CaverDock: a novel method for the analysis of
1
School of Chemistry, Newcastle University, Newcastle upon Tyne, the ligand transport processes based on
United Kingdom, 2Scho, Newcastle upon Tyne, United Kingdom iterative docking
O. Vavra1, J. Filipovic2, S. Marques1, J. Plhak2, D. Bednar1,
IDPs, or Intrinsically Disordered Proteins, are a class of proteins
J. Brezovsky1, L. Matyska2, J. Damborsky1
which have only partial tertiary structure or no tertiary structure 1
Loschmidt Laboratories, Brno, Czech Republic, 2Institute of
at all. This characteristic gives this class of proteins a high level
Computer Science, Brno, Czech Republic
of molecular plasticity. Also, this natural flexibility creates a high
level instability, creating a high level of aggregation and Protein tunnels, ligand access pathways, play an important role
oligomerization to attain thermodynamic stability. The event of in the function, stability or substrate specificity of enzymes. The
oligomerization and aggregation is related to several pathological tunnels enable the transport of ligands from the outside environ-
conditions including Neurodegenerative diseases such as Parkin- ment into the buried active site inside of the protein. Here we
son’s disease, making this set of molecules important targets for present CaverDock, a software implementing a novel method for
the development of small molecules for therapeutic intervention. the analysis of the ligand transport processes. The input for
To develop therapeutical drugs, one of the main methods consists CaverDock calculations requires tunnel geometry obtained from
in the use of computational tools, such as Molecular Dynamics CAVER and setup for molecular docking. Our method lies in
softwares, which uses force fields to characterize protein models. between of geometrical approaches and molecular dynamics sim-
These databases are mainly biased to structured proteins, creat- ulations. Contrary to geometrical methods, it provides an evalua-
ing a lack of proper parameters for IDPs. So, a iterative MC fit- tion of chemical forces and better description of the studied
ting code had been developed. Using GROMACS, the dihedral system. On the other hand, it is not as setup-intensive and com-
parameters for 8 disorder inducing residues (A, R, Q, E, G, K, P putationally demanding as the methods based on molecular
and S) are randomly sampled and a comparison between experi- dynamics. The method uses molecular docking to iteratively place
mental and theoretical SAXS are assessed to check the improve- the ligand along the tunnel with preservation of continuous
ment in the simulation by the changes in the parameters. A movement of the ligand and minimization of its binding energy.
second approach was studying the effect of the TIP3P model on The output of the calculation is the ligand trajectory and the
the ensemble generated, as we observe a improvement on sam- energy profile of the transport process. CaverDock uses a modi-
pling for the proteins at hand. fied version of the algorithm from AutoDock Vina for ligand
placement and implements a parallel heuristic algorithm to search
the space of possible trajectories. The duration of the simulations
P.22-009-Wed takes from several minutes to few hours, allowing parallelization
Raman spectroscopy and multidimensional for virtual screening of a large pool of molecules. We demon-
data analysis of biological fluids strate CaverDock usability by i) comparison of the results with
L. Halamkova1, J. Halamek2, I. K. Lednev1 other available tools for the simulation of ligand unbinding, ii)
1
SUNY at Albany, Albany, NY, United States of America, analysis of the ligand trajectories in different tunnels of wild type
2
SUNY at Albany, Albany and engineered proteins and iii) calculation of the energetic pro-
files for ensembles of substrates. The software CaverDock is
Raman spectroscopy contributes to the various application areas freely available at https://loschmidt.chemi.muni.cz/caverdock/.
and has advanced in recent years becoming increasingly impor-
tant for different application areas. This is mainly due to techni-
cal improvement, the versatility of sampling methods and the
availability of advanced methods that helps in the data analysis.
Over the last years, advances in the instrumental design of
Raman spectroscopy overcame the problem with undesired fluo-
rescence, low sensitivity and reproducibility. Raman spectro-
scopic data gives the vibrational characteristics of analyte, which
can be understood as “fingerprint” type of information. The
impact of chemometrics to the application of Raman spec-
troscopy for forensic purposes and medical diagnostics directly
from body fluids will be discussed in this contribution. Raman
data is characterized by high dimensionality. The high complexity
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 449
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Computational biology POSTERS
450 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Computational biology
P.22-014-Tue and typical objects are selected, they are used to estimate the
statistics of single cell sizes. In the second step, the area of the
Type II toxin-antitoxin systems for
raw image exceeding the chosen threshold is divided by the typi-
metagenomic studies cal cell size this way providing the estimate of the cell count in a
K. Klimina1,2, E. Poluektova1, A. Kudryavtseva3, A. Kasianov1, given sample. The proposed approach is an excellent solution
V. Danilenko1 when direct count of cells by flow cytometry is not available, like
1
Vavilov Institute of General Genetics Russian Academy of analyzing the fractions of differentially stained subpopulations in
Sciences, Moscow, Russia, Moscow 119991, Russia, 2Federal tissue samples and bacterial biofilms. The performance of the
Research and Clinical Center of Physical-Chemical Medicine of suggested tool has been confirmed using examples of eukaryotic
Federal Medical Biological Agency, Moscow, Russia, 3Engelhardt and bacterial cells with different morphology. The software is
Institute of Molecular Biology, Russian Academy of Sciences, entitled BioFilmAnalyzer, designed for Windows 32 bit architec-
Moscow, Russia, Moscow, Russia ture and can be freely downloaded by the following link https://
The study of the microorganism diversity in human gut micro- bitbucket.org/rogex/biofilmanalyzer/downloads/, utilized and
biota (GM) is actual task. More detailed studies revealed that a redistributed. The financial support of this work was provided by
person’s metagenome depends on the country, urban or rural the Ministry of Education and Science of the Russian Federation
dwelling, age group, diet preferences, general health state or vari- (assignment 2.5475.2017/6.7) and Program of Competitive Devel-
ous diseases. Today two main approaches are used to character- opment of Kazan Federal University
ize taxonomic diversity: whole metagenome shotgun analysis and
analysis of PCR amplicons from the 16S rRNA gene. The most
comprehensive information is provided by metagenomic analysis. P.22-016-Mon
Most of similar software tools based on the presence of marker Potential binding site of two noninhibitory
genes like MetaPhlAn or MG-RAST have resolution at most at chaperons of human glucocerebrosidase
the species level. These approaches based on gene copy number G. N. Rychkov1,2, A. K. Emelianov1, S. N. Pchelina1
variation, require very deep metagenome sequencing and long 1
Petersburg Nuclear Physics Institute, NRC Kurchatov Institute,
analysis. We suppose that toxin-antitoxin systems (TAS) can pro- Gatchina, Russia, 2Peter the Great St. Petersburg Polytechnic
vide additional markers for metagenomic analysis of species and University, Saint-Petersburg, Russia
strain diversity. The objective of this work was to use polymor-
Gaucher disease (GD) is caused by mutations in the glucocere-
phism in TA genes to analyze representation of specific species
brosidase gene, leading to misfolding of the enzyme and substan-
and strains in human GM. TAS are present in genomes of the
tial reduction of its activity in lysosomes. N370S amino acid
majority of bacteria and archaea. Previously we have shown that
substitution in glucocerebrosidase (GBase) is frequent among
in Bifidobacterium and Lactobacillus sp. the TAS may be used as
GD patients. In contrast to enzyme replacement and substrate
functional biomarkers to differentiate these groups of bacteria at
reduction therapies, development of small chemical chaperons
the species and strain levels. We expanded the database of TAS
which increase enzyme stability and catalytic activity seems to be
of RelBE and MazEF superfamily and included bacteria inhabit-
a promising approach for disease treatment. Recently, two chemi-
ing the GM and shown that distribution of toxins and antitoxin
cal compounds NCGC00241607 [1] and NCGC00188758 [2] were
genes in these bacteria are species and strain specific too and
found to increase an activity of mutant N370S GBase (Aflaki
each strain is associated with the specific set of genetic markers.
et al. Sci. trans. med., 2014, 6(240), 240ra73-240ra73., Aflaki
Thus we created the pipeline TAGMA and tested it on metagen-
et al. J. Neurosci., 2016, 36(28), 7441–7452). Here, we used
omes samples from healthy people. The results of analysis were
molecular modeling approach to identify potential binding site of
compared with results of well-known programs PhymmBL and
compounds [1] and [2] on a surface of mutant N370S GBase.
MetaPhlAn. Based on a limited number of well selected markers
Among four revealed binding sites we chose the one located near
TAGMA displays good time performance taking several hours to
N370 residue. N370S amino acid substitution leads to formation
analyze a single metagenome as compared with several days
of a cavity on the molecular surface of the enzyme. Molecular
required for MetaPhlan2 or PhymmBL.
docking showed that both compounds: i) can partially embed
their chemical groups into the cavity ([1] benzene, [2] pyra-
P.22-015-Wed zolopyrimidine), ii) can form up to three hydrogen bonds with
amino acid residues of the enzyme. Obtained complexes of two
Quantification of cell cultures by combination
compounds remained stable during 100 ns molecular dynamics
of object counting and statistical analysis simulations in explicit water. The next stage of our work assumes
methods utilization of revealed binding site for virtual screening of high
M. Bogachev1,2, V. Volkov1, O. Markelov1, E. Trizna2, affinity chemical compounds, having potential to act as molecu-
D. Baidamshina2, A. Kayumov2 lar chaperons of mutant N370S GBase. The results of the work
1
St. Petersburg Electrotechnical University, St. Petersburg, Russia, were obtained using computational resources of Peter the Great
2
Institute of Fundamental Medicine and Biology, Kazan Federal Sainte-Petersburg Polytechnic University Supercomputing Center
University, Kazan, Russia (www.scc.spbstu.ru). The work was carried out with funds from
We present a universal tool that implements an original semi- the Russian Science Foundation (project No. 17-75-20159).
automatic cell count approach suitable for while not limited to
the analysis of microscopic images of bacterial cells and tissue
samples, bacterial colonies, histological images. Our approach
consists of two consecutive steps. In the first step, objects of
interest, e. g. single cells, are selected from representative sample
images. This selection is performed using multi-threshold adjust-
ment followed by size histogram analysis. Each local size his-
togram maximum corresponds to certain typical object size for a
given threshold value. Once the appropriate maximum is found
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 451
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Computational biology POSTERS
452 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Computational biology
P.22-021-Wed and magnetic circular dichroism spectra agree well with the exper-
imental ones. For the absorption and CD spectra lines of the
Free energy calculation of transition state
transition to each exciton state were non-empirically broadened
analogues binding by carboxypeptidase B taking into account electron-phonon interaction with the protein
mutants environment. We used conventional approach in which system
D. Podshivalov1,2,3, V. Akparov4, V. Timofeev1,3, I. Kuranova1,3, was separated on the quantum subsystem (chromophores) and
I. Khaliullin2,5, V. Svedas2, T. Rakitina3,6 thermal bath (protein). Hamiltonian of system-bath interaction
1
Shubnikov Institute of Crystallography of Federal Scientific was constructed based on the exciton wave functions of bacteri-
Research Centre “Crystallography and Photonics”, Russian ochlorophyll system and normal modes of the protein. The calcu-
Academy of Sciences, Moscow, Russia, 2Belozersky Institute of lated spectral density of system-bath interaction was used for the
Physicochemical Biology, Lomonosov Moscow State University, calculation of dynamics of excitation energy transfer and exciton
Moscow, Russia, 3National Research Centre “Kurchatov Institute”, states population. We used a theory of quantum dissipative
Moscow, Russia, 4State Research Institute for Genetics and dynamics with non-equilibrium thermal bath which was previ-
Selection of Industrial Microorganisms (GosNIIgenetika), Moscow, ously applied by our group to the process of primary charge sepa-
Russia, 5Moscow Institute of Physics and technology, Dolgoprudny, ration in the reaction center of Rba. sphaeroides.
Russia, 6Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
of the Russian Academy of Sciences, Moscow, Russia
P.22-023-Tue
Carboxypeptidase B (CPB) has a high specificity for positively
charged C-terminal amino acid residues. CPB is involved in the Identification of target-specific autophagy
process of insulin production, so the study of CPB has both sci- modulators for therapeutic intervention in
entific and practical significance. This study is devoted to the cancer
investigation of a number of CPB mutations by computer simula- W. Fewell1, L. Venables1, O. Tastan-Bishop2, T. Koekemoer1, M.
tion methods. The calculation of the free binding energy by the van de Venter1
1
MM_PB/GBSA method implemented in the Amber14 program Department of Biochemistry and Microbiology, Nelson Mandela
was used. The analogues of the transition state for the residues University, Port Elizabeth, South Africa, 2Department of
of phenylalanine and arginine of N-sulfamoyl-L-phenylalanine Biochemistry, Rhodes University, Grahamstown, South Africa
(sphe) and N-sulfamoyl-L-arginine (sarg), respectively, were used.
Modulating autophagy using small-molecule inhibitors or activa-
Charges for inhibitor molecules were determined by QM opti-
tors represents one of the most promising therapeutic strategies in
mization in R.E.D. tools. Parameters for atoms are selected from
the fight against cancer and various major human diseases. Until
the GAFF force field. The protein and mutant molecules are
now only a limited number of pharmacological agents suited for
parametrized in the FF14SB force field. As new, there are four
this purpose have been identified, hence restricting therapeutic
single and two double mutations of CPB: L203Q, A250N,
exploitation of autophagy. Given the considerable progress made
A250S, T268V, T268N, S207G G243N, S207G G243S. For each
in the elucidation of crystallographic structures of components
of the mutants in the complex, binding energies with sphe and
core to autophagy regulation, the present study employed several
sarg were calculated. According to the results of the work, it is
structure-based drug discovery techniques and computational
shown that mutants A250S and S207G G243N are the most
approaches to identify new target-specific autophagy modulators.
promising for further research. This work has been carried out
The aim of the present study was to identify potent inhibitors of
using computing resources of the federal collective usage center
mammalian target of rapamycin complex 1 (mTORC1) as lead
Complex for Simulation and Data Processing for Mega-science
compounds for the development of next-generation autophagy
Facilities at NRC “Kurchatov Institute” and supported by the
modulators with potential clinical utility. Structure-based virtual
Russian Science Foundation (Project 17-14-01256).
screening (SBVS) was employed for mTORC1 and the South Afri-
can Natural Compound Database (SANCDB) selected for screen-
P.22-022-Mon ing. A total of four hit compounds were identified from virtual
screening and further analysed for their autophagy-inducing
Protein environment influence on the energy
capacity using in vitro cell-based systems. Autophagy activity was
transfer and quantum dynamic in LH1-RC monitored by acridine orange (AO), monodansylcadaverine
complex of Thermochromatium tepidum (MDC), LC3B antibody detection, which identified promising
I. Glebov, M. Kozlov autophagy-modulating effects for selected hit compounds. In con-
M. V. Lomonosov Moscow State University, Moscow, Russia clusion, the present study provides a firm foundation for contin-
Theoretical description of pigment-pigment and pigment-protein ued experimental analysis regarding the autophagy-modulating
interaction is a question of high importance for the understanding effects of candidate compounds specifically targeting mTORC1.
of high efficiency of the photosynthetic processes. Pigment-pig-
ment interaction in the supramolecular chromophore-protein
P.22-024-Wed
complexes provide an effective light absorption and selective
interaction with the protein environment ensures excitation energy Prediction of the binding modes of
transfer to the reaction centers where charge separation process antidepressants in the active-site gorge of
occurs. In the current work we report the calculation of spectral acetylcholinesterase or butyrylcholinesterase:
properties of circular LH1-RC complexes of purple bacteria Ther- a protein–ligand docking and interaction
mochromatium tepidum and rates of excitation energy transfer and
profiling study
exciton relaxation. To calculate stationary spectra we used
O. Dalmizrak, K. Terali, H. Ogus, N. Ozer
method of exciton Hamiltonians with transition energies calcu-
Department of Medical Biochemistry, Faculty of Medicine, Near
lated by CASSCF/XMCQDPT2 method and exciton couplings
East University, Nicosia, Mersin 10, Turkey
calculated by TrCAMM decomposition. To take into account the
site heterogeneity we carried out a separate calculation for each of Categorized into diverse classes of drugs with somewhat different
the bacteriochlorophyll. Calculated absorption, circular dichroism mechanisms of action, antidepressants are used by many clinicians
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural bioinformatics POSTERS
worldwide in the treatment of major depressive disorder. The abil- is valuable for increasing our knowledge concerning the use of
ity of antidepressants to readily penetrate the blood–brain barrier chitin deacetylases for the enzymatic conversion of chitin to chi-
means that they may interfere with a number of neurobiochemical tosan, as the currently used chemical procedure is toxic for the
processes. Cholinesterases play multiple roles in the brain and environment. Despite the reduced sequences identity for investi-
periphery, carrying serious implications for human health and dis- gated chitin deacetylases, there are amino acids belonging to their
ease. In this study, a tricyclic antidepressant (amitriptyline) and active sites that are strongly conserved reflecting the common
two selective serotonin reuptake inhibitors (fluoxetine and sertra- biological function of these enzymes. Furthermore, the catalytic
line) were separately docked in the active-site gorge of acetyl- cavities of investigates enzymes reveal almost similar electrostatic
cholinesterase or butyrylcholinesterase based on kinetic properties, all of them being negatively charged. There are some
measurements previously taken by our laboratory and other local properties that are distinct for the bacterial and fungal
groups. Redocking calculations with the bona-fide inhibitors hupr- chitin deacetylases. The hydrophobicity profiles of the catalytic
ine W and tacrine were also performed to evaluate the power of the cavities of fungal CDAs are distinct by comparison to bacterial
methodology to correctly recognize the active-site gorge of choli- CDAs, fungal CDAs revealing a local region with higher
nesterases and reliably predict the experimentally determined struc- hydrophobicity that seems to be important for the catalytic effi-
tures of cholinesterase–inhibitor complexes. Docking simulations ciency. The loops surrounding the catalytic cavities expose dis-
and subsequent calculations of noncovalent interactions between tinct flexibility and hydrophilicity indexes. These local variations
the protein and the ligand revealed that each antidepressant could could be responsible of the distinct catalytic efficiencies of the
be accommodated well in the active-site gorge of the tested choli- investigated enzymes.
nesterase and that they necessarily interact with at least one of the
several aromatic residues from the choline-binding subsite in addi-
tion to key residues from various subsites other than the choline- P.23-002-Tue
binding subsite (such as the peripheral anionic site or the esteratic Proteogenomics of ADAR-mediated RNA
subsite) depending on the orientation of the drug in the active-site editing in mouse brain
gorge. These findings highlight the increased likelihood of acquired A. Kliuchnikova1,2, K. Kuznetsova1, L. Levitsky3, D. Karpov1,4,
cholinesterase deficiency due to the long-term use of antidepres- M. Ivanov3,5, M. Gorshkov3,5, S. Moshkovskii1,2
sants and may also facilitate the design of new reversible cholines- 1
Institute of Biomedical Chemistry (IBMC), Moscow, Russia,
terase inhibitors as treatments for Alzheimer disease. 2
Pirogov Russian state medical university, Moscow, Russia,
3
Institute of Energy Problems of Chemical Physics, Moscow,
Russia, 4Engelhardt Institute of Molecular Biology, Moscow,
P.22-025-Mon Russia, 5Moscow Institute of Physics and Technology,
Development of a new classification approach Dolgoprudny, Russia
for detecting the heterogeneity in cell
Adenosine-to-Inosine (A-to-I) editing is a posttranscriptional
population modification of RNA molecule catalyzed by ADAR enzymes. It
S. Aburatani1, T. Tamura2 occurs mainly in neural tissue, where, as a result of the modifica-
1
National Institute of Advanced Industrial Science and Technology, tion, adenosine is converted to inosine in particular sites of
Tokyo, Japan, 2National Institute of Advanced Industrial Science RNA, some of them code amino acid substitutions. We present a
and Technology, Sapporo, Japan bioinformatic study of this phenomenon on publicly available
Biological data is considered to compose heterogeneous popula- deep shotgun proteomes of brain regions and acutely isolated cell
tions in usual. To clarify the mechanism in the living cell by com- cultures of C57BL/6 mice using proteogenomic approach. The
putational approach, dividing such heterogeneous data is method includes database generation for proteomics search of
important. In this study, we developed a new classification algo- edited sites based on specialized genome-wide databases of RNA
rithm based on Latent Profile Analysis (LPA) combined with Con- editing events. All datasets were searched using X!Tandem soft-
firmatory Factor Analysis (CFA) to divide mixed data into classes, ware with a group-specific false discovery rate for edited peptides
depending on their heterogeneity. In general cluster analysis, the with MP Score post search validation. For data filtering the
number of measured points is a constraint, and thereby the data Open search strategy was used. In total, 14 edited proteins were
must be classified into fewer groups than the number of samples. identified in 10 proteins, such as Gria2, Gria3, Gria4, Grm4, neu-
By our newly developed method, the measured data can be divided ral proteins Flna, Flnb, Cyfip2, Cadps and Ube2o. Four identi-
into groups depending on their latent effects, without constraints. fied edited proteins belonged to AMPA glutamate receptor
Our method is useful to clarify all types of omics data, including complex which played a significant role in excitatory synaptic
transcriptome, proteome and metabolic information. transmission. Identification of RNA editing in these proteins was
in good agreement with background works. The signal from pep-
tides resulted from A-to-I editing was strongest in early stages of
Structural bioinformatics development such as young cell culture of microglia and oligo-
dendrocytes. Therefore, editing can play a significant role in for-
P.23-001-Mon mation of nervous system. It was also noted that the neurons
Structural bioinformatics characterization of such as cortical and cerebellar granule neurons, are subjected to
editing more than glial cells such as astrocytes and oligodendro-
chitin deacetylases secreted by marine
cytes. The results presented above have a good correspondence
organisms with the literature data. Interestingly, both the edited form of the
D. L. Roman1,2, M. Roman1,2, V. Ostafe1,2, A. Isvoran1,2 peptide and the wild-type peptide can be presented in one sam-
1
West University of Timisoara, Timisoara, Romania, 2Advanced ple, which requires further research. Thus, it was shown that a
Environmental Research Laboratories, Timisoara, Romania proteogenomic approach is a potential tool for the analysis of
The purpose of this work is to use structural bioinformatics tools RNA editing at the shotgun proteome level.
to characterize and compare the structures of chitin deacetylases
(CDAs) belonging to fungi and marine bacteria. This information
454 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural bioinformatics
P.23-003-Wed P.23-005-Tue
Selection of bifunctional tyrosyl-DNA A machine learning approach towards
phosphodiesterase 1 inhibitors through sequence based RNA binding protein sites
molecular modeling prediction in human proteome
I. Gushchina1, V. Svedas 1,2
, D. Nilov2 A. Agarwal, S. Sivanandan, S. Mukherjee, N. Chandran, R.
1
Lomonosov Moscow State University, Faculty of Bioengineering P. Bahadur
and Bioinformatics, Moscow, Russia, 2Lomonosov Moscow State Indian Institute of Technology Kharagpur, Kharagpur, India
University, Belozersky Institute of Physicochemical Biology,
The availability of human genome sequence and expansion of
Moscow, Russia
structural data in the Protein Data Bank (PDB) has transformed
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a DNA repair the biomedical research over the past decade. Analysis of the
enzyme and a promising target for anticancer treatment. It cleaves complete human genome sequence has thus far led to the identifi-
chemotherapy-induced stalled topoisomerase-DNA complexes, cation of approximately 20,687 protein-coding genes, although
where a catalytic tyrosine is linked to the 30 -terminal phosphate, the annotation still continues to be refined. RNA-Protein interac-
thus maintaining the DNA structure in cancer cells. There are sev- tions play crucial roles in driving the cellular machinery as evi-
eral compounds known to suppress TDP1 activity, but their inhibi- dent from their active participation in regulation of gene
tory mechanisms and specific interactions are still to be uncovered. expression, protein synthesis, mRNA processing, mRNA assem-
We identified two binding cavities in the active site of the con- bly, ribosome function and eukaryotic spliceosomes. RNA bind-
structed molecular model of human TDP1: one for the phosphoty- ing proteins (RBPs) are known to regulate all aspects of RNA
rosine and the second for the oligonucleotide moiety of the biogenesis and various post-transcriptional gene regulation pro-
substrate. The binding site of the first (from the 30 -terminus) phos- cesses and their dysfunction can lead to several diseases ranging
phate group of the substrate is located in the phosphotyrosine cav- from neurological disorders to cancer. Thus, accurate prediction
ity and comprises residues Lys265, Asn283, Lys495, Asn516, as of RNA-binding residues (RBRs) in proteins using innovative
well as a conserved water molecule. The second phosphate group computational techniques is indispensable to comprehend the
binds in the oligonucleotide cavity forming interaction with time consuming and cost-ineffective experimental methods and to
Ser400, Ser518, and two conserved water molecules. Virtual screen- apprehend the functional implications of specific protein-RNA
ing was performed to identify bifunctional inhibitors targeted interactions, to perform site-directed mutagenesis and to develop
towards both phosphate binding sites and able to form specific novel targeted drug therapies. We have thereby, developed a
hydrogen bonds with the above-mentioned residues and water method to identify the RBRs from a given protein sequence
molecules. Since sulfo group is a structural analogue and isostere employing a machine learning approach, using global and local
of the phosphate group, we retrieved sulfonates from the ZINC sequence based features derived from known crystal structures of
database (2227 compounds) and docked them into the TDP1 active protein-RNA complexes deposited in the PDB. A Random For-
site using the Lead Finder software. Docking poses were then fil- est based classifier is used for the prediction. The analysis of the
tered using an in-house script to identify compounds capable of results on the training dataset using leave-one-out cross-valida-
forming the specified interactions. Several potential TDP1 inhibi- tion experiments gave an accuracy of 98.4%, specificity of 99%,
tors containing two sulfo groups joined together via a linker (e.g. sensitivity of 72.7% and MCC of 0.82, performing better than
dimesna) were selected for experimental evaluation. The flexible few existing methods. Our prediction model will be further
linker allowed functional substituents to occupy both the phospho- extended to validate the known RBPs and prepare a catalogue of
tyrosine and oligonucleotide cavities and form hydrogen-bonding RBRs in entire human proteome. An online accessible RBR pre-
networks with the phosphate binding sites. The work was sup- diction server is under development.
ported by a grant of the Russian Foundation for Basic Research
(18-315-00389).
P.23-006-Wed
Investigating the heterogeneity of pathogenic
P.23-004-Mon bacterial genomes using computational
An automatically refreshed public list of approaches
amyloid and pre-amyloid structures from the A. Shelenkov, D. Shagin
PDB Central Research Scientific Institute of Epidemiology, Moscow,
K. Takacs, B. Varga, V. Grolmusz Russia
E€
otv€
os University, Budapest, Hungary
Bacterial populations are known to be heterogeneous, which can
The Protein Data Bank is the most important resource of protein provide them with evolutionary advantages during changes of
structural information today, containing 130 000 entries. The their environmental conditions, including development of resis-
proper mathematical analysis of this dataset may yield significant tance to antibiotics. In some cases, as it has been already shown
biochemical results. In the present work we constructed the PDB for Mycobacterium tuberculosis isolates, heterogeneity exists at
Amyloid List, an automatically updated list of PDB entries, the genomic level, so that significant variations can be revealed
which contain amyloid structures, or substructures, which have even within a bacterial population seeded by a single cell. Cur-
the potential of becoming amyloids. Our algorithm examines the rently, next generation sequencing (NGS) is extensively used for
geometrical properties of the polypeptide chains of the PDB defining the structures of bacterial genomes, so it is very impor-
entries, and finds parallel beta-sheets, satisfying some strict addi- tant to ensure that NGS is able to identify minor clonal variants
tional properties. The resulting list of PDB entries is publicly in population, which should be distinguished from sequencing
available at https://pitgroup.org/amyloid. errors. Moreover, it is possible that in some cases the output of
NGS data assembler software would represent an “averaged”
genome sequence consisting of a mixture of several different bac-
terial genomes. Obviously, these issues complicate further analy-
sis of such genomes. We have developed a heterogeneity measure
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 455
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural bioinformatics POSTERS
456 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural bioinformatics
ageing. The species with this ontogenetic property include mam- P.23-011-Tue
mals with a greater lifespan than could be expected from their body
Application of a generic platform for the
size: mole rats and primates (including the naked mole rat, Damar-
aland mole rat, capuchin monkeys, gibbons, western gorilla, discovery and optimization of bio-active
bonobo, chimpanzee, human). The list of lost mouse genes includes molecules
Smpd5, Spint5, Ttc41, Wap, and 2310003L06Rik expressed in the G. Nicolaes1, K. Wichapong1, B. Zarzycka1, G. Vriend2,
tongue, and certain vomeronasal and olfactory receptor genes. T. Seijkens3, T. Hackeng1, C. Weber4, W. Mulder3,
Overall, the revealed mouse genes demonstrate specific expression O. Soehnlein4, E. Lutgens3
1
in reproduction-associated tissues, which agrees with the Williams’ Cardiovascular Research Institute Maastricht, Maastricht,
hypothesis concerning the reallocation of physiological recourses. Netherlands, 2RadboudUMC, Nijmegen, Netherlands, 3Academic
The loss of some revealed vomeronasal and olfactory receptor Medical Center, Amsterdam, Netherlands, 4Institute for
genes in human and naked mole rat conforms to their special Cardiovascular Prevention (IPEK), Ludwig-Maximilians-
anatomical features. For this data, we suggest that the loss of cer- University, Munich, Germany
tain genes in evolution is a determinant of lifespan elongation and In today’s molecular medicine, information exchange between
ageing deceleration including neoteny. The research was supported experimental and computational approaches is vital for the
by the Russian Science Foundation, pr.14-50-00150. rational study of clinically relevant proteins. The structural
knowledge of such proteins provides the rational context that not
only leads to a more profound understanding of the activities of
P.23-010-Mon
such proteins, but also allows exploitation of such knowledge to
Bioinformatics analysis of a transgenic provide novel means of diagnosis or therapy. We have built a
personalized murine model of refractory generic platform that combines experimental approaches (i.e.
epilepsy basic biochemistry, molecular biology, peptide chemistry, surface
V. Sukhorukov1, V. Kalmykov1, A. Sharkov1,2, P. Kusov1,3 plasmon resonance and ITC) with structural bioinformatics
1
Institute of Gene Biology, Russian Academy of Sciences, Moscow, approaches (docking, molecular modeling, virtual ligand screen-
Russia, 2Research and Clinical Institute for Pediatrics at the ing, molecular dynamics simulations and free energy calcula-
Pirogov Russian National Research Medical University, Moscow, tions). With this platform, a team of experimentalists and
Russia, 3Skolkovo Institute of Science and Technology, Moscow, bioinformaticians jointly address common research questions and
Russia adhere to a structure-driven approach to guide and rationalize
functional work. This allows the identification and optimization
GRIN2A is a protein coding gene, which encoded NR2A subunit
of biologically active molecules that are active in vivo and that
of NMDAR. A mutation in the exon 14 of the human GRIN2A
may be used as novel therapeutics or form the basis of novel
gene was found in the patient (chr16: 9857449; G>G/A) and was
modes of diagnosis. We have successfully identified small mole-
associated to refractory epilepsy. In this study, we applied a
cules and peptides against a variety of protein targets, both intra-
bioinformatics approach to model murine mutated GRIN2A pro-
and extracellular, including some from the fields of thrombosis,
tein and appreciate possibility to create a murine model of this
atherosclerosis, inflammation and antibiotic resistance. The vari-
disease. The BLAST was used to find homologous mutation in
ety of targets illustrates the synergistic power of our approach
murine GRIN2A gene. X-ray resolved structural models of the
and moreover, we have shown that it is possible to obtain highly
NR2A segments were used as source for M4T Raptor algorithm
active molecules that express their activities also in vivo, via a
to generate predicted structure of mutated protein. The position
cost-effective optimized non-traditional route of discovery and
of homologous point mutation in murine GRIN2A gene was
optimization.
revealed by the BLAST. Sequence alignment of the murine and
human NR2A had 98% similarity, allowing to operate X-ray
resolved structural models of the NR2A segments. It was shown P.23-012-Wed
that the replacement of Val440 to Ile440 amino acid via single
The reliability of loop rebuilt by homology
nucleotide mutation, was located in S1 domain (404–539) region,
forming loop called “loop 1” functioning as the NR1 NR2 modelling in context of protein functionality
ABD–ATD junction and heterodimer structural interface. Both and active site accessibility
residues were aliphatic and it was claimed, that this particular K. Mitusinska, A. G
ora
site is conservative mutation site. Nevertheless, observed changes Biotechnology Centre Silesian University of Technology, Gliwice,
in mutated protein model involve beta strands 2- and 3-elongated Poland
regions of beta sheets could be part of the structural compensa- The reliability of study based on protein structures strongly
tion due to steric effect of bulkier than natural valine isoleucine depends on quality of models and flexibility of particular com-
residue. It is possible to assume, that NR2A disfunction can pos- partments of macromolecules. Flexible and solvent-exposed
sibly lead to NMDAR hypofunction and neuron excitatory prop- regions like loops are prone to be poorly solved what hinders fur-
erties changes, causing higher nervous functions malfunction and ther protein analysis. Homology modelling is one of the widely
further severe symptoms. It was shown that homologous muta- used approach for reconstruction of missing residues in incom-
tion in human and murine GRIN2A gene led to the same confor- plete structures. The quality of obtained model is determined
mational change in the protein structure and formed the mutated mostly by sequence similarity between used templates and size of
NR2A subunit. Thus, it is possible to create murine personalized the missing fragment, however scoring functions assessing accu-
model of the human refractory epilepsy. This study was sup- racy of obtained models were not designed for flexible and sol-
ported by Russian Science Foundation (Grant #17-75-20249). vent-exposed fragments of proteins. The structure of Aspergillus
niger epoxide hydrolase is a good example: each crystal structure
deposited at PDB database (Protein Data Bank) is missing a
nine-amino-acids-long loop: 320TASAPNGAT328. This missing
loop is located at the protein surface adjacent to the entrance of
a buried active site in proximity of the catalytic pocket and thus
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 457
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Structural bioinformatics POSTERS
it can play a major role in regulating access to the active site. P.23-014-Tue
The reconstruction of this particular loop is not trivial since in
Bioinformatic structural analysis of a
comparison with other members of the epoxide hydrolase family,
Aspergillus niger epoxide hydrolase has an additional N-terminal transgenic personalized murine model of
meander domain and extended cap domain. The missing loop is epileptic encephalopathy
located just between the cap domain and the protein core. There- P. Kusov1,2, V. Kalmykov1, A. Deikin1
1
fore it is a perfect model for detailed study of the importance of Institute of Gene Biology RAS, Moscow, Russia, 2Skolkovo
reconstructed model quality and reliability of the most often used Institute of Science and Technology (Skoltech), Moscow, Russia
approach. We decided to verify obtained models using molecular GNAO1 is a Guanine nucleotide-binding protein G(o) subunit a
dynamics simulations and analyse the water accessibility into the coding gene. Being part of heterotrimeric G proteins, Ga subunit
active site using AQUA-DUCT software. The extended analysis transduce signal from GPCRs to cytoplasmic effectors. Single
suggests that the active site accessibility is quite similar although nucleotide change in GNAO1 cause 203G>R aminoacid replace-
the pathways used by water molecules depend highly on the loop ment. We built structural model of murine mutated Ga protein
model used for analysis. We were able to discard poor models segment to analyse possible structural-activity change and to sub-
easily and propose a general approach which can be applied for stantiate creation of a murine model of this human disease.
loops reconstruction. Homologous mutation in murine GNAO1 gene was found by
BLAST. X-ray resolved structural model of the murine Ga were
used as template for M4T Raptor algorithm to generate pre-
P.23-013-Mon
dicted structure of mutated protein. Structural models were visu-
Creation of a personified model of alised and aligned for analysis in PyMol (Schr€ odinger, Portland,
atherosclerosis in genetically modified mice OR). 203G being changed to arginine, which is negatively
M. Kubekina1, P. Kusov2, V. Kalmykov3 charged and sterically much bulkier, located in immediate prox-
1
Institute of Gene Biology RAS, Moscow, Russia, 2Skolkovo imity to the ligand binding site, in conserved switch II region pre-
Institute of Science and Technology, Moscow, Russia, 3Institute of dicted to dramatically decrease GTP binding and activation of
gene biology, Moscow, Russia signal transducing intracellular effector proteins. This mutation,
According to the WHO CVD are the leading cause of early death which human-mice homologicity was determined by BLAST,
worldwide. Atherosclerosis is a chronic artery disease character- possibly lead to severe physiological neurological disorders, called
ized by the accumulation of lipoproteins in the vessel wall. Gen- epileptic encephalopathy. Human/murine GNAO1 203>G
eral theory of atherosclerosis is still not developed. 9 mutations replacement mutation homology allows us to claim, that murine
in the mitochondrial genome associated with atherosclerosis were epileptic encephalopathy model should be established for further
found in a group of 190 patients. So, there is an actual problem investigations for diseased people treatment search. Structural
of creation genetically modified mouse model of atherosclerosis, changes in mutated protein being described could be useful for
which has homologous mutations. To solve this problem, we optimal therapy development. This study was supported by Rus-
chose mutations located in structural genes. There are MT-ND6 sian Science Foundation (Grant # 17-75-20249).
gene (mt:14459; G>A) and MT-CYTB gene (mt:14846; G>A).
The MT-ND6 gene encodes the 6th core subunit of the ETC 1st
P.23-015-Wed
complex – NADH-dehydrogenase, and the MT-CYTB gene
encodes the catalytic subunit of the 3rd complex – cytochrome- Bioinformatic analysis of a transgenic
bc1-oxidase. We studied the spatial structures of these proteins personalized murine model of Lesch-Nyhan
using bioinformatic programs PyMOL and Geneious. In ND6 syndrome
protein the amino acid alanine72 is replaced by valine72, and in V. Kalmykov1, P. Kusov2, A. Deikin3
1
CYTB protein amino acid glycine34 is replaced by serine34. It IGB, Moscow, Russia, 2Skolkovo Institute of Science and
was shown that mutation in the MT-ND6 gene leads to a change Technology, Moscow, Russia, 3Institute of Gene Biology RAS,
in the ubiquinone-binding site of NADH-dehydrogenase, making Moscow, Russia
it more sensitive to ubiquinone, which is able to inhibit complex
HPRT1 is a Hypoxanthine-guanine phosphoribosyltransferase
I. In the protein MT-CYTB mutant serine is sterically bulkier
(HGPRT) coding gene, mutations of which cause, among other
then native glycine. Apparently, this leads to incorrect assembly
diseases, Lesch-Nyhan syndrome. LNS is a severe X-linked reces-
and a decrease of the enzymatic function. We assume that in this
sive neurological disorder. In this study, we built structural
case, an electron leak occurs during oxidative phosphorylation,
model of mutated murine HGPRT segment, with Val8del to
resulting in oxidative stress, which is known to correlate with the
compare it with native form of the protein by bioinformatic
course of CVD. It was shown that homologous mutations in the
tools. Alignment shows 98.6% homology of human and murine
appropriate genes of human and mouse lead to the same confor-
protein sequences. By this, we assume that murine model of this
mational changes. Thus, a mouse model of mutations in the
human disease should be developed. The BLAST was used to
genes MT-ND6 and MT-CYTB is suitable for the human, and
find homologous mutation in murine HPRT1 gene comparing to
these mutations were chosen to create a genetically modified
Human one. X-ray crystallographic structural model of the
model of atherosclerosis in mice. This study was supported by
HPRT1 was used as template for M4T Raptor algorithm to gen-
Russian Science Foundation (Grant # 17-75-20249).
erate predicted structure of mutated protein. Models were visual-
ized in PyMol (Schr€ odinger, Portland, OR). Lack of enzymatic
activity of the HGPRT could be caused via troubled homodimer-
ization. Deletion is found to be located in the HGPRT monomer
surface close to the dimer junction. Absence of the aliphatic
Valine could be the reason of the hindered monomers interaction.
Even such small structural changes in this region could be the
reason of enzyme disfunction due to folding, functional activity
checkpoint in endoplasmatic reticulum by ER quality control
458 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Structural bioinformatics
(ERQC) system. Homologous mutation in human and murine chain. GoF ADAMTS13 mutations were in silico introduced to
HPRT1 gene resulting into comparable conformational change in final poses and both WT- and GoF- ADAMTS13 were subjected
the protein model structure was revealed, so murine personalized to binding free energy calculation with AMBER16 over 100 ns
model of the Lesch-Nyhan syndrome could be developed. Struc- molecular dynamics simulation. Subsequently, these poses were
tural changes can be further studied to provide treatment strategy investigated to reveal which antibody residues contribute to
for people suffering from Lesch-Nyhan syndrome. This study was ADAMTS13 binding. A pose with relatively higher binding affin-
supported by Russian Science Foundation (Grant # 17-75-20249). ity to WT- and lower binding affinity to GoF-ADAMTS13 was
used to predict which residues participate in autoantibody bind-
ing. These residue predictions will be subjected to in vitro muta-
P.23-016-Mon tion studies to test changes on ADAMTS13 conformation, its
DNA structural alphabets proteolytic activity and resistance against autoantibodies. We
J. Cerny, B. Schneider used structural bioinformatics tools to predict the nature of
Institute of Biotechnology CAS, v. v. i., BIOCEV Research autoantibody binding which renders the human ADAMTS13 pro-
Center, Vestec, Czech Republic tein inactive. This knowledge can be further applied on designing
peptide inhibitors of autoantibody binding to ADAMTS13.
DNA is a structurally plastic molecule that enables its biological
function by adapting to its binding partners. A possible approach
to comprehend the structural base of biomolecular recognition and P.23-018-Wed
function is to translate complicated three-dimensional structures
Artifact-resistant bioinformatics pipeline for
into a linear code using so called structural alphabets, employing a
limited set of building blocks that can be symbolically represented detection of somatic retroelement insertions
by alphabet letters. The approach is used fairly routinely for pro- in human genome
tein structures defining an alphabet using oligopeptide segments, G. Nugmanov1, A. Komkov2, M. Saliutina2,3, Y. Lebedev2,
the approach is however new in analysis of DNA structures. The I. Mamedov2
1
prevailing DNA architecture, a right-handed double helix com- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
posed of sequentially complementary antiparallel strands accom- Russian Academy of Sciences, Moscow, Russia, 2Shemyakin-
modates large conformational changes by forming kinks or bends Ovchinnikov Institute of Bioorganic Chemistry of Russian
or undergo radical rearrangements into loops or folded forms such Academy of Sciences, Moscow, Russia, 3Lomonosov Moscow State
as quadruplexes. These conformational changes cannot be under- University, Moscow, Russia
stood without going beyond the “A-B-Z classification” tradition-
The high-throughput sequencing (HTS) technologies opened
ally used to describe the DNA structural diversity. The resulting exceptional opportunities to analyse genomic diversity of somatic
DNA structural alphabet CANA (Conformational Alphabet of cells in health and disease. However, due to the high sensitivity
Nucleic Acids) accessible at the web site dnatco.org is successfully of HTS, the goal to distinguish the true signals from errors
used to analyze the structural properties of typical DNA struc-
became a real challenge for bioinformatics. The main types of
tures. The alphabet also allows quantitative assessment and valida- errors include single nucleotide errors appearing during HTS, chi-
tion of DNA structures and their subsequent analysis by means of meric molecules formed during library preparation and incorrect
pseudo sequence alignment.
genome mapping of HTS data. Here we describe the advanced
bioinformatics pipeline for error-resistant identification of one of
P.23-017-Tue the main source of human genome instability – somatic retroele-
ment (RE) insertions. Our pipeline consists of Python scripts set
Structural bioinformatics prediction of assembled in Jupyter Notebook interface. The pipeline includes:
autoantibody binding to ADAMTS13 genome mapping of HTS data, removal of known RE, errors fil-
J. Hrdinova1,2, B. Ercig1,2, N. A. Graca1,3, K. Wichapong2, tration, comparison of target and control HTS libraries and iden-
C. Reutelingsperger2, K. Vanhoorelbeke4, J. Voorberg1, tification of target site duplications – characteristic signs of
G. Nicolaes2 transposition events. For the genome mapping we tested two
1
Sanquin, Amsterdam, Netherlands, 2Maastricht University, widely used programs: BWA and Bowtie2. Our results indicated
Maastricht, Netherlands, 3Icosagen Cell Factory OU,€ Tartu, 4% rate of false-positive somatic RE generated by BWA and
Estonia, 4KU Leuven Campus KULAK, Kortrijk, Belgium absence of these errors in case of Bowtie2. Artifacts filtration step
In most patients with acquired TTP, autoantibodies target a contains removal of chimeras produced during adapter ligation,
cryptic epitope in the spacer domain of ADAMTS13. Based on template switching and incorrect priming in PCR amplification.
the findings, auto-antibody resistant, gain-of-function variant Thresholds for artifacts filtration were determined by statistical
(GoF) of ADAMTS13 was designed on the antibody epitope of models based on training sets of fixed and polymorphic RE. The
spacer domain with the following amino acid substitutions: accuracy of our pipeline was tested on series of sequenced RE
R568K/F592Y/R660K/Y661F/Y665F. Functional studies libraries with artificially generated imitations of RE insertions
revealed that GoF-ADAMTS13 can bypass the binding of used as spike-in. During this tests, we correctly detected above
autoantibodies while the enzyme gains a higher proteolytic activ- 80% of added spike-ins. Thereby, our pipeline is one of the first
ity than its WT form. Our aim was to employ modeling to iden- artifact-resistant bioinformatics method for detection of somatic
tify structural determinants in autoantibody binding and provide RE insertions. It can by applied for analysis of various type of
a knowledge necessary for the design of small compounds that HTS data including RE enriched libraries and whole-genome
inhibit autoantibody binding to ADAMTS13. Rosetta Antibody data. Funding: RFBR grants № 17-04-01280, № 16-04-00779 and
and Bioluminate Antibody modules were employed to model the RSF grant № 18-14-00244.
Fab fragments of ADAMTS13 autoantibodies II-1 and I-9.
Autoantibody binding onto the spacer domain was studied by
HADDOCK protein-protein docking where the structures were
constrained according to major epitope residues in ADAMTS13
Spacer domain and the CDR3 residues of the antibody heavy
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 459
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Omics technologies POSTERS
Omics technologies ageing. For that, we took use of nuclear magnetic resonance-
based metabolomics to determine the testicular metabolic profiles
of rats from 3 to 24 months-of-age. Moreover, as testicular func-
P.24-001-Mon tion is highly susceptible to oxidative stress levels, due to the high
Subfractionation of Cronobacter membranes metabolic rates, testicular oxidative damages were also evaluated.
and proteins characterization by mass Several small metabolites were determined and creatine signals
spectrometry and immunoblotting were the most predominant in testis up to 9 months-of-age.
y, L. Le
J. Novotn nkova, J. Vlach, B. Svobodova, M. Krausova, There was an age-associated decrease in most antioxidant
L. Karamonova, L. Fukal metabolites, like betaine, creatine and oxidized glutathione. Ami-
Department of Biochemistry and Microbiology, University of noacid content changed as early as 6 months-of-age, with an
Chemistry and Technology Prague, Technick a 3, 166 28, Prague 6, increase in branched chain and aromatic amino acids, accompa-
Prague, Czech Republic nied by decrease of nucleotide synthesis (IMP, CMP, ATP). On
contrast, the testicular content of phosphatidylcholine and phos-
Cronobacter spp. are opportunistic foodborne pathogens. These phatidylethanolamine increased with advanced age, with a con-
bacteria can cause severe necrotizing enterocolitis and meningitis, current decrease on the levels of their phosphorylated products,
especially to immunosuppressed people and newborns with low illustrating lower phospholipid synthesis. The contents of other
birth weight with an alarming lethality up to 80%. The pathogene- phospholipid precursors, myo-inositol and glycerol, also
sis processes remains unclear, even though several virulence factors increased in the testis of the same animals, further suggesting
have been already described. Identification of potentially virulent compromised spermatogenesis. This is the first metabolomics
proteins could help to clarify the process of Cronobacter pathogen- study in testicular tissue of aged rats. We were able to identify
esis. Subsequently, specific proteins could also be used to develop metabolites associated with reproductive maturity from the onset
specific immunochemical identification methods, which can help to senescence. Our results provide evidence for an influence of
with early identification to prevent the spread of disease. In this ageing on testicular metabolome, as early as 6 months-of-age,
work, the method for isolation of membrane fractions and purifica- with a profound alteration on several key metabolic pathways
tion of proteins for further mass spectrometry based analyses was associated with the male reproductive potential.
established. The isolation of periplasm, outer and inner membrane
fraction of 7 representatives of Cronobacter spp. and 3 strains of
other related genera was performed in triplicates. These fractions P.24-003-Wed
were characterised by SDS-PAGE and immunoblotting using anti- Identification of potential molecular targets by
bodies against the whole Cronobacter cells and against particular
quantitative phosphoproteomics in sorafenib
membrane fraction of virulent Cronobacter strain. Proteins from
membrane fractions were identified using mass spectrometry cou- resistant hepatocellular carcinoma cells
pled with liquid chromatography (LC-MS). Altogether over 8000 L. Chow, C. Chen
proteins have been identified and subcellular localization was deter- Institute of Biochemistry & Molecular Biology, College of
mined by in silico analysis. Finally, the potential virulent function of Medicine, National Taiwan University, Taipei, Taiwan
the identified proteins was determined. Hepatocellular carcinoma (HCC) is one of the most common can-
cer mortality and is often been diagnosed with advanced stages.
Many efforts had been made for the development of effective HCC
P.24-002-Tue therapy, but sorafenib is to date the first and only FDA approved
The impact of ageing on the metabolic profile targeted therapy for advanced HCC. While many patients showed
of rat testicular tissue: from the onset to the good initial response rate, it was inevitable for the development of
senescence drug resistance soon after the treatment. Due to the lack of other
S. De Almeida1,2, I. JaraK2,3,4, R. A. Carvalho3, A. Barros1,5,6, novel effective therapeutic agents, sorafenib is still the only option
M. Sousa2, M. G. Alves2, P. F. Oliveira1,2,7 for advanced HCC. Therefore, comprehensive understanding of
1
i3S – Instituto de Investigac~
ao e Inovac~
ao em Sa
ude, the underlying mechanism of sorafenib resistance in HCC and try-
Universidade do Porto, Porto, Portugal, Porto, Portugal, ing to identify the potential therapeutic protein targets is of great
2
Department of Microscopy, Laboratory of Cell Biology and Unit importance. To elucidating the signaling changes during the forma-
for Multidisciplinary Research in Biomedicine (UMIB), Institute tion of drug resistance in HCC, we adopted quantitative phospho-
of Biomedical Sciences Abel Salazar (ICBAS), University of proteomic approach to the analysis of the difference in
Porto, Porto, Portugal, Porto, Portugal, 3Department of Life phosphoproteome between parental HuH-7 and sorafenib resistant
Sciences, Faculty of Sciences and Technology and Centre for HuH-7 (HuH-7R) cell lines. In current study, we identified and
Functional Ecology (CFE), University of Coimbra, Coimbra, quantified 884 phosphoproteins among which most are up-regu-
Portugal, Coimbra, Portugal, 4CICS – UBI – Health Sciences lated in serine and threonine sites. Global bioinformatics analysis
Research Centre, University of Beira Interior, Covilh~
a, Portugal, of the significantly (>1.5-fold) up-regulated 569 phosphoproteins
Covilh~a, Portugal, 5Centre for Reproductive Genetics Prof. Alberto revealed that many signaling pathways involved in cell growth,
Barros, Porto, Portugal; 4-Department of Genetics, Faculty of motility, adhesion, protein synthesis and metabolism, comprised of
Medicine, Porto, Portugal, Porto, Portugal, 6Centre for certain important molecules such as RPS6, 4EBP1, PXN, EPHA2
Reproductive Genetics Prof. Alberto Barros, Porto, Portugal, and PFKP were altered during resistance development. From in sil-
Porto, Portugal, 7Department of Genetics, Faculty of Medicine, ico interaction linkage analysis, we found that an Akt-centered sig-
Porto, Portugal, Porto, Portugal naling network is highly activated in HuH7R cells. Among the
altered phosphoproteins, some important proteins which are impli-
Delayed parenthood has renewed the interest in age-related cated in close cross-talk with Akt. In conclusion, by quantitative
decline of male fertility. Ageing is often associated with testicular phosphoproteomics we figured out a drug-resistance induced Akt
morphological and functional alterations that result in the declin- signaling activation in HCC cells and also identified some potential
ing of its functioning. Still, little is known about the molecular targets for further evaluation of the effect on intervention in dys-
mechanisms associated with testicular senescence. Herein, we regulated resistant signaling.
evaluated the testicular metabolic alterations associated with
460 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Omics technologies
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 461
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Omics technologies POSTERS
protozoa. The availability of tick in vitro culture systems has of mRNA expression. Usually the major SP from each group of
facilitated many aspects of tick-borne pathogen research, includ- T. molitor and T. castaneum SP were not orthologous. Some
ing proteomics. One of the opportunities is a study of tick tissue- SPH had rather high levels of mRNA expression, and this
specific responses to an infection, as these cell lines are derived implies that they have important physiological functions. This
from embryonic cells and can differentiate to the various cell work was supported by RFBR grants 17-54-61008 Egypt_a, 18-
types differing in morphology and behavior. Nevertheless, the 04-01221_a.
nature of tick cell lines remains unclear. Thus, the aim of the pre-
sent study was the proteomic comparison of different tick cell
lines. Three cell lines obtained from the hard tick Ixodes ricinus, P.24-009-Wed
and two from I. scapularis were investigated. An Intact mass A digestive system microbiota of medicinal
spectrometry (IC MS) approach was used, as well as classical leech Hirudo medicinalis
proteomic workflows, including 2DE. The IC MS spectra were N. Polina1, V. Babenko1, E. Grafskaia1,2, O. Podgorny1,3,
very similar between all analyzed cell lines. The comparison of P. Bobrovsky1, D. Kharlampieva1, A. Belova1, D. Shirokov1,4,
IC MS spectra of the cell lines and I. ricinus tick’s organs O. Miroshina1,2, V. Manuvera1,2, V. Lazarev1,2
revealed 15, 12, 7, and 2 shared peaks between cell lines and 1
Federal Research and Clinical Center of Physical-Chemical
ovaries, salivary glands, Malpighian tubes, and gut, respectively. Medicine of Federal Medical Biological Agency, Moscow, Russia,
Two cell line peaks at m/z 6154 and 6822 were detected only in 2
Moscow Institute of Physics and Technology (State University),
the ovaries spectra, and peak at m/z 8451 only in the salivary Dolgoprudny, Russia, 3Koltzov Institute of Developmental Biology
glands. The peak at 6935 m/z was detected in all analyzed sam- of Russian Academy of Sciences, Moscow, Russia, 4K. I. Skryabin
ples. Despite the high similarity of tick cell lines according to the Moscow State Academy of Veterinary Medicine and
IC MS, some changes in their protein profiles were determined Biotechnology, Moscow, Russia
by 2DE. This study was supported by the Ministry of Education,
Youth and Sports of the Czech Republic project Post- Recent omics studies have attracted attention to the role of the
dok_BIOGLOBE (CZ.1.07/2.3.00/30.0032) and the Grant Agency microbial community for maintaining host organism. This is par-
of the Czech Republic (15-03044S, 18-27204S). Access to instru- ticularly true for hematophagous due to different molecular
ments and other facilities was supported by the Czech research mechanisms and evolution adaptations caused by blood feeding.
infrastructure for systems biology C4SYS (project no These adaptive mechanisms may include a signal transduction
LM2015055). cascades and pathways related to immune response, blood diges-
tion and storage processes. A combination of laser microdissec-
tion, sequencing, and bioinformatics allowed us to determine the
P.24-008-Tue bacterial composition of morphologically and functionally dis-
A set of serine peptidases from the S1A tinct parts of the leech digestive system. Analysis of 16S rRNA
data obtained for different parts of the leech digestive system
chymotrypsin family in Tenebrionidae beetles
revealed that bacterial community of crop and coeca differs from
E. N. Elpidina1, A. G. Martynov2, A. O. Abd EL-Latif3
1 intestine. In a H. medicinalis metagenome we identified taxo-
Belozersky Institute of Physico-Chemical Biology, Lomonosov
nomic groups of bacteria relevant for leeches. Besides the genera
Moscow State University, Moscow, Russia, 2Center for Data-
of bacteria (Mucinivorans, Aeromonas, Niabella) that have been
Intensive Biomedicine and Biotechnology, Skolkovo Institute of
described for leeches earlier, we determined several bacterial taxa
Science and Technology, Moscow, Russia, 3Department of Plant
that have not seen in the leech metagenome before. Some of
Protection, Faculty of Agriculture, Sohag University, Sohag, Egypt
them were also described in other hematophagous. We found
The stored product pests Tenebrio molitor and Tribolium casta- that microbiome of the digestive system supports the synthesis of
neum are model beetles from the family Tenebrionidae. We iden- vitamins and water-soluble cofactor, fixation of heme compounds
tified, annotated and compared predicted sets of serine peptidases and excessive iron metabolism. Surprisingly we identified genes
from the S1A chymotrypsin family (SP) in the gut transcriptomes involved in antibiotic resistance and genes encoding multidrug
of T. molitor and T. castaneum larvae and evaluated relative efflux pumps. Thus, the sequencing data obtained in our study
expression levels of their mRNA. In T. molitor larvae we found allows for the analysis of the metabolic potential of microbiota
183 sequences similar to SP, but only 87 represented active pepti- and determination of the contribution of individual bacterial spe-
dases. The residual 96 sequences had substitutions in the catalytic cies and strains to metabolic pathways. Detailed studies are rele-
triad and were classified as inactive homologs of serine peptidases vant not only for bloodsucking leeches but other hematophagous
(SPH). 31 sequences of active SP were related to trypsins, two to to determine the role of microbiota in the host organism adapta-
chymotrypsins A/B, one to chymotrypsin C, 13 were chy- tion to blood nutrition. This work was supported by the Russian
motrypsin-like peptidases, 12 pancreatic elastases I, and the speci- Science Foundation (project № 17-75-20099).
ficity of 28 peptidases was uncertain due to unusual residues in
the substrate binding site. T. castaneum contained 178 SP
sequences, of which 98 represented active peptidases, and 80
SPH. T. castaneum contained many more trypsin sequences – 51,
and fewer chymotrypsin sequences than T. molitor: one chy-
motrypsin A/B, one chymotrypsin C, and only five chy-
motrypsin-like peptidases. Three sequences were annotated as
pancreatic elastases I and 37 peptidases were with uncertain
specificity. These data are reflective of a highly complex digestive
organization in these larvae, presumably a result of high selection
pressure of cereal inhibitors. Analysis of the relative expression
levels of SP mRNA revealed that each group of SP usually con-
tained one major SP with the expression level exceeding 50% of
the total expression of the whole group, a number of peptidases
with medium level of expression, and many SP with minor level
462 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Omics technologies
P.24-010-Mon P.24-011-Tue
A genome-wide association study of muscle Proteomics for the detection of protein
fibers composition revealed 5 SNPs associated interactors of vascular endothelial growth
with slow-twitch fibers factor in age-related benign prostatic
O. Borisov1, E. Semenova2, E. Miyamoto-Mikami3, hyperplasia rats
H. Murakami4, H. Zempo5,6, E. Kostryukova1, N. Kulemin1, M. Dıaz-Lobo1, M. Vilanova1, M. Gay1, J. E. Rodriguez-Gil2,
A. Larin1, M. Miyachi4, E. Ospanova1, A. Pavlenko1, J. M. Fernandez-Novell3, M. Vilaseca1
E. Lyubaeva7, D. Popov7, O. Vinogradova7, E. Boulygina8, 1
Mass Spectrometry and Proteomics Core Facility, Institute for
H. Kumagai6,9, R. Kakigi10, H. Kobayashi11, H. Naito6, Research in Biomedicine (IRB Barcelona). The Barcelona Institute
E. Generozov1, V. Govorun1, N. Fuku6, I. Ahmetov1,12 of Science and Technology (BIST), Barcelona, Spain,
1
Federal Research and Clinical Center of Physical-Chemical 2
Department of Animal Medicine and Surgery, Faculty of
Medicine, Moscow, Russia, 2Department of Biochemistry, Kazan Veterinary Medicine, Autonomous University of Barcelona,
Federal University, Kazan, Russia, 3Department of Sports and Life es), Barcelona, Spain, 3Department
Bellaterra (Cerdanyola del Vall
Science, National Institute of Fitness and Sports in Kanoya, of Biochemistry and Molecular Biomedicine, Faculty of Biology,
Kagoshima, Japan, 4Department of Physical Activity Research, University of Barcelona, Barcelona, Spain
National Institutes of Biomedical Innovation, Health and Nutrition,
Tokyo, Japan, 5Japan Society for the Promotion of Science, Benign prostatic hyperplasia (BPH) is an age-related disease
Tokyo, Japan, 6Graduate School of Health and Sports Science, affecting the majority of elderly men worldwide. BPH involves
Juntendo University, Chiba, Japan, 7SSC RF Institute for an inflammatory process that can later lead to the development
Biomedical Problems of the Russian Academy of Sciences, of prostate tumors. Inflammation is mediated by pro-inflamma-
Laboratory of Exercise Physiology, Moscow, Russia, 8“Omics tory cytokines, chemokines and prostanoids, and growth factors
Technologies” OpenLab, Kazan Federal University, Kazan, Russia, such as vascular endothelial growth factor (VEGF). VEGF stim-
9
Research Fellow of Japanese Society for the Promotion of ulates the proliferation and survival of endothelial cells and pro-
Science, Tokyo, Japan, 10Faculty of Medicine, Juntendo motes angiogenesis and vascular permeability. VEGF participates
University, Tokyo, Japan, 11Department of General Medicine, in tumor metastasis and intraocular neo-vascular syndromes.
Mito Medical Center, Tsukuba University Hospital, Ibaraki, Here we combined the Surface Acoustic Wave (SAW) technique
Japan, 12Laboratory of Molecular Genetics, Kazan State Medical with proteomic approaches for the direct characterization of
University, Kazan, Russia interacting proteins related to the same metabolic pathways in
biological samples. This novel combination was used to study the
Biochemical properties of muscle tissue are linked to a specific pathway of VEGF protein and its receptor. Briefly, the antibody
muscle fibers type. Predominance of slow-twitch muscle fibers against VEGF was immobilized on the surface of a SAW biosen-
predispose to more efficient endurance performance while fast- sor chip. Prostate homogenates of 12-month-old rats with age-
twitch fibers are crucial for sprinting activity. Since the composi- related hyperplasia were injected and passed through the chip
tion of muscle fibers is known to be influenced by heredity up to surface. Affinity binding phenomena of prostatic homogenate
45%, we aimed to identify genetic factors associated with both proteins with the immobilized anti-VEGF antibody and also with
muscle fibers structure and athletic performance. We compared its receptor were first measured via the SAW biosensor. Interact-
genome-wide genotyping profiles (HumanOmni1-Quad) of 89 ing proteins were then eluted from the chip surface, collected and
Russian sprinters and 103 endurance athletes. High throughput analysed by classical bottom-up proteomics. All the proteins
genotyping (OmniExpressExome-8) and investigation of muscle identified were related to VEGF and VEGFR metabolic path-
fibers composition using immunohistochemical analysis were per- ways and were previously reported to play crucial roles in inflam-
formed for another group of 45 male athletes. To validate the mation diseases, cancer cell proliferation and metastasis when
results, we used a Japanese athletes sample (54 sprinters, 60 they are overexpressed in cells. The biosensor was also coupled
endurance athletes). Data analysis, processing and annotation online to MS using a dedicated interface (MSI). Proteins of 19–
were done using Plink, R, and Ensembl VEP. Russian sprinters 23 kDa corresponding to various isoforms of rat VEGF-A and
and endurance athletes genotypes were compared using a case- VEGF-B were detected by online combination SAW-MS. This
control design: all together and after excluding sub-elite athletes. approach allows the detection, quantification and mass spectro-
The results were intersected with a study of similar design that metric structural characterization of affinity-bound biopolymers.
included Japanese athletes and resulted in 201 common SNPs PRB3 (IPT17/0019-ISCIII-SGEFI/ERDF), BMBS COST Action
(P < 0.05). After performing regression analysis of muscle fibers BM 1403 and FBG309273.
composition (percentage), we matched the findings and identified
5 SNPs associated with both predominance of slow-twitch fibers
in males and endurance athlete status. Two SNPs (rs4561252, P.24-012-Wed
rs7080650) belong to phosphodiesterase superfamily and are Adaptation of Acholeplasma laidlawii to
involved in cardiovascular function while other three (rs6789275,
stressors: changes in genome, cell and
rs7525663, rs992297) are intergenic and require further functional
investigation. None of the 5 SNPs were described in the vesicular proteomes and mutagenicity to
NHGRI-EBI GWAS Catalog earlier. This study has revealed 5 human cells
SNPs that connect the slow-twitch muscle fibers profile with the E. Medvedeva1,2, N. Baranova1,2, T. Malygina1,
predisposition to endurance sport performance. The work was A. Mouzykantov1,2, M. Davydova1, O. Chernova1,2,
supported by the Russian Science Foundation, Grant No. 17-15- V. Chernov1,2
1
01436. Kazan Institute of Biochemistry and Biophysics, Federal Research
Center “Kazan Scientific Center of RAS”, Kazan, Russia, 2Kazan
(Volga Region) Federal University, Kazan, Russia
Acholeplasma laidlawii (class Mollicutes) is widespread in nature
(it can be found in humans, animals, plants); is the main contam-
inants of cell cultures and vaccine preparations, being a danger
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 463
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Omics technologies POSTERS
to human health. Solution of the problem controlling the molli- with TLR4 activation. Both agonists induce shift of PUFAs
cute infections turns out to be connected with elucidating the metabolism from proinflammatory to resolution substances. Sup-
molecular bases of adaptation of these microorganisms to ported by RSF (№ 16-15-10298).
adverse environments that determine a broad distribution of the
bacteria in nature, formation of the “parasite-host” system
in vivo and in vitro, and realization of virulence. The omics tech- P.24-014-Tue
nologies have opened new opportunities for carrying out the Metabolic profile analysis of a Drosophila
appropriate studies. To select A. laidlawii PG8B strains with dif- model of Parkinson’s disease
ferential sensitivity to adverse environments (long-term exposure F. J. Sanz Lopez1, C. Solana Manrique1, V. Mu~ noz Soriano1,
to low temperature, substrate limitation), to antimicrobials M. Palomino Sch€atzlein2, A. Pineda Lucena2, N. Paricio1
(ciprofloxacin, tetracycline, melittin) used for suppressing molli- 1
Departamento de Gen etica and Estructura de Recerca
cutes, and to perform the comparative analysis of the complete Interdisciplinar en Biotecnologia i Biomedicina (ERI
genomes, cell and vesicular proteomes and virulence of the BIOTECMED), Valencia, Spain, 2Joint Research Unit in Clinical
strains was the object of the study. Adaptation to low tempera- Metabolomics, Centro de Investigaci on Prıncipe Felipe/Instituto de
ture, substrate limitation and antimicrobials in A. laidlawii Investigaci
on Sanitaria La Fe, Valencia, Spain
proved to be accompanied by multiple changes in genomic pro-
file, cell and vesicular proteomes as well as level of extracellular Parkinson’s disease (PD) is the second most common neurode-
vesicles production. In all cases the processes proved to be generative disease in the world. Its symptomatology is due to a
accompanied by mutations in genes for targets of antimicrobials selective loss of dopaminergic (DA) neurons in the substantia
of different classes, including those indifferent for these bacteria. nigra pars compacta. Currently, diagnosis of PD is mainly based
The mutant genes were detected in the A. laidlawii extracellular on the presence of motor alterations, which appear at late stages
vesicles, which can provide gene distribution in bacterial popula- when there is up to 70% DA neuron loss. Therefore, it is impor-
tions via horizontal transfer. A significant part of the stress-reac- tant to find new biomarkers that will allow early diagnosis and
tive genes and proteins proved to be related to virulence. It was treatment of PD patients. Recent studies have revealed links
found that the strains adapted to the stressors exhibit mutagenic- between glucose metabolism and alterations in cellular bioener-
ity to human cells – induce hypoploidy and total premature cen- getics, redox homeostasis and cell death progression induced by
tromere separation in peripheral blood lymphocytes in vitro. PD-related risk factors. To study the possible link between
Work was supported by grant MK-1099.2017.4; RFBR 18-04- altered metabolism and PD, we used a Drosophila model of PD
00660. based on inactivation of the DJ-1ß gene (ortholog of the DJ-1
human gene). DJ-1 is involved in early-onset recessive PD and
encodes a multifunctional protein that plays an important role in
P.24-013-Mon antioxidant responses but also regulates central metabolism. Con-
Comparison of TLR3- and TLR4-mediated sistent with this, studies performed in our laboratory have shown
that several proteins related with metabolic pathways are highly
signaling cascades in glial cells
carbonylated in mutant flies. To further investigate the impact of
D. Chistyakov1,2, N. Azbukina3, A. Astakhova1, V. Chistyakov4,
DJ-1ß loss on cellular metabolism, we characterized the meta-
S. Goriainov4, M. Sergeeva1
1 bolic profile of polar and non-polar extracts from 15-day-old DJ-
A.N. Belozersky Institute of physico-chemical biology MSU,
1ß mutant and control flies by high resolution nuclear magnetic
Moscow, Russia, 2Pirogov Russian National Research Medical
resonance (NMR) spectroscopy. These analyses have revealed the
University, Moscow, Russia, 3Department of Bioengineering and
presence of metabolic changes in PD model flies that could be
Bioinformatics, Lomonosov Moscow State University, Moscow,
relevant to understand the pathophysiology of PD. We will pre-
Russia, 4Peoples’ Friendship University of Russia (RUDN
sent the results obtained in these studies, which have allowed to
University), Moscow, Russia
the identification of metabolic pathways altered in mutant flies.
Toll-like receptor (TLR) mediated signaling cascades play crucial Our results will be discussed in the context of how these meta-
role in neuroinflammation and therefore may be targets for bolic alterations could help to understand the pathophysiology of
manipulation with therapy purposes. Although many efforts have PD and may lead to the discovery of biomarkers for PD diagno-
made, molecular mechanisms of TLR-mediated signaling in sis.
astrocytes is largely unknown, especially relative to regulation of
signaling lipids release. Many of signaling lipids belong to a fam-
ily of resolution of inflammation processes factors, and low P.24-015-Wed
molecular weight substances can regulate their synthesis, there- Restoration of the NEPHGE two-dimensional
fore the aim of our study was comparison of signaling lipids syn- electrophoresis method
thesis in course of stimulation by lipopolysaccharide (LPS, a R. Zinkeviciut_e, R. Slibinskas
TLR4 agonist) and poly I:C (PIC, a TLR3 agonist). Primary Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
astrocytes from newborn rats were cultured 12 days before exper-
iments; Western blot was used for detection of TLR signaling Two-dimensional gel electrophoresis (2DE) is one of the most
pathway proteins and enzymes of lipid synthesis. Cell-free culture widely used techniques for the global protein separation.
media were taken for solid-phase lipid extraction; lipid mediators NEPHGE (non-equilibrium pH gradient electrophoresis) based
(25 lipids) were analyzed by 8040 series UPLC-MS/MS (Shi- 2DE is a carrier ampholytes-based isoelectric focusing (IEF) tech-
madzu) with 4 MS standards. Time-course cellular responses (1, nique that failed to achieve widespread application due to its
2, 4, 6, 8, 12, 24 h) for stimulation with LPS and PIC were com- labor intensiveness but is used in more specialized research. It
pared. We obtained that three pathways of polyunsaturated acids provides better separation of highly basic proteins, has a higher
(PUFAs) transformation (COXs, 12/15 LOX, cytP450) are protein capacity with good reproducibility and quality of spots at
involved in astrocytes responses. 13 substances are released in sig- high protein load when compared with a widespread IPG (immo-
nificant amounts. Releases of PUFAs or their non-enzymatic bilized pH gradient) 2DE technique. The equipment and solu-
derivatives are similar for both tested agonists. TLR3 activation tions needed for performing NEPHGE technique were
significantly induces enzyme-mediated metabolism in comparison commercialized by WITA GmbH as a “WITAvision” 2DE
464 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Omics technologies
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 465
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Omics technologies POSTERS
466 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Omics technologies
similar proteomic analysis of M-PMV virions was performed to skeletal muscle. However, there are currently no effective and
identify host cell proteins that are incorporated into newly formed common therapeutic approaches to prevent or delay the progres-
particles. Identification of reproducibly incorporated proteins into sion of these diseases. Recent studies revealed important regula-
the virions could help to clarify the mechanisms of morphogenesis tory roles for small noncoding RNAs, called microRNAs
or virulence of M-PMV. About sixty of host cell proteins were sta- (miRNAs), in skeletal muscle function under physiological and
bly identified in whole M-PMV particles produced in human pathological conditions. In this study, we aim to identify com-
embryonic kidney cells. From those, the proteins participating in mon miRNA signatures associated with etiopathogenesis of dif-
clathrin-mediated endocytosis, which has been found to be used by ferent neuromuscular diseases (Duchenne Muscular Dystrophy,
HIV-1 when entering the host cell, or proteins of the ESCRT com- Megaconial Congenital Muscular Dystrophy (CMD), Ullrich
plex, which HIV-1 uses to bud from the host cell, were identified. CMD and alpha-dystroglycanopathy), each caused by mutations
Additionally, another identified proteins are involved in COPI and in different nuclear genes encoding proteins with distinct roles.
COPII mediated endosomal transport, which could be important For this purpose, skeletal muscle biopsies from selected NMDs
for transport of M-PMV proteins within the host cell. presenting mitochondrial damage (n = 12, 3 from each group)
and control individuals (n = 3) were analyzed by using Affyme-
trix GeneChip miRNA 4.0 Array. To identify differentially
P.24-022-Mon expressed miRNAs in patients, raw data was analyzed by two
Discriminating sub-population responses of a different programs, MeV-SAM and Affymetrix TAC. Differen-
mixture of human cell lines by proteogenomics tially expressed miRNAs whose expression were found to be sta-
C. Almunia1, Y. Cogne1, O. Pible1, C. Lepleux2, F. Chevalier2, tistically significant by both programs (miRNAs that showed an
J. Armengaud1 increase/decrease by 2 fold in patient samples compared to the
1
CEA/DRF/LI2D, CEA Marcoule PRAE Marcel boiteux, Bagnols control group) were identified as candidates. We then identified
eze, France, 2CEA/DRF/iRCM/LARIA, Campus Jules
sur C potential target genes of these candidate miRNAs by using miR-
Horowitz, Caen, France Walk and classified them by using GENE ONTOLOGY-
PANTHER databases. Our results revealed that 17 miRNAs
Monitoring proteome dynamics from different human cell types were differentially expressed in patients and 5 of these miRNAs
present concomitantly in a given sample is of great interest and are likely involved in skeletal muscle differentiation. Our com-
could be applied to ultra-precise molecular characterization of monality approach will provide contribution to the literature by
complex tissues. Here, we propose a proteogenomics-based strat- identifying common potential therapeutic targets and/or
egy to point at cell line molecular signatures. For this, the pro- biomarkers related to different rare NMDs.
teome is analyzed by high-throughput shotgun mass spectrometry
and specific bioinformatics search are performed. First, mRNA
from chondrosarcoma cells (SW1353 cell line) and immortalized P.24-024-Wed
chondrocytes (T/C28A2 cell line) were sequenced by RNAseq for Quantitation of drugs using for treatment of
establishing the most appropriate protein sequence database. For
essential hypertension in dried blood spots by
this an innovative cascade search allows to conciliate de novo and
mapping RNAseq assemblies and the Human SwissProt database. LC-MS/MS and correlation with dried serum
A set of 2 million of discriminating peptide sequences of the two spots
cell lines are then identified. From them, 480 peptide sequences A. Chernonosov1, M. Kasakin1, V. Koval1,2
1
were detected and monitored based on extracted ion chro- Institute of Chemical Biology & Fundamental Medicine,
matogram (XIC) signals recorded by tandem mass spectrometry. A Novosibirsk, Russia, 2Novosibirsk State University, Novosibirsk,
list of 55 peptides was used for quantitating the ratio of each cell Russia
type in a given co-culture sample with high precision, selected with
Mortality from cardiovascular diseases in Russia is in the first
cell lines mixed at 2:1, 1:1; and 1:2 ratio. This new methodology place (Russian Statistical Yearbook, 2015). As a rule, the cause
was used to analyze the bystander effect generated by irradiated of this is complications of coronary heart disease leading to ang-
chondrosarcoma cells (SW1353 cell line) on immortalized chondro-
ina, myocardial infarction, cardiac arrhythmia, sudden cardiac
cytes (T/C28A2 cell line) in co-culture conditions. Such strategy death (McMurray J. J 2012), slowing of blood flow, formation of
could be applied to investigate intercellular interactions between intravascular thrombus (Uster V 1997). In about half the cases,
different cell types, paving the way to new insights into the molecu- the disease begins without previous symptoms with unstable ang-
lar mechanisms of crosstalk between human cells.
ina or myocardial infarction and immediately changes into a
chronic form (RI Litvinenko, 2014). Often the complications lead
P.24-023-Tue to increased physical and psychological stress. The development
of new tests for determining the pathologies of the cardiovascular
Common miRNA signatures in a group of rare system consists in finding new biomarkers and improving existing
neuromuscular disorders methods by increasing the sensitivity and accuracy of the analy-
E. Aksu1, Y. Z. Akkaya-Ulum1, B. Balci-Peynircioglu1, sis. The ease of collection and storage of samples makes the choice
D. Dayangac-Erden1, A. Yuzbasioglu1, B. Bakir-Gungor2, of dry blood spots (DBS) and dry blood plasma spots (DPS)
B. Talim3, B. Balci-Hayta1 attractive as a source of biological material collection for biomedi-
1
Hacettepe University, Faculty of Medicine, Department of cal research and analysis, especially outside the traditional medical
Medical Biology, Sihhiye 06100, Ankara, Turkey, 2Abdullah Gul environment (for example, at home, in remote locations, limited
University, Faculty of Engineering and Natural Sciences, resources) (McDade TW, 2007). Unlike standard blood plasma
Department of Computer Engineering, 38039, KAYSERI, Turkey, samples, samples on dry spots do not require freezing, can be
3
Hacettepe University, Faculty of Medicine, Department of stored and transported in simple folders or envelopes at room tem-
Pediatrics, Pathology Unit, Sihhiye 06100, Ankara, Turkey perature or with cooling to 4°C. If the stability of the substances
Neuromuscular disorders (NMD) are heterogeneous group of is proved, then such samples can be sent by regular mail. In this
genetic diseases that encompasses many different syndromes and study, we propose to develop methods for quantitation of drugs
diseases that either directly or indirectly impairs the function of using for treatment of essential hypertension in dried blood spots
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 467
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Omics technologies POSTERS
by LC-MS/MS and correlation with dried serum spots. The genome_c.php); 130 samples (only derived from Caucasian
research was supported by Program of RAS “Fundamental patients) of LAPC without lymphatic dissemination were
research for biomedical technology” 2018–2020. divided into two groups depending on the cancer prognosis (fa-
vorable or unfavorable). In the analysis, the genes previously
identified by published data as potential prognostic biomarkers
P.24-025-Mon were considered. Additional eight genes, that are characterized
MS-key to unlocked bacteria resistant to beta- by differential expression, were found. Increased expression of
lactam antibiotics TWIST1, TUBB3, and CHAT and decreased expression of
E. Ilgisonis1, M. Rubtsova2, G. Presnova2 CYP1B1, IGSF1, EDN3, MSMB, and SERPINA3 were detected
1
V. N. Orekhovich Institute of Biomedical Chemistry, Moscow, in the group of patients with poor prognosis in comparison to
Russia, 2Lomonosov Moscow State University, Moscow, Russia one with favorable prognosis. These genes are involved in the
key processes of carcinogenesis, such as angiogenesis, prolifera-
In recent years, multidrug resistant strains are actively spreading, tion and migration of tumor cells (CYP1B1, EDN3, TWIST1,
which are resistant to several or even all antibiotics. Resistance TUBB3, and CHAT), as well as with disturbance of regulation
to antibiotics is a big botnet in the selection of personalized ther- of signaling cascades (IGSF1, SERPINA3, and MSMB). Thus,
apy. In this regard, there is a problem of identifying bacteria that we identified a number of genes as potential prognostic
are resistant to beta-lactam antibiotics. The main mechanism of biomarkers associated with poor prognosis of LAPC without
this type of resistance is the production of beta-lactamase lymphatic dissemination. This work was funded by the Program
enzymes. They are extremely different in structure and substrate of fundamental research for state academies for 2013–
specificity. In this work several approaches have been developed 2020 years (№ 01201363819) and by RFBR according to the
for the MS profiling of beta-lactamases to determine the family research project no. 17-29-06083.
and type, depending on which antibiotic therapy is selected.
From the UniProt and literary sources amino acid sequences
belonging to mutant forms of beta-lactamases (1) of different P.24-027-Wed
types (2)were loaded. Using Skyline virtual trypsinolysis was per- Empowering shotgun analysis with 2DE data:
formed and using BLAST-search proteotypic peptides (specific
story of HepG2 proteome exploration
for each form) were determined for subsequent mass spectromet-
O. Kiseleva, S. Naryzhny, V. Zgoda, A. Lisitsa, E. Poverennaya
ric analysis. Proteotypic peptides for several TEM, SHV, CTX-M
Institute of Biomedical Chemistry, Moscow, Russia
families were chosen. Methods for their detection using SRM
technology were developed. It was shown that this peptides can Proteome heterogeneity is unavoidable during proteomic studies.
be used for mass-spectrometric identification basing on more Investigation of alternative splicing, single amino acid variations
than 50 experiments in mass-spectromic repositories. At the same and posttranslational modifications is crucial because of signifi-
time, it was not possible to select specific peptides that distin- cance of these events in expression and functional properties of
guish mutant forms TEM-1 from TEM-84. Bioinformatic analy- proteins. Our research is devoted to studying proteome of HepG2
sis of mutant forms of beta-lactamases allowed to determine cell line. Basing on the results of RNASeq analysis of HepG2 cell
specific peptides, taking into account the changes in the line, we created a transcriptome-specific library, containing 52
nucleotide sequence for the subsequent creation of a set of thousand protein sequences, encoded by 12 thousand genes. Such
peptides and their mass spectrometric identification in the library allows to take into account individual variations and
samples. An experimental verification of the proposed helps to avoid uncontrolled extension of search area by popula-
approach presupposes a directional mass spectrometric analy- tional data and reduce FDR. One of the most popular methods
sis of beta-lactamases in the periplasmic fraction. Ek.I. of proteomic analysis – shotgun mass spectrometry – is charac-
acknowledge the Leading Scientific School of Prof. Andrey terized by low (ca. 20%) coverage of protein sequence. Short
Lisitsa (№ NSh6313.2018.4). peptides, detected with shotgun MS, do not allow to distinguish
highly homologous proteoforms. To empower shotgun approach,
we added two dimensional electrophoresis (2DE). After 2DE
P.24-026-Tue fractioning we cut the gel in 96 cells and analysed every cell by
Potential prognostic markers for locally MS. Fractioning of protein mixture before MS allows to enrich
advanced prostate cancer without lymphatic the results with coordinates of proteoform on the gel (pI and
MW). 2DE profiling with further MS analysis allowed to dis-
dissemination
cover over 2614 proteoforms (1972 of which aberrant) encoded
E. Pudova1, E. Lukyanova1, K. Nyushko2, A. Snezhkina1,
by 2326 genes. Without 2DE we identified only 925 proteoforms
M. Kiseleva2, A. Kaprin2, A. Kudryavtseva1,2
1 because of sample heterogeneity and lack of knowledge of physi-
Engelhardt Institute of Molecular Biology, Russian Academy of
cal–chemical parameters of proteoforms, used for specification of
Sciences, Moscow, Russia, 2National Medical Research
certain proteoform. Effective tandem of 2DE/MS allows us to
Radiological Center, Ministry of Health of the Russian Federation,
forecast modifications, which can change physical–chemical
Moscow, Russia
parameters (and the location of protein spot on the gel, conse-
Prostate cancer (PC) is a major cause of cancer-related deaths in quently). Obtained results consist not only of evaluation of prote-
men worldwide. Locally advanced prostate cancer (LAPC) is oforms implemented at the protein level, but also of
characterized by invasion of the prostatic capsule without evi- improvement of experimental approaches to cell proteotyping.
dence of nodular or distant metastatic spread. Patients within O.K. and Ek.P. acknowledge the Leading Scientific School of
this clinical category have different risks of recurrence. Our Prof. Andrey Lisitsa (NoNSh-6313.2018.4).
study is aimed at identifying novel prognostic biomarkers for
LAPC, which will lead to optimization of treatment and devel-
opment of appropriate clinical recommendations. We performed
bioinformatics analysis of The Cancer Genome Atlas (TCGA)
project RNA-Seq data using the computational resources of
EIMB RAS “Genome” center (http://www.eimb.ru/rus/ckp/ccu_
468 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Omics technologies
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 469
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Omics technologies POSTERS
470 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Omics technologies
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 471
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Nanoworld POSTERS
472 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Nanoworld
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ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Nanoworld POSTERS
474 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Nanoworld
nanostructures. Our data suggest that nanostructured gold can substrate (e.g. ascorbic acid (AA). Metalloporphyrins contained a
inhibit vessel density, total vessels network length, total branch- transition metal firstly were investigated as components for bin-
ing point and total vessel segment in VEGF induced angiogenesis ary catalytic therapy. However, poor water-solubility of metallo-
on CAM. More importantly, we found that anti-angiogenic porphyrins limits its use in binary catalyst therapy. Thus,
effects of AuNPs are size dependent and 4 nm AuNPs had com- biodegradable metalloporphyrins-loaded nanoparticles based on
parable higher anti-angiogenic effects than 20 nm sized ones. poly(D,L-lactide-co-glycolide) (PLGA) were obtained. Here,
Thus, these materials could be potential therapeutic platforms for nanoparticles of two transition metal, tetraphenyl porphyrins,
treatment of pathological angiogenesis. (Fe3ClTPP, Mn3ClTPP), were prepared by one-step emulsifica-
tion method and were optimized for particle size and entrapment
efficiency. The results showed that size of optimized nanoparticles
P.25-011-Tue were in the range of 190–230 nm and the loading contents were
Surface modification with polyallylamines for 7.9% and 5.2% for Fe3ClTPP and Mn3ClTPP, respectively. The
immobilization of biopolymers and cells results of MTS assays showed that nanoparticles in combination
N. Barinov, O. Morozova, O. Levchenko, Z. Cherpakova, with AA possess cytotoxicity in chronic myelogenous leukemia
O. Volosneva, V. Prokhorov, E. Pavlova, K. Aldarov, (K562) cell line. It was found that such combination exhibits
D. Bagrov, E. Obraztsova, K. Prusakov, A. Belova, antitumor properties against K562 cell lines, due to the interac-
V. Podgorsky, D. Basmanov, V. Lazarev, D. Klinov tion of metalloporphyrins and AA, which led to the formation of
Federal Research and Clinical Center of Physical-Chemical reactive oxygen species. The results revealed that free substance
Medicine, Moscow, Russia and AA in this concentration range has no effect on the viability
of the cells. As a control, K562 cell line was treated with 3-
Surface modification for bio-medical, environmental, agricultural amino-1,2,4-triazole, a catalase inhibitor, which increased the
and security applications can be performed physically and chemi- cytotoxic activity of the Fe3ClTPP and Mn3ClTPP with AA
cally by surface coverage or grafting with polymers and conjugat- against K562 cell line.
ing ligands. The immobilized substances must retain their
structures and not desorb from the functionalized surfaces.
Hydrophobic, electrostatic and van-der-Waals interactions, coor- P.25-013-Mon
dination and covalent binding are involved in the adsorption. Intrathecal injection of Foxp3 encoded
Our aim was the development of the functionalization method
plasmids-encapsulated PLGA nanoparticles
for immobilization of native proteins and viable cells. Cationic
polymers with NH2-groups were used for functionalization of attenuate spinal nerve ligation-induced
charged and uncharged surfaces of planar slides, wells of plates neuropathic pain behavior
and spherical nanoparticles. Poly(allylamine)s (PAA) and J. Shin, D. W. Kim, J. Hong
polylysines (pLys) of different molecular weights formed inter- Chungnam National University, Daejeon, South Korea
faces of the thicknesses ~1.5-2 nm for PAA 65 kDa as measured
Neuropathic pain from damage or dysfunction of the nervous
by: (1) the atomic force microscopy on mica slides; (2) the label- system is a highly debilitating chronic pain state and is often
free biosensor with registration of the critical angle of total inter- resistant to currently available treatments. It has become clear
nal reflection on photonic crystal surface covered with SiO2. that neuroinflammation mediated by proinflammatory cytokines
Sorption capacity of 0.1 mg/ml PAA 65 kDa exceeded the values
and chemokines from microglia plays an essential role in the
of other polyamines with different concentrations. Physisorption development of central sensitization during the establishment and
of proteins on PAA-layer is reversible and up to 70% of attached maintenance of neuropathic pain. Here, we succeeded in alleviat-
proteins can be removed. Glutaraldehyde provides stable chemi-
ing spinal nerve ligation (SNL)-induced neuropathic pain by
cal cross-linking of the compounds containing primary NH2-
inhibiting the microglia activation through Foxp3 overexpression
groups with the aminated surfaces. The proteins immobilized on with PLGA nanoparticles. To relieve SNL-induced pain behav-
the pAA-covered surface retained their ability to bind with speci- ior, plasmids encoding Foxp3-loaded PLGA nanoparticles (Fox-
fic monoclonal and polyclonal antibodies. Bacterial cells attached
p3-PLGA-NP) were prepared and characterized. We revealed
to PAA65-covered surfaces could express the green fluorescent that the Foxp3-PLGA-NP decreased the expression of pro-
protein (GFP) gene under control of the inducible lac promoter. inflammatory genes such as TNF-alpha, IL-1beta and iNOS in
Eukaryotic cells also remained alive on PAA-treated slides as BV2 cells incubated with ATP. Moreover, neuropathic pain gen-
shown by fluorescent staining. Thus, treatment of plain and
erated by SNL was effectively diminished in rats administrated
spherical surfaces with PAA is convenient for attachment of intrathecally with the Foxp3-PLGA-NP in behavioral test. Simi-
biopolymers and viable cells. The authors acknowledge funding larly, the loss of microglial activity was also confirmed during
from the Russian Science Foundation [17-75-30064 to D.V.K.]. histological and cytokine analysis. Taken together, these data
suggest that Foxp3 overexpression in spinal microglia with
PLGA nanoparticles efficiently relieves SNL-induced neuropathic
P.25-012-Wed
pain, and it would be therapeutic value for treating neuropathic
Cytotoxic activity evaluation of pain.
metalloporphyrins in binary catalyst system
M. Faustova1, M. Mollaev1, O. Zhunina2, E. Nikolskaya2,
A. Lobanov3, V. Shvets1, N. Yabbarov2
1
Moscow Technological University, Moscow, Russia,
2
Biotechnology Laboratory Russian Research for Molecular
Diagnostics and Therapy, Moscow, Russia, 3Semenov Institute of
Chemical Physics, Moscow, Russia
Binary catalyst therapy is a promising method in cancer treat-
ment that based on the production of reactive oxygen species
stimulated by catalyst (transition metal complex) and oxidation
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 475
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Nanoworld POSTERS
476 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Nanoworld
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 477
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Nanoworld POSTERS
478 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Nanoworld
P.25-023-Tue P.25-025-Mon
Label-free, multiplexed, single-molecule Biophysical aspects of fullerenols interaction
characterization of protein-DNA complexes with peripheral blood mononuclear cells
with biological nanopores subjected to ionizing radiation
G. Celaya, J. Perales-Calvo, M. Ruiz de Gauna, A. Muga, A. Lichota, A. Krokosz
F. Moro, D. Rodriguez-Larrea Department of Molecular Biophysics, University of Lodz, Lodz,
Biofisika Institute (CSIC, UPV/EHU), Leioa, Spain Poland
Protein-DNA interactions are key regulators of gene expression Exposure to ionizing radiation, for example during radiotherapy
and replication. Therefore, they are good targets in cancer therapy and accidental exposures, causes damage to normal tissues. In
and against antibiotic resistance. Beyond its importance, the study order to reduce dangerous radiation effects on healthy tissues
of the interaction between these two macromolecules is challeng- and organs the radioprotective compounds are searched for. Full-
ing. Nanopore technology measures the ionic currents passing erenols C60(OH)x, the water-soluble derivatives of fullerenes, are
through a single nanopore embedded in a lipid bilayer. It detects being intensively studied in the context of the possibility of their
single molecules passing through the pore by the decrease in the application in the biomedicine. The aim of this study is to exam-
ionic current. The measurements have high temporal resolution ine the mechanism of the influence of fullerenol C60(OH)x, x>30
and sensitivity. Indeed, it is used for next-generation DNA on peripheral blood mononuclear cells (PBMCs) subjected to ion-
sequencing, as each nucleotide of a single-stranded DNA causes a izing radiation. Studies on PBMCs will also assess the possibility
different ionic current drop when passing through the nanopore. of intravenous administration of this compound. Peripheral
Here we present an approach that allows to measure single mole- blood mononuclear cells (PBMCs) were obtained from leukocyte
cules of protein-DNA complexes and to obtain the kinetic and platelet concentrates from healthy donors. Leukocyte platelet
affinity constants of the interaction. It works with an oligonu- concentrates were purchased from Regional Blood Donation and
cleotide reporter that self-hybridizes in the 30 -end forming the bind- Transfusion Center in Lodz. PBMCs were separated from ery-
ing site of a target protein, followed by a single-stranded DNA throcytes and granulocytes by density gradient centrifugation.
that pulls the reporter through the nanopore detector. We take The impact of fullerenol on the level of radiation damage to
advantage of the sequencing ability of the pore to distinguish dif- PBMCs was analyzed inside the cell and within plasma mem-
ferent DNA sequences and using different reporters we multiplex brane. The level of oxidative stress inside the cells was measured
the measurements. We use two oligonucleotides with distinct by fluorescent label H2DCF-DA and DNA fragmentation by
ssDNA tails and protein-binding sites that allow us to identify TUNEL assay. The lipid peroxidation in plasma membrane was
which protein-DNA complex molecule is passing the nanopore assessed by fluorescent label C11-BODI-PY581/591. The
detector. We also show that it is possible to analyze the inhibitory obtained results will be discussed in the context of the influence
effect of small compounds on each protein-DNA complex. Our of cellular localization of fullerenol on its radioprotective proper-
current work aims to expand the multiplexing capability of our ties. In addition, the genotoxicity of fullerenol to PBMCs will be
approach by generating a single-molecule barcoding system. This discussed.
would allow simultaneous measurements of hundreds of different
protein-DNA interactions within the same sample.
P.25-026-Tue
Biochemical study of selenite bioconversion by
P.25-024-Wed Azospirillum brasilense
The evaluation of gold nanoparticles and A. Tugarova, P. Mamchenkova, Y. Dyatlova, A. Kamnev
carbon dots as vaccine nanocarrier system Institute of Biochemistry and Physiology of Plants and
E. Yavuz1, S. Dinc1, M. Kara1, E. U. Bagriacik2 Microorganisms, Russian Academy of Sciences, Saratov, Russia
1
Selcuk University, Konya, Turkey, 2Gazi University Faculty of
Bacteria play an important environmental role in transforming
Medicine, Ankara, Turkey
selenium compounds, particularly in reducing selenium oxyanions
Recently, different antigen carrier systems are being explored to into less toxic and insoluble elementary selenium. The exact
increase the effectiveness of vaccines to fight infectious diseases mechanisms of this phenomenon are yet not fully clear. Different
and cancer. Especially nanoparticle-based delivery systems are cellular biochemical processes can be involved: anaerobic respira-
being widely investigated. Biocompatible gold nanoparticles and tion, denitrification, detoxification, etc. We present here the
carbon dots have been commonly used as potential delivery vehi- results of a study of selenite reduction by rhizobacteria of the
cles. Recently, gold nanocages (AuNCs), a special design with species A. brasilense with the formation of Se nanoparticles
ultra thin porous walls and hollow interiors, have successfully (SeNPs). A mutant unable to synthesise nitrite reductase showed
been used in the fields of image-guided delivery systems. Here, a higher resistance to selenite as compared to the parent strain
our aim is to use the freshly synthesized AuNCs and carbon dots (A. brasilense Sp245) but was still capable of reducing SeO32–.
extracted from sugar beet molasses for the delivery of specific This indicates that both the denitrification system and other
cargo and to compare their toxicological and inflammatory routes are involved. We optimised the synthesis conditions for
potential in innate immune cells (i.e. macrophages) in vitro. Here, these bacteria to produce extracellular homogeneous SeNPs. The
Au nanocages and carbon dots were used to explore their effect involvement of proton motive force-dependent transport in the
on the metabolic activity, uptake efficiency and immune extracellular synthesis of SeNPs was demonstrated: after treat-
responses of antigen presenting macrophage cells by different ment with the efflux pump inhibitor carbonyl cyanide m-
techniques such as Real-Time Cell Analysis System, Flow Cytom- chlorophenylhydrazone (CCCP), TEM images showed the forma-
etry and ELISA. The in vitro tracking of AuNC and carbon dot- tion of intracellular Se crystallites only. Extracellular SeNPs were
based nanocarrier systems was performed by confocal micro- isolated and characterised. Using FTIR spectroscopy it was
scopy imaging. We believe that construction, characterization shown that SeNPs are associated with proteins, polysaccharides
and biotesting of different nanocarrier systems might increase and lipids, while TEM images showed a thin layer over the NPs.
their promising potential in nanomedicine more. SDS-PAGE study of proteins associated with SeNPs showed the
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 479
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Nanoworld POSTERS
480 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Chemistry of food and environment
in cultured melanoma cells, HEK293 and LNCap cancer cell. Chemistry of food and environment
Upon penetration of the cells the anodic current quickly increases
followed by equilibration to a level above the one measured in
the cell media. A cell can withstand multiple penetrations and we
P.26-001-Mon
measured a substantial difference between the electrochemical sig- Bioactive molecules isolated from olive
nal measured inside and outside the cell. We believe these results pomace extract protect cells from calcium
show the potential of functional nanoelectrode to probe endoge- mediated damages
nous species into cells and with further improvements they may A. Franchi, M. Averna, A. Martines, M. Pedrazzi, R. De Tullio,
allow the study of oxidative stress under influence of different B. Sparatore, E. Melloni
drugs. The research was carried out at the expense of the Russian University of Genova, Genova, Italy
Science Foundation grant (project No. 17-75-10065) - Skolkovo.
In this study we show that molecules extracted from olive
pomace prevent cell death induced by Ca2+-overloading. Expo-
P.25-030-Wed sure of human neuroblastoma SKNBE, mouse brain endothe-
Electrospinning p-tert-butylcalix[4]arene lioma bEnd5 and human peripheral blood mononuclear cells to
these molecules counteracts the Ca2+-induced cell damages by
scaffolds manufacture for three-dimensional
reducing the activation of the Ca2+-dependent protease calpain.
colon cancer cell culture and their applications Indeed, this proteolytic enzyme is involved in key cellular pro-
in drug discovery cesses including apoptosis. In order to characterize this cell pro-
€
P. Uyar Arpacı1,2, F. Ozcan 2,3
tective effect, we purified the olive pomace extract and
1
Department of Biotechnology, Selcuk University, Konya, Turkey, concentrated the phenolic fraction, which contained mainly tyro-
2
Advanced Technology, Research and Application Center, Selcuk sol, caffeic acid, oleuropein and apigenin. However, none of these
University, Konya, Turkey, 3Department of Chemistry, Selcuk polyphenolic compounds, separately tested as purified molecules,
University, Konya, Turkey showed the protective effect on cell viability following Ca2+-
overloading. Hence, to identify the relevant bioactive molecules
A growing body of evidence has suggested that 3D cell culture
in the purified olive pomace extract, we performed a further frac-
systems, in contrast to the 2D systems, represent more accurately
tionation by RP-HPLC. Only one of the fractions obtained,
the actual microenvironment where cells reside in tissues. There-
maintained the ability to protect the cells from damages induced
fore, researchers have concentrated on developing realistic
by Ca2+-overloading. This finding indicates that the cell protec-
in vitro 3D cell culture models that actually resemble the complex
tive effect depends of molecules different from the main polyphe-
environment of native tissues. p-tert-butylcalix[4]arene, similar to
nols present in the purified olive pomace extract. We hypothesize
a ring basket, represent a third generation of supramolecular
that the molecular characterization of these molecules would
hosts. Synthesis of calix[4]arene nanofibers by electrospinning will
eventually define new strategies for therapeutic applications in
arise innovative approaches in biomedical applications to estab-
pathologies characterized by alterations of the intracellular Ca2+
lish in vitro cell-based systems that can more realistically mimic
-homeostasis. These promising starting points prompted us to
the in vivo cell behaviors and provide more predictable results to
plan further investigations aimed to identify the chemical nature
in vivo tests. 5,11,17,23-Tetra-tert-butyl-25,27-bis (3-aminomethyl-
of the bioactive molecules contained in this extract and the speci-
pyridineamido)-26,28-dihydroxycalix[4]arene functionalized with
fic cell target sites.
a pyridinium group on position 3 were synthesized by appropri-
ate procedures and the nanofibers were withdrawn by electrospin-
ning. Surface characterization of the nanofibers was done by P.26-002-Tue
SEM, TEM and AFM analysis. Caco-2 cells (1 9 105) were cul-
tured on nanofibers and after 48 h incubation, cells were treated
Effects of gluten-free buckwheat cookies on
with 240 lM irinotecan for 24 h. Cell growth/proliferation analy- plasma phospholipid fatty acid profile in
sis were done by XTT assay and fluorescent microscopy analysis healthy women
with DAPI stain. SEM/EDS measurement was used to character- M. Kojadinovic1, A. Arsic1, T. Popovic1, J. Debeljak-Martacic1,
ize the morphology of the attached cells. Cell attachment kinetics E. Stokic2, A. Mandic3
1
revealed that the Caco-2 cells attached to the nanofibers of p- Institute for Medical research Belgrade, University of Belgrade,
tert-butylcalix[4]arene at the same rate as to tissue culture plates. Belgrade, Serbia, 2Faculty of Medicine, Novi Sad, Serbia,
3
3D tumors on p-tert-butylcalix[4]arene scaffolds, it was found Institute of Food Technology, University of Novi Sad, Novi Sad,
that 3D Caco-2 cells were significantly more sensitive to irinote- Serbia
can than 2D Caco-2 cells, with a 10-fold disparity in the IC50 val-
Balanced and healthy diet, regular physical activity and promo-
ues. In the present study, p-tert-butylcalix[4]arene 3D cell culture
tion of healthy dietary habits with quality intake of carbohy-
systems for anticancer drug screening systems have been devel-
drates, fat and fatty acid, is very important not only for saving
oped as suitable platforms for drug screening and are served as
health care costs, but also for preventing many chronic patholog-
more reliable models for in vitro testing, compared to 2D.
ical conditions such as obesity, atherosclerosis, insulin resistance,
diabetes type 2 and cancer. In the present study we investigated
whether gluten-free buckwheat cookies with added blueberry and
raspberry powder can modulate plasma phospholipids fatty acid
(FA) composition in healthy subjects. In this study we included
20 healthy woman from 30 to 50 years old, which consumed glu-
ten-free buckwheat cookies during 4 weeks. Formulation of glu-
ten-free buckwheat cookies mixture, substituted with blueberry
and raspberry, was made in Institute for Food Technology.
Plasma lipids were extracted by a solvent mixture chloroform/
methanol (2:1). Plasma phospholipid fatty acid methyl esters
were analyzed by gas–liquid chromatography. Our results have
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 481
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Chemistry of food and environment POSTERS
482 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Biochemical education
different ages (0+, 1+, and 2+) and smolts (4+) exhibited differences Biochemical education
in the levels of the essential 18:2ɷ-6 and 18:3ɷ-3, with a higher pro-
portion of 18:2ɷ-6 and a higher 18:2ɷ-6/18:3ɷ-3 ratio among fish
in all of the age groups studied. This is connected to the abun-
P.27-006-Wed
dance, availability and digestion effectiveness of certain food types The necessity of biochemistry and molecular
that are, for the most part, rich in 18:2ɷ-6. The high content of biology for being pharmacist – a students’
22:6ɷ-3 among juvenile (at 0+ and 1+) is a mechanism of biochemi- opinion
cal adaptation related to the features of habitat use and environ- A. Dugonjic Okrosa1, S. Supraha Goreta2, L. Bach-Rojecky1,
ment (mainly, the current flow and rate): the motor activity of fish J. Dumic2
increases and the processes of elongation and desaturation of ɷ- 1
University of Zagreb Faculty of Pharmacy and Biochemistry,
3PUFAs derived from food are accelerated. In tributaries, juvenile Department of Pharmacology, Zagreb, Croatia, 2University of
trout (at 0+ and 1+) have active life styles (inhabit places with rapid Zagreb Faculty of Pharmacy and Biochemistry, Department of
currents) and actively feed at night, in contrast to older age groups. Biochemistry and Molecular Biology, Zagreb, Croatia
It was supposed that the content of 22:6ɷ-3 in structural phospho-
lipids reflects the degree of metabolic and physiological activity; in Without any doubt, the knowledge in biochemistry and molecu-
particular, the correlation of 22:6ɷ-3 to the degree of fish mobility, lar biology (BMB) is a prerequisite for understanding expert
i.e., their functional activity, was demonstrated. The research was knowledge in pharmaceutical sciences. Yet do Pharmacy students
supported by the Russian Science Foundation № 14-24-00102. shear that opinion? This research aimed to explore Pharmacy stu-
dents’ opinions regarding the necessity of the knowledge in BMB
for understanding of professional subjects. We also examined
P.26-006-Wed whether pharmacy students find the knowledge in BMB necessary
“Lipid sac”: physiological and biochemical for their future jobs and beneficial for their future career. This
survey was conducted among 100 pharmacy students of the 4th
adaptation of daubed shanny Leptoclinus
year, at the University of Zagreb Faculty of Pharmacy and Bio-
maculatus (Stichaeidae family) in Svalbard chemistry. As expected, the survey confirmed that most students
waters agree that the knowledge in BMB is necessary for understanding
S. N. Pekkoeva1, S. A. Murzina1, Z. A. Nefedova1, pharmacodynamics, pharmacokinetics, and mechanism of action
S. Falk-Petersen2, J. Berge3, O. J. Lønne4, N. N. Nemova1 of drugs and that this fundamental knowledge is a prerequisite
1
IB KarRC RAS, Petrozavodsk, Russia, 2Akvaplan-niva AS, Fram for apprehension of drug metabolism and biotransformation, and
Centre, Tromsø, Norway, 3The Arctic University of Norway molecular basis of disease. The vast majority of the respondents
(UIT), Tromsø, Norway, 4The University Centre in Svalbard, also indicated that it is essential to combine the fundamental
Longyearbyen, Norway knowledge in BMB with the expert knowledge fields. Around
one-third of students agreed that the acquired knowledge in
Fatty acids (FAs) of lipids are the most important structural and
BMB is necessary for their future job and that this knowledge
functional components of all living systems. Individual FAs and
will be beneficial for their future career. The conclusion of this
their combinations represent as biomarkers and as indicators
survey is that the students consider the knowledge in BMB neces-
reflecting the nutritional needs and physiological condition respec-
sary for understanding of the expert knowledge in general and
tively of fish during ontogeny. The reserve lipids (triacylglycerols
that these two should be combined. Moreover, it is very impor-
(TAG)) have in their structure mainly dietary FAs that allow con-
tant that the basic subjects contain in addition to fundamental
sider them as trophic biomarkers. Daubed shanny, Leptoclinus
principles their practical applications so the students could testify
maculatus, is a circumpolar fish and ecologically important as inter-
the importance of basic knowledge for understanding profes-
mediate link in the Arctic trophic food webs. A unique feature of its
sional subjects. This could make the process of studying easier
postlarvae is the provisional organ – a «lipid sac» that accumulates
and basic subject more interesting and their usefulness and appli-
a large amount of reserve lipids and maintains the buoyancy of
cability clearly visible.
pelagic postlarvae. FAs profile of lipid sac and flesh of the daubed
shanny during early ontogenesis were analyzed using the Equip-
ment Sharing Centre KarRC RAS. Monounsaturated FAs Miscellaneous
(MUFA) were dominating in TAG in the lipid sac. During early
ontogenesis, it was determined the shifts in major individual
P.28-001-Mon
MUFAs: hatched postlarvae had abundant amount of 18:1(n-9)
due to feeding on phytoplankton while older developmental stages Remodelling of the bacterial ribosome during
inhabiting pelagic zone showed large amounts of 20:1(n-9) and 22:1 transition into stationary growth phase
(n-11) FAs – biomarkers for zooplankton Calanus. These FAs due S. Lilleorg1, K. Reier1, L. Peil2, A. Liiv1, J. Remme1
1
to their physical and chemical properties maintain phase of lipids in Institute of Molecular and Cell Biology, University of Tartu,
the lipid sac and it is appropriate for developing pelagic larvae Tartu, Estonia, 2Institute of Technology, University of Tartu,
faced such severe conditions as low temperatures, starvation and Tartu, Estonia
change in mode of life from pelagic to bottom during development.
Ribosomes are essential enzymes that participate in gene expres-
Interesting, the lipid sac in adults, which descent to the bottom,
sion by conducting protein synthesis in every cell. Ribosomes
resolve and in muscles of adult L. maculatus, certain 18:1(n-9), 18:1
consist of two subunits both comprising ribosomal RNA and
(n-7) and 16:1(n-7) FAs dominated showing the feeding by benthos.
proteins. In general, ribosomes are considered as macromolecular
The research was made in the frame of the budgetary theme №
complexes with homogeneous and stable composition. This
0221-2017-0050 and was supported by RFBR No. 17-04-00466.
implies that all ribosomes are functionally equivalent and their
structure is rather insensitive to environmental changes. However,
growing experimental evidence indicates ribosome heterogeneity,
i.e. that there are structurally different ribosome subpopulations
in eukaryotic and bacterial cells. This notion has motivated the
study of ribosomes from the perspective of potential translation
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 483
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Biochemical education POSTERS
regulator rather than passive protein factory. This study investi- P.28-003-Wed
gates changes in bacterial ribosomal protein composition during Ribosome-associated quality control (RQC) in
cell growth focussing on Escherichia coli ribosomes. Quantitative
mass spectrometry analysis of ribosomes from different growth
Saccharomyces cerevisiae upon oxidative
phases has shown that bacterial ribosomal protein composition is stress
generally stable. However, both paralogs of a ribosomal protein M. Pietrzyk, A. Chacinska
(named bL31 and encoded by two paralogous genes with 45% Centre of New Technologies University of Warsaw, Warsaw,
identity) are simultaneously present in E. coli ribosomes in vivo Poland
demonstrating ribosome heterogeneity. Interestingly, their abun- Over senescence number of processes in the living cell become
dance changes remarkably as cells transition from fast growth to dysfunctional and lead to protein homeostasis imbalance. One of
stationary growth phase. The abundancy of bL31A paralog the key impairments is mitochondria dysfunction that may result
decreases remarkably concurrent with the respective increase of in reactive oxygen species (ROS) production. It is hypothesized
bL31B. Next, in vitro assays demonstrated that under acidic pH that increased levels of ROS lead to production of aberrant pro-
bL31A preferentially dissociates from ribosomes and that bL31B teins resulting in the ribosome stalling. With the use of model
replaces bL31A on ribosomes. In conclusion, we have demon- substrates it has been shown that ribosome-associated quality
strated on the example of bL31 paralogs that E. coli ribosomes control (RQC) is a crucial mechanism for clearance of non-stop
are heterogeneous at ribosomal protein level, their protein com- or no-go proteins. Here, we investigate the influence of RQC on
position changes at the end on fast growth and it is partially the protein homeostasis upon oxidative stress. We have con-
caused by protein exchange on ribosomes. firmed that cells lacking the RQC components (DLtn1, DRqc2),
show growth defect under translation inhibiting conditions (hy-
gromycin B or cycloheximide treatment). Interestingly, deletion
P.28-002-Tue
of Ltn1 or Rqc2 in yeast causes accumulation of dysfunctional
Profile of mitochondrial biogenesis markers proteins synthesized upon oxidative stress, which was mimicked
and acrosomal reaction are disturbed in by H2O2 treatment. Furthermore, the oxidative stress increases
spermatozoa from stressed adult rats number of ubiquitinated proteins. Taken together, these results
I. Starovlah1, S. Radovic1, D. Patricio2, T. Kostic1, S. Andric1 suggest that under conditions of increased ROS production aber-
1 rant nascent proteins are released from the ribosome through the
Department of Biology and Ecology, Faculty of Sciences,
University of Novi Sad, Novi Sad, Serbia, 2University of Aveiro, RQC pathway and are further ubiquitinated by Ltn1 for protea-
AVEIRO, Portugal somal degradation. The work was founded by National Science
Centre, Poland 2015/18/A/NZ1/00025.
Although psychophysical stress is the most common stress in
human society and major contributor to wide variety of patho-
logical conditions, the molecular adaptation of spermatozoa from P.28-004-Mon
stressed males were not described well. The aim of this study was Deletion of insulin and IGF1 receptors genes
to determine the functionality and molecular adaptation of sper-
matozoa from stressed rat by applying in vivo and in vitro
disturbs expression of the key mitochondrial
approach. For in vivo experimental model, psychophysiological biogenesis regulators in mouse prepubertal
stress by immobilization (IMO), was performed for 3 h in differ- steroidogenic cells of testes but not ovaries
ent time during the day (03:00 h, 07:00 h, 15:00 h), for one S. Radovic1, I. Starovlah1, S. Nef2, T. Kostic1, S. Andric1
1
(1xIMO) or ten (10xIMO) consecutive days. For in vitro Department of Biology and Ecology, Faculty of Sciences,
approach, epididymal spermatozoa from undisturbed rats were University of Novi Sad, Novi Sad, Serbia, 2Department of Genetic
stimulated with stress hormones adrenaline and cortisol for Medicine and Development, Faculty of Medicine, University of
30 min. Results showed that number of spermatozoa significantly Geneva, Geneva, Switzerland
decreased in all 10xIMO rats comparing to control. Acrosomal
Controlled changes in mitochondrial biogenesis and morphology
status (response to acrosome-reaction-inducer progesterone) sig-
are required for cell survival and homeostasis, but the molecular
nificantly decreased in spermatozoa from 1xIMO and 10xIMO
mechanisms are largely unknown. Here, the male and female
rats comparing to control. The same effect was observed in sper-
knock out prepubertal mice (P21), with insulin and IGF1 recep-
matozoa stimulated in vitro with stress hormones. Preliminary
tors deletion in steroidogenic tissues (Insr/Igf1r-DKO), were used
RQ-PCR results revealed that transcription of the main mito-
to investigate transcription of key regulators of mitochondrial
chondrial biogenesis markers Nrf1, Ppara and Ppard decreased in
biogenesis (Ppargc1a, Ppargc1b, Pparg, Nrf1, Tfam) in Leydig
spermatozoa from 10xIMO rats. In the same spermatozoa sam-
cells, ovaries and adrenals as well as mitochondrial architecture
ples the similar effect was registered for Ucp2, the mediator of
(Mfn1, Mfn2, Opa1) in Leydig cells. Accordingly, gene expression
regulated proton leak. Oppositely, the significant increase of Cytc
was followed employing RQ PCR, proteins expression detected
transcription was registered in spermatozoa from 10xIMO rats.
by western blot, while hormones level was measured by radioim-
Incubation of spermatozoa with adrenaline decreased level of
munoassay. Results showed that expression of PGC1, the master
Ppargc1a and Nrf2a transcripts, while cortisol decreased expres-
regulator of mitochondrial biogenesis and integrator of environ-
sion of mitochondrial transcription factor TFAM. Repeated psy-
mental signals, as well as its downstream target Tfam, signifi-
chophysical stress decreased the number and functionality of
cantly decreased in testosterone-producing Leydig cells from Insr/
spermatozoa and disturbed transcriptional profile of their mito-
Igf1r-DKO animals. This was followed with reduction of Mtnd1,
chondrial biogenesis markers.
the core subunit belong to the minimal assembly required for
catalysis. Transcription of mitochondrial biogenesis markers
remained unchanged in ovaries. Differently, in adrenals, the pat-
tern of mitochondrial markers transcripts was the same in both
sexes and, besides Pparg and Tfam, opposite from Leydig cells.
The transcription level of mitochondrial architecture markers
(Mfn1, Mfn2) was significantly increased in Leydig cells from
484 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Miscellaneous
Insr/Igf1r-DKO mice suggesting that the mitochondrial architec- also demonstrate that AKT phosphorylation and expression of
ture and mitochondrial phase of steroidogenesis was affected in the neuronal marker protein are increased when JAB1 is overex-
males. Our results are the first to show that insulin and IGF1 pressed in CPNE1 high expressed cells. Moreover, overexpression
receptors are important for mitochondrial biogenesis in gonadal of both CPNE1 and JAB1 effectively increased neurite out-
steroidogenic cells of prepubertal males, but not females, as growth. Collectively, our findings suggest that JAB1 activates the
important regulators of both mitochondrial biogenesis and archi- neuronal differentiation ability of CPNE1 through the binding of
tecture markers transcription. C2A domain in CPNE1 with MPN domain in JAB1.
P.28-005-Tue P.28-007-Mon
Autophagy in corpus luteum as a possibly Insertion-deletion mutations of a single
critical mechanism for rodent pregnancy and nucleotide in the rRNA large subunit
parturition methyltransferase gene rlmAII sways the
S. Kurusu, Y. Ishiwata, Y. Oishi, S. Oka, H. Yoshio, susceptibility of the honeybee pathogen
R. Terashima, M. Kawaminami Melissococcus plutonius to mirosamicin
Kitasato University School of Veterinary Medicine, Towada, Japan D. Takamatsu1, E. Yoshida2, E. Watando3, Y. Ueno1,
Ovarian corpus luteum (CL) is an ephemeral tissue whose regu- M. Kusumoto4, M. Okura1, M. Osaki1, K. Katsuda1
1
lated secretion of progesterone is essential for maintenance and/ National Agriculture and Food Research Organization, Tsukuba,
or timely termination of pregnancy in rodents. Our previous find- Japan, 2Iwate Prefectural Chuo Livestock Hygiene Service Center,
ing that CL of pregnant rats without Fas/FasL system (an Takizawa, Japan, 3Aichi Prefectural Chuo Livestock Hygiene
authentic apoptotic system) suggests that this tissue may alterna- Service Center, Okazaki, Japan, 4National Agriculture and Food
tively undergo autophagic cell death during regression. Here we Research Organization, Kagoshima, Japan
investigated the presence of autophagic system in CL and its any
American foulbrood (AFB) and European foulbrood (EFB)
implications in rodent pregnancy and parturition. LC3 (–I and – caused by Paenibacillus larvae and Melissococcus plutonius,
II) expression in CL was estimated by Western blot analysis in respectively, are two major bacterial infectious diseases of honey
comparison with progesterone secretion and luteal mass through-
bee broods. Although the 16-membered ring macrolide antibi-
out pregnancy. LC3 distribution was tested by immunocytochem- otics, mirosamicin and tylosin, have been used to prevent AFB in
istry. While the expression of cytosol-associated LC3-I was not Japan, macrolide-resistant P. larvae has yet to be found. M. plu-
significantly altered throughout pregnancy, that of autophago- tonius is not the target pathogen of these drugs; however, since
some-associated LC3-II increased significantly from day 15,
these macrolides are used as prophylactic medicines, M. plutonius
showed a peak on day 21, and decreased on day 23 (day of nor- may have also been exposed to these drugs in the field. In the
mal parturition). LC3-II/I ratio had positive correlations with present study, we revealed for the first time the presence of
steroidogenic activity and cell size. Immunoreactive LC3 was mirosamicin-specific macrolide-resistant strains in Japanese
found to be present in the cytosol of steroidogenic cells and its
M. plutonius and that the gene encoding the rRNA large subunit
aggregation was marked on day 21. Functional implication of methyltransferase A (rlmA2) was disrupted exclusively in
autophagy in CL tissue was examined by local treatment in vivo mirosamicin-susceptible strains due to the same single nucleotide
with Bafilomycin A1 (autophagy and V-ATPase inhibitor,
insertion. Primer extension assays demonstrated that M. plutonius
6.23 pg/0.1 ml/ovary) on day 15 or 19. The drug treatment on RlmA2 methylates G748 in hairpin 35 in domain II of 23S
day 15 resulted in altered timing of delivery associated with sig- rRNA, and the deletion of rlmA2 resulted in increased suscepti-
nificant reduction in steroidogenic cell size, but not progesterone bility to mirosamicin and the loss of methylation at G748 of 23S
secretion, compared to vehicle-treated control groups. We con-
rRNA, suggesting that methylation at G748 by RlmA2 confers
clude that the autophagy parameter is temporally matched with mirosamicin-specific resistance in M. plutonius. The single nucleo-
further structural and functional activation of CL and that Cl tide mutation in rlmA2 of mirosamicin-susceptible strains was
autophagy may contribute to activation, but not regression, of easily repaired by spontaneous deletion of the inserted nucleotide
rodent CL and thus their female reproduction.
under laboratory conditions, resulting in the acquisition of resis-
tance to mirosamicin; nevertheless, intact rlmA2 was only found
P.28-006-Wed in Japanese M. plutonius. In a Paraguayan strain tested and all
of the whole-genome sequenced European strains in the database,
JAB1 regulates CPNE1 function via direct rlmA2 was disrupted due to the same point mutation, suggesting
binding to CPNE1 that these strains are mirosamicin-susceptible. Since mirosamicin
J. C. YOO1, N. Park2, H. Y. Choi3, Y. H. Lee1, G. Yi1 has been used in apiculture only in Japan, its use as a prophylac-
1
KAIST, Daejeon, South Korea, 2Inje University, Busan, South tic medicine for AFB may have influenced the macrolide suscepti-
Korea, 3Gyeongsang National University Hospital and College of bility of the causative agent of EFB in this country.
Medicine, Jinju, South Korea
Copine1 (CPNE1), has tandem C2 domains and an A domain.
P.28-008-Tue
We previously demonstrated that CPNE1 directly induces neu-
ronal differentiation via Protein kinase B (AKT) phosphorylation Nicotine metabolic genes in Arthrobacter:
in the hippocampal progenitor cell line, HiB5. To better under- pAO1 vs AK-YN10
stand its cellular function, we carried out a yeast two-hybrid R. S. Boiangiu1, A. Andrei2, M. Mihasan1
1
screening to find CPNE1 binding partners. Among the identified Alexandru Ioan Cuza University of Iasi, Iasi, Romania, 2Albert-
proteins, Jun activation domain-binding protein 1 (JAB1) Ludwigs-University Freiburg, Freiburg, Germany
appears to directly interact with CPNE1. Between CPNE1 and The accumulation of sequence data through various sequencing
JAB1, the physical interaction was confirmed in vitro and in vivo. projects allows us nowadays to screen and identify metabolic
In addition the specific binding regions of CPNE1 and JAB1 was pathways in bacterial genomes. We are particularly interested in
confirmed with truncated mutant assay. Furthermore, our results
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 485
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Miscellaneous POSTERS
486 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Miscellaneous
that protective antioxidant molecules are present in the fraction been proposed that hydroxyl group at C33 position of the OlgA
containing low molecular weight metabolites (under 10 kDa). side chain might have an influence on its binding with the target.
Additional HPLC analysis resulted in the identification of five Also, recently we have found that modifications of OlgA macro-
fractions with anti H2O2 protective properties which are currently lactone core led to significant changes in its biological properties.
evaluated for their metabolite composition. This work was sup- In order to investigate mitochondrial ATP-synthase inhibition by
ported by the Russian Science Foundation project 16-14-10200 oligomycins more closely, a series of semi synthetic OlgA deriva-
and performed in accordance with the Russian Government Pro- tives with site-selective modifications were synthesized and their
gram of Competitive Growth of Kazan Federal University. inhibitory activity on the FoF1 ATP synthase were determined on
inverted membrane vesicles obtained from cells of Streptomyces
fradiae ATCC 19609 (strain, supersensitive to OlgA). It has been
P.28-012-Wed found that nitron-oligomycin, modified at positions C7 and C3
Identification of fucoidan sulfatases using into intramolecular heterocycle and 2,3,16,17,18,19-hexahydrooli-
bioinformatics and functional screening gomycin A with reduced double C-C bonds didn’t inhibit ATP-
approaches synthesis in the vesicles, probably due to significant change of
macrolactone geometry and, consequently, decreasing the affinity
A. Silchenko, A. Zueva, A. Rasin, M. Kusaykin, S. Ermakova
to the target. These data were in agreement with results of molec-
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far
ular modelling of binding of OlgA and its derivatives to the
Eastern Branch of the Russian Academy of Sciences, Vladivostok,
intracellular target. In striking contrast, 33-azido-33-deoxy-oligo-
Russia
mycin and 33-O-mesyl-oligomycin inhibited ATP synthesis more
Sulfatases play a key role in the catabolism of various sulfated potently than parent antibiotic. Finally, 33-dehydrooligomycin
polysaccharides (ulvans, carrageenans, agarans, fucoidans, etc.) and 33-deoxy-33-thiocyanato-oligomycin were slightly less active
of marine organisms. The variety of sulfated polysaccharide than OlgA. Thus, we can conclude that structural changes in
structures implies a large amount of sulfatases with different sub- macrolactone cycle can be critical for binding affinity of OlgA to
strate specificity. Despite advances in processing and annotating ATP synthase and functional groups at C33 position indeed play
the genomic and metagenomic data of marine microorganisms, the important role in ATP-synthase inhibition by OlgA. This
the correct annotation of carbohydrate sulfatases is still difficult. work was supported in part by Russian Science Foundation
Due to the fact that sulfatases acting on many marine polysac- (agreement № 15-15-00141).
charides have been poorly characterized, and some have not yet
been discovered. To date, only a few carrageenan sulfatases and
agaran sulfatases have been biochemically characterized. There P.28-014-Tue
are only fragmentary data about fucoidan sulfatases. Amino acid Relationship between iFGF23, iPTH and
sequences, specificity, mode and mechanism of action of fucoidan phosphorus in renal transplant patients
sulfatase are still unknown. We analyzed the genome of the A. Senelmis1, O. T. Pasaoglu2, U. Derici3, O. Helvaci3,
fucoidan degrading marine bacterium Wenyingzhuangia fucanilyt- H. Pasaoglu1
ica CZ1127 and identified 80 hypothetical sulfatases genes. Six 1
Gazi University, Institute of Health, Medical Biochemistry,
hypothetical sulfatases genes, named by us as swf1-6 were located Ankara, Turkey, 2Gazi University, Health Services Vocational
in close proximity to genes of fucoidanases (107 family of gly- School, Ankara, Turkey, 3Gazi University, Department of Internal
coside hydrolases CAZy). We assumed that the presence of sulfa- Medicine (Nephrology), Faculty of Medicine, Ankara, Turkey
tases coding genes in the same locus with fucoidanases indicates
their participation in the catabolism of fucoidans. To confirm IFGF23, a bone-produced hormone, plays a key role in phospho-
their function, genes of sulfatases were cloned and proteins were rus metabolism. The aim of this study is to investigate the rela-
produced in Escherichia coli cells. Functional screening among tionship between iFGF23, iPTH and Phosphorus in renal
hypothetical sulfatases using sulfated fucooligosaccharides and transplant patients. 40 males (age 39.28 11.01) and 20 female
fucoidans resulted to identification of two fucoidan sulfatases (age 40.05 12.09) renal transplant patients who are followed in
SWF1 and SWF4. Specificity and some catalytic features of sul- the transplantation polyclinic of Gazi University Medical Faculty
fatases were determined using various sulfated fucooligosaccha- Hospital were included in the study. The control group consisted
rides. Based on the substrate specificity, the enzymes are of 18 males (age 41.22 6.34) and 16 healthy females (age
classified as fucoidan exo-2O-sulfatase (SWF1) and fucoidan exo- 41.88 8.24) were included in the study. The mean transplant
3O-sulfatase (SWF4). This work was supported by the Russian year was 6.95 5.44. The median (min–max) values of serum
Science Foundation (Project No. 18-04-00905). iFGF23 in renal transplant patients were found to be 219.67
(93.7–652.83) pg/ml respectively, while serum iPTH levels were
83.6 (16.52–278.3) pg/ml, and serum Phosphorus was found to be
P.28-013-Mon 3.195 (1.6–5.7) mg/dl. Serum iFGF23 levels were found to be
ATP-synthase inhibition by semi-synthetic 119.67 (52.39–361.74) pg/ml in the control group, while serum
oligomycin A derivatives iPTH levels were 38.05 (3.41–109.1) pg/ml serum Phosphorus
3.52 (2.54–4.62) mg/dl respectively. Serum iFGF23 and iPTH
O. Omelchuk1,2, D. Mavletova3, T. Koshenko3, L. Lysenkova3,
levels were statistically significantly higher in the patient group
O. Bekker3, A. Vatlin3, V. Danilenko3, A. Shchekotikhin1,2
1 than the control group (P < 0.05). Whereas serum Phosphorus
Gause Institute of New Antibiotics, Moscow, Russia, 2D. I.
levels were statistically significantly lower in the patient group
Mendeleev University of Chemical Technology of Russia, Moscow,
than the control group (P < 0.05). Correlation study of trans-
Russia, 3Vavilov Institute of General Genetics RAS, Moscow,
plant patients showed a strong positive correlation between
Russia
serum iFGF23 and serum iPTH (P < 0.01) but no correlation
Macrolide antibiotic oligomycin A (OlgA) is a high-active ATP- between iFGF23 and Phosphorus (P > 0.05). There was no cor-
ase inhibitor, which is widely used for biochemical studies of relation between iFGF23 and iPTH and Phosphorus in the corre-
mitochondrial FoF1 ATP synthase. In micromolar concentra- lation study performed in the control group (P > 0.05).
tions, OlgA binds to Fo c-subunit and blocks proton transloca-
tion, resulting in disruption of bioenergetic metabolism. It has
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 487
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
Miscellaneous POSTERS
488 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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including 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, chronic inflammation and involvement of the migration and pro-
and numerous postlarval stages. To assess the larval variations in liferation of keratinocytes, among other factors such as infection,
the activity of trypsin, chymotrypsin and metalloproteinases along poor angiogenesis and increased oxidative stress. Keratinocytes
larval development, we measured the proteolytic activity using play a central role in healing, maintaining skin integrity, and par-
zymograms and specific fluorogenic substrates. Trypsin reached its ticipating in tissue repair through the release of cytokines,
maximum activity at the beginning of postlarval stage, while chy- chemokines and growth factors. Although SAA is involved in
motrypsin and metallopeptidase activity increased sharply at the skin disorders, the mechanisms of action of this protein are
third protozoea stage; in general, protease activity decreased signif- unclear. Our objective was to study the effect of SAA on the
icantly on subsequent stages. Using class-specific inhibitors and release of TNF-a, IL-1b, IL-6, IL-8, VEGF and FGF in cultured
mass spectrometry followed by bioinformatics analysis allowed us HaCaT keratinocytes. To define the toxic dose of SAA in ker-
to distinguish and identify protease bands on zymograms. It was atinocytes, we analysed the number of viable cells by MTT assay.
clear that each type of showed a particular pattern of appearance Cytokines and growth factors were quantified by ELISA in the
and disappearance during larval development, indicating that such supernatant of keratinocytes treated with SAA. After 24 h, the
peptidases fulfill a role in larval development yet to be investigated. supernatants were collected, and cytokines were determined by
ELISA. In the concentration range studied, SAA (0, 5, 10, 20
and 40 lg/ml) was not toxic to keratinocytes. A dose-dependent
P.28-022-Mon increase in TNF-a (r = 0.99, P < 0.05), IL-1b (r = 0.99,
Anti-inflammatory effect of palmitoleic acid on P < 0.05), IL-6 (R = 0.99, P < 0.05), IL-8 (r = 0.99, P < 0.05)
effector mechanisms of neutrophils and VEGF-a (r = 0.97, P < 0.05) by keratinocytes incubated in
E. Weimann1, B. Belmiro1, M. B. B. Silva1, G. M. Murata1, the presence of SAA was observed. In the studied conditions,
J. R. Bortolon1, A. Dermargos1,2, E. Hatanaka1 SAA did not alter the production of FGF-1. Our results point to
1
Cruzeiro do Sul University/Institute of Physical Activity and SAA as an important protein involved in signalling for healing,
Sport Sciences, S~ao Paulo, Brazil, 2Paulista University, S~ao scarring, angiogenesis and fibrosis. Added SAA could contribute
Paulo, Brazil to psoriasis pathogenesis via keratinocyte activation, maintaining
a cutaneous inflammatory and proliferative environment. Know-
Neutrophils play a pivotal role orchestrating the inflammatory ing the mechanisms by which SAA interferes with signalling for
response through migration to the inflammatory focus, phagocy- cutaneous healing is essential for the development of new thera-
tosis of microorganisms and/or cellular debris, and release of peutic agents. Financial Support: FAPESP (Process 16/24758-8)
cytokines, reactive oxygen species and other inflammatory media- and CNPq (Process 307769/2014-3).
tors. Palmitoleic acid, an n-7 monounsaturated fatty acid that
occurs in plant oils from macadamia nuts and sea-buckthorn, is
found in the sebum cutaneum of young humans and decreases P.28-024-Wed
with age. Recent studies have reported that palmitoleic acid is a Detection and Chlamydia trachomatis genovar
useful tool in treating disorders related to skin inflammation, but
distribution in blood and genital samples of
the mechanisms of action are unclear. Herein, an investigation
was made into the effects of palmitoleic acid in neutrophil death Chlamydia patients
(apoptosis and necrosis), migration to the inflammatory focus V. Feodorova1,2, Y. Saltykov1,2, S. Zaytsev1,
and cytokine release. In the concentration range studied (0– E. Sultanakhmedov3, S. Utz3, V. Motin4
1
200 lM), palmitoleic acid is not toxic to neutrophils as observed Federal Research Center for Virology and Microbiology, Saratov,
by the maintenance of membranes and DNA integrity in palmi- Russia, 2Saratov State Agrarian University, Saratov, Russia,
3
toleic acid treated neutrophils. In in vivo assays for neutrophils, Saratov State Medical University, Saratov, Russia, 4University of
including migration and the formation of exudate in a sterile Texas Medical Branch (UTMB), Galveston, United States of
inflammatory air pouch, it was observed that palmitoleic acid has America
potent anti-inflammatory activity, inhibiting LPS-induced TNF-a Zoonotic Chlamydia could be routinely detected in blood of
(73%, P ≤ 0.05), IL-1b (66%, P ≤ 0.001), IL-6 (75%, P ≤ 0.001), infected animals as well as in both their genital and extra-genital
MIP-3a (70%, P ≤ 0.05) and l-selectin (16%, P ≤ 0.05) release. sites. In contrast, C. trachomatis (CT) is typically found in the
Palmitoleic acid also inhibited LPS-stimulated neutrophil migra- urogenital, rectal or pharyngeal sites of Chlamydia patients. The
tion. In vitro, palmitoleic acid inhibited LPS-stimulated cytokine main goal of this study was to investigate a possibility of detect-
production by cultured human neutrophils. We concluded that ing CT DNA in the blood samples of Chlamydia patients who
palmitoleic acid has anti-inflammatory effects, decreasing the have demonstrated a positive response for the presence of CT
neutrophil inflammatory response. Further studies are needed to DNA in their genital samples. In total, clinical specimens from
elucidate the mechanisms involved in these effects. Financial Sup- genital sites (urethra and cervix) and blood from 22 individual
port: FAPESP (Process 14/03947-1 and 11/15360-7) and CNPq Chlamydia patients (males and females), mean age 26 years, and
(Process 307769/2014-3). 22 healthy subjects (males and females), mean age 25 years, were
enrolled in this study. DNA samples from both cohorts of
Chlamydia patients and healthy controls were amplified by PCR
P.28-023-Tue with commercial duplex TaqMan PCRs followed by sequencing
Serum amyloid a induces cytokine and growth of the ompA gene for genovar typing. Our results demonstrated
factor production in HaCaT keratinocytes the presence of CT DNA in the blood samples of 21/22 (95,4%)
B. Belmiro1, E. Weimann1, A. Dermargos1,2, E. Hatanaka1 and in genital specimens of 19/22 (86,3%) of Chlamydia patients,
1
Cruzeiro do Sul University/Institute of Physical Activity and respectively. There were CT genovars E (%), F (%) and G (%)
Sport Sciences, S~ao Paulo, Brazil, 2Paulista University, S~ao in the blood and CT genovars D (%), E (%) and G (%) in geni-
Paulo, Brazil tal samples. Based on the CT ompA single nucleotide polymor-
phisms (SNP), identical SNPs at positions 995 bp and 997 bp of
Serum amyloid A (SAA), an acute phase marker with potent
the genovar E were found in the samples from both blood and
proinflammatory activity, can modulate regenerating tissue, influ-
genital sites. The data obtained from DNA testing were con-
encing cutaneous healing. Poor wound healing is associated with
firmed when each CT-positive blood and genital specimen was
490 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
POSTERS Miscellaneous
examined by slide technique and direct immune-fluorescent test for 1 h at room temperature. The reduced aptamer was then
with CT A-L serovar-specific monoclonal antibodies. No positive dropped on a gold electrode. After incubation for certain time,
responses were seen in any specimen from healthy controls. This the electrode was incubated with 6-mercapto-1-hexanol (6-MCH)
is the first study that examined the impact and efficiency of for complete blocking of the electrode surface. For determination
detection of CT DNA in blood samples of Chlamydia patients of optimum measurement conditions, different incubation times
compared with their genital sites. This work was supported by with different concentrations of aptamer and 6-MCH were tested.
the Russian Science Foundation (Project No. 17-16-01099). In addition, the effects of buffer type and pH were investigated.
17 h of aptamer incubation, 1 h of 6-MCH incubation, 1 lM of
aptamer, and 1 mM of 6-MCH were identified as optimum con-
P.28-025-Mon ditions. The EIS measurements were recorded in 5 mL of 5 mM
Saliva extract of Dermacentor marginatus [Fe(CN)6]3/4 in 0.01 M phosphate buffer (pH 7.5) under the
(Acari: Ixodidae) leads to apoptotic cell death following conditions: a potential of 0.223 V, the frequency range
S. Demirkesen, S. Kar, C. Aral from 10 kHz to 100 mHz, and an amplitude of 0.01 V. A linear
Namik Kemal University, Faculty of Arts and Sciences, detection range between 5 pM and 500 nM of sarcosine was
Department of Molecular Biology and Genetics, Tekirdag, Turkey achieved. Preliminary results show that aptamer-based biosensor
is promising an alternative method for simple, reliable, sensitive
Physiologically, biologically and morphologically distinct, about and specific detection of sarcosine in urine. The forthcoming
900 different tick species have been reported worldwide. Some experiments will focus on further characterization of biosensor
properties such as antioxidant, anticoagulant and anti-angioge- and measurements in artificial urine samples. We thank the finan-
netic activities of tick-saliva have been shown in the studies con- cial support of TUBITAK (grant number 215Z182).
ducted on some tick species. In this study, it was aimed to
determine the effects of salivary gland extract of Dermacentor
marginatus, tick species which has not been studied for such a P.28-027-Wed
purpose, on the cell culture in some aspects. For this purpose, The effect of vitamin d on ovarian
adult male and female ticks were fed on the rabbits using ear bag
inflammation in experimental polycystic
method, and at the eighth day of feeding/in the fast engorgement
phase, ticks were detached. Of those, the female ticks were ovarian syndrome in rats
weighed, washed with distilled water, and salivary glands were S. Yıldırım-Tan1, G. Abban-Mete1, S. Tan1, N. Çil1, M. Secme2,
dissected and washed three times with phosphate-buffered saline. Y. Dodurga2, E. Mete3
1
Total protein extraction was done in PBS via mechanical disrup- Pamukkale University, School of Medicine, Department of
tion and protein content was determined by Bradford’s assay. Histology and Embryology, Denizli, Turkey, 2Pamukkale
Different doses of the extracts were added to MEFs and cell via- University, School of Medicine, Department of Medical Biology,
bility tested by MTT assay as well as microscopic examination. Denizli, Turkey, 3Pamukkale University, School of Medicine,
Apoptosis-related proteins (such as c-PARP, caspases) were Department of Medical Microbiology, Denizli, Turkey
determined by immunoblotting. As MTT assay is a marker of Polycystic ovary syndrome (PCOS) is a common endocrine disor-
mitochondrial activity, oxygen consumption rates were deter- der in women of reproductive age. In addition to diagnostic crite-
mined by using Clark-type electrode in digitonin-permeabilized ria such as chronic anovulation, infertility, hyperandrogenism,
cells. As a result, cell viability was decreased in a dose and time- hirsutism; it is associated with diseases such as insulin resistance,
dependent manner. A significant PARP cleavage via caspase obesity, endothelial dysfunction and metabolic syndrome. In
pathway induction was also found. Oxygen consumption rate of recent years, the relationship between PCOS-associated diseases
the cells treated with saliva extracts were found to be lower than and PCOS has been thought to be chronic inflammation and
the control group. After these initial findings, effects of tick sal- vitamin D deficiency. However, the results of studies investigating
iva on different cancer cell lines examined. Our preliminary data the association of vitamin D with PCOS are contradictory, and
evidenced that saliva of D. marginatus induces apoptotic cell immunohistochemical studies have not been found to explain
death. how the cytokines IL-1 beta IL-6 and TNF-alpha, which are
thought to increase in the case of inflammation, affect the ovar-
ian tissue. In this study, short-time and mid-term effects of vita-
P.28-026-Tue min D on IL-1b, IL-6 and TNF-a in the ovary were investigated
Optimization of aptamer-based impedimetric in dehydroepiandrosterone-induced PCOS rat model. We investi-
biosensor for detection of sarcosine gated whether pre-administration (daily from 2 h before PCOS
Z. Celik Canbay1, C. Ozyurt2, U. Mengulluoglu2, E. Dinckaya2, induction) and post-administration (DHEA injection daily for 20
S. Evran2 consecutive days and 20 days after the first injection of DHEA,
1
Ege University, Faculty of Science, Biochemistry Department, daily from 2 h before DHEA injection) of vitamin D could have
Izmir, Turkey, 2Ege University, Izmir, Turkey effect dehydroepiandrosterone-induced PCOS rat model. Vitamin
D has not been shown to have a significant effect on the IL-1 IL-
Sarcosine, a natural amino acid, is an intermediate of glycine
6 and TNF-a markers in the short-term in the ovary, but it has
metabolism. Recent studies revealed the role of sarcosine in acti-
been shown to reduce the number of cystic follicles. However,
vation of prostate cancer. Therefore, sarcosine has been identified
increased expression of IL-6, IL-1b and TNF-a was observed in
as a potential biomarker for monitoring the prostate cancer pro-
the longer exposure group to DHEA.
gression to metastasis. In this study, the interaction between sar-
cosine-specific DNA aptamer and sarcosine was monitored by
electrochemical impedance spectroscopy (EIS). Specific DNA
aptamer was selected using graphene oxide-assisted systemic evo-
lution of ligands by exponential enrichment (GO-SELEX). Then,
the aptamer was obtained in 50 -TTTTTT-thiol modified form.
Before each measurement, the thiolated aptamer was treated with
10 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP)
FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 491
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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492 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 493
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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494 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453 495
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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P.28-040-Mon Biotechnology
Effects of mutations mimicking
phosphorylation of myosin essential light P.07-063-Wed
chain on the actin-myosin interaction in Novel cucumovirus as a candidate for plant
optical trap assay biotechnology applications
D. Logvinova1,2, A. Matyushenko1,2, S. Nabiev1, O. Hertsen1, S. Silaev, T. Gasanova, P. Ivanov
L. Nikitina1, S. Bershitsky1 Lomonosov Moscow State University, Moscow, Russia
1
Institute of Immunology and Physiology, Ural Branch of Russian
The Baltic isolate of Gayfeather mild mottle virus (GfMMV),
Academy of Sciences, Yekaterinburg, Russia, 2A.N. Bach Institute
belonging to the genus Cucumovirus, family Bromoviridae, was
of Biochemistry, Research Center of Biotechnology of the Russian
isolated from Liatris spicata plant and characterized earlier by
Academy of Sciences, Moscow, Russia
one of the NGS methods (454 pyrosequencing of total RNA,
It is known that myosin essential light chain (ELC) can be phos- Adams et al., 2009). This approach is intended for rapid sequence
phorylated at two sites, Thr68 and Ser194, in skeletal and cardiac determination without purification of virions, especially in cases
muscles. However, till now it is unknown how the ELC phospho- of mixed infections. Unfortunately, it does not allow sequencing
rylation can affect the molecular mechanism of muscle contrac- of satellite RNAs that are characteristic for this viral group and
tion. In the present work, we tried to elucidate how the ELC significantly affect the development of infection; analysis of
phosphorylation affect the mechanical properties of the myosin encapsulated subgenomic RNA(s) is not possible as well. In our
motor, i.e., the isolated myosin head (myosin subfragment 1, S1). laboratory three genomic RNA sequences of a new Moscow iso-
For this purpose, we produced, using site-directed mutagenesis, late (GfMMV-Mo) discovered in Arctium lappa were defined by
three recombinant ELC constructs with mutations T65D, S193D, conventional Sanger method. Sequences of all terminal genome
and S193D/T65D, which mimic the naturally occurring ELC segments had noticeable discrepancies with the data published in
phosphorylation, and these ELC mutant constructs were then the GenBank. Exact determination of the 50 -proximal and 30 -
introduced into the S1 molecule. We applied an optical trap tech- proximal parts of the genome is necessary for cloning cDNA
nique to investigate the effects of these ELC mutations on the copies of genomic RNAs. Comparison of the gene sequences
mechanical characteristics of the S1 interaction with actin fila- responsible for replication revealed the high level of homology
ment. This method makes it possible to measure both step size of (99.1% to 99.5%). Phylogenetic analysis demonstrated an early
a single S1 molecule during its interaction with actin filament divergence of GfMMV from other Cucumovirus group members.
and duration of the step. It has been shown that all these ELC Moscow isolate was capable of establishing systemic infections in
mutations have no appreciable influence on the step size of S1 Nicotiana clevelandii, N. tabacum and N. benthamiana; the last
(from 8 to 11 nm). Mutations T65D and S193D/T65D signifi- two species are widely used in “green” biotechnology. Infection
cantly decreased the duration of the S1 step, by 30%, from 26– of Baltic isolate in N. tabacum was limited to inoculated leaves
28 ms to 19 ms, whereas the ELC mutation S193D had no effect (Adams et al., 2009). GfMMV-Mo is easily transmissible by
on the step duration. This implies that mutation T65D, but not mechanical inoculation and accumulation level of coat protein in
S193D, can affect the lifetime of the S1-actin complex during the Nicotiana spp. was about 3–4 g/kg of fresh upper leaves. Thus,
ATPase cycle, thus making faster the ATP-dependent interaction GfMMV-Mo is a promising object for plant biotechnological
of the myosin head with actin filament. These results indicate for applications such as constructing a viral vector(s) designed for
the first time that ELC phosphorylation at Thr65 may affect the expression of target protein in plants as well as assembly of
kinetic characteristics of the interaction of the myosin heads with genetically or chemically modified icosahedral nanoparticles or
actin filament in the absence of load and may play an important nanocontainers.
role in actin-myosin interaction during muscle contraction. This [Correction added on 14 September 2018, after online publica-
work was supported by the Russian Science Foundation (grant tion: abstract P.07-063-Wed has been added in this version.]
16-14-10044).
496 FEBS Open Bio 8 (Suppl. S1) (2018) 107–496 DOI: 10.1002/2211-5463.12453
ª 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.