************************************************************************************************************ Thanks to M.

Ambati for her time and dedication towards putting this material together for her DES sessions. Please use this material as a supplement to the primary material/notes given to you in the course, and not as a sole authoritative source for studying. ************************************************************************************************************

MEMBRANES
functions: compartmentalization, identity (self vs. non-self), transport mechanisms, signal transduction, energetics structure: phospholipid bilayer with hydrophobic core and hydrophilic heads fluid-mosaic model: lipid and protein components move around membrane through lateral diffusion (no flipping between internal and external faces) o NB: different population of phospholipids and other molecules present in internal face vs. external face

other molecules: sphingolipids, lipids, cholesterol (not in mitochondrial membranes), proteins o integral membrane proteins have hydrophobic cores and are usually glycosylated externally e.g. glycophorin found in erythrocyte cell membrane (single membrane spanning -helix with 19 residues)
NB: normally integral membrane proteins have 7 membrane-spanning regions

O-limited (attached by OH to serine or threonine) carries carbohydrate groups for blood group antigenicity

peripheral membrane proteins bound to membrane through electrostatic connections; eluted with salt e.g. mitochondria creatine kinase proteins attached via lipid or glycolipids anchors

lipid anchors saturated FA or propenyl chain (C15-farnesyl or C20-geranylgeranyl) is covalently linked to protein via thioether linkage GPI anchors (~glycosyl-PtdIno) long FA of inositol + sugar residues + ethanolamine attaches to C-terminus of protein to outer surface of plasma membrane e.g. lipoprotein lipase, acetylcholinesterase

protein:lipid ratio varies between membranes composition affects functions inner mitochondrial membrane = >50% protein (rich in cardiolipin)

nature of lipid composition affects fluidity:

saturated FA= fluidity unsaturated FA = fluidity inner mit. membrane 0 45 24 18 0 6

Plasma membrane cholesterol PtdCho (~lecithin) PtdEth Cardiolipin Sphingomyelin PtdIno (~signal transduction) 30 18 11 0 14 4

Lipid / membrane rafts: microdomains in membranes e.g. areas rich in cholesterol and sphingolipids o o o Tm and fludiity many proteins present with lipid or GPI anchors ~ signal transduction Caveolae invaginated lipid raft domains of plasma membrane that have roles in cell signaling and membrane internalization cholesterol + sphingolipids + caveolin (in cytosolic leaflet of plasma membrane in caveolae; creates a functional compartment that helps to hold proteins tied in signal transduction)

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Paroxysmal Nocturnal Hemoglobinuria genetic, but not inherited o o intravascular hemolysis and venous thrombosis caused by somatic mutations involved with assembling GPI anchors hematopoeitic stem cells attacked by immune system as a result

TRANSPORT
I. Passive Diffusion goes down concentration gradients a. Simple diffusion small, non-polar molecules (~soluble in lipids) i. b. e.g. CO2, NH3, ethanol no protein carrier necessary carrier proteins have binding sites and transport substrate via conformational changes (e.g. glucose transporter) channel proteins open to form pores in membranes 1. 2. allows substances of certain size ranges often gated with specificity for anions or cations iii. ligand-gated open when receptor is bound; e.g. IP3-gated Ca++-channel in ER for signal transduction voltage-gated open when voltage present; e.g. Ca++-channel in plasma membrane

Facilitated diffusion involved protein molecules that help to transport molecules down gradients across membranes i. ii.

conductance of channel:carrier proteins = 106:103

II.

Active Transport goes against concentration gradients a. Primary active transport uses ATP hydrolysis directly to drive transport of cations i. b. e.g. Na+/K+-ATPase (3 Na+ out, 2 K+ in) conformational change occurs with phosphorylation by ATP
+ Secondary active transport energy created by Na -gradient is used to co-transport other molecules = symport

i. ii.

e.g. brush borders of kidneys and small intestines accumulate glucose and amino acids, despite concentration gradient e.g. in parietal cells of stomach: H+-ATPase transporter exchanges H+ out and K+ in via established K+-gradient

FACILITATED DIFFUSION saturated kinetics specificity for solutes can be inhibited moves down concentration gradient to equilibrium no input of external energy

ACTIVE TRANSPORT saturated kinetics specificity for solutes can be inhibited solutes can go against concentration gradient NEEDS external energy input

Cardiac glycosides (~digitalis) inhibits Na+/K+-ATPase that increases force of contraction in failing hearts o after depolarization, there is [Na+] inside the cell without active Na+/K+-ATPase, Na+ can only exit cell via a Na+/Ca+++ ++ ++ exchanger (3Na out, 1Ca in) accumulates more Ca inside cell produces stronger contraction on subsequent depolarization

Cystic Fibrosis disease of membrane Cl- transporters o o o genetic mutation in CFTR prevents its transport from ER to plasma membranes salt content of secretions thick mucous vulnerability to bacterial infections blocks airways and pancreatic duct

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SIGNAL TRANSDUCTION PATHWAYS

hormones coordinate metabolic regulation between tissues; only tissues with appropriate receptors can respond to hormones in carbohydrate metabolism: o o o -cells (pancreas) secrete insulin -cells (pancreas) secrete glucagon acts on liver adrenal medulla secretes epinephrine produces similar response as glucagon

NB: insulin opposes actions of glucagon/epinephrine which act via protein kinase A (PKA) o insulin stimulates protein phosphatases and cAMP-phosphodiesterases cAMP and phosphorylated proteins

KNOW how glucagon/epinephrine interact with G-protein to cAMP o o o o glucagon/epinephrine bind receptor receptor binds -subunit of G-protein causes G-protein to exchange GDP for GTP dissociation of complex into bound receptor + -subunit + -subunit of G-protein with GTP -subunit of G-protein with GTP binds adenylate cyclase Gs (stimulatory G-protein) activates adenylate cyclase when it binds to it cAMP Gi (inhibitory G-protein) inhibits adenylate cyclase when it binds to it cAMP

Cholera toxin covalently modifies Gs so adenylate cyclase is always active cAMP Pertussis toxin inhibits Gi cAMP PHOSPHATIDYLINOSITOL BISPHOSPHATE PATHWAY (PIP2) o o o vasopressin, epinephrine, GH binds -adrenergic receptor activates G-protein activates phospholipase C (PLC) PIP2 (in membrane) (PLC) IP3 (released) + DAG (within membrane) IP3 binds to ER surface ER releases Ca++ Ca++ binds to calmodulin and activates calmodulin kinase II Ca++ also activates membrane-bound protein kinase C (PKC) o DAG binds to and activates PKC phosphorylates serine and threonine residues (e.g. MAP kinase pathway) depending on FA residues on PIP2, different DAGs are made phorbol esters mimic DAG keeps PKC on promotes cell growth (~tumor-promoter agent)

appreciate phosphoinositide cycle regeneration of PIP2 Ca++ binds to calmodulin activates Ca++/calmodulin dependent protein kinase conformational change exposes catalytic site o o activates myosin light chain kinase smooth muscle contraction vasoconstriction activates CaM-kinase II autophosphorylates itself to remain active autophosphorylated CaM-kinase II has an open catalytic domain with decreasing [Ca++], Ca++ diffuses off causing dissociation of Ca++ and calmodulin form complex molecular memory allows kinase to stay active even without Ca++ present until phosphatase comes and cleaves phosphate off inactivates CaM-kinase II

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MECHANISM OF INSULIN ACTION

Global insulin responses: glucose uptake, glycogen synthesis, protein synthesis, fat synthesis gluconeogenesis, glycogenolysis, lipolysis altered gene expression to support above responses

insulin binds receptor tyr kinase domain autophosphorylates tyr residues phosphorylates insulin receptor substrate (IRS) recruits PI3K to membrane NB: PI3K has SH2 domains (~sarc homology) that are essential for metabolic effects of insulin

PIP2 (PI3K) PIP3 (PTEN phosphotase) PIP2 o PIP3 activates PDK-1 activates protein kinases stimulates nutrient uptake and regulates cell metabolism phosphorylates PKB

stimulates nutrient uptake and regulates cell metabolism phosphorylates p70s6 (controls ribosomal activity) stimulates transcription and gene expression phosphorylates and inactivates GSK-3 (~glycogen synthase) stimulates gene expression, glycogen synthesis, and protein synthesis

UNDERSTAND how Ras acts like PKC MAP kinase activation

AMP-activated protein kinase (AMPK) great sensitivity and allows cell to respond to changes in energy-status

o o o

[AMP] activates AMPK in muscle (~exercise) and liver (~fasting) know [cytosolic citrate] is a measure of fuel excess (indirect measure of acetyl-CoA via malate-citrate shuttle) while [cytosolic citrate] is a measure of fuel deprivation [AMP] fuel deprivation [AMPK] inhibits ACCase [AMP] fuel excess [AMPK] stimulates ACCase malonyl-CoA

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DNA and RNA

DNA know structures of bolded items o o o o o Adenine and Guanine (~purines) and Cytosine, Thymine, and Uracil (~pyrimidines) other purines of importance: xanthine, hypoxanthine, urea (this comes back later in the term) nucleoside = base + ribose or deoxyribose (know difference between 2) nucleotide = base + sugar + phosphate know how to read DNA/RNA (5 3) and structure of phosphodiester linkage
NB: in the past, Chadwell has not tested any historical information

adenine makes 2 H-bonds to thymine, while guanine makes 3 H-bonds to cytosine

DNA exists in B-form (right-handed), 10 bases per turn (34 total), hydrophobic bases are inside, phosphate groups are outside know Watson-Crick model o DNA stabilization occurs via H-bonding and base stacking forces

understand what semi-conservative replication means (new DNA contains one template strand of old DNA and one new strand made during replication) denaturation/melting (dsDNA strands separate into ssDNA) when temperature increases; at Tm, 50% of dsDNA has separated o higher GC content, higher Tm DNA absorbs UV light at 260nm (ssDNA absorbs more than dsDNA hyperchromic effect) know other forms of DNA: B-form, A-form (R-handed, doesnt exist in nature; 11 bases/turn), Z-form (L-handed, 12 bases/turn, alternating sequence of pyrimidines and purines)

RNA higher mutation rate and more prone to cleavage at 2-OH exists primarily as single strands (double stranded regions caused by hairpin loops between sequences on same RNA strand) uracil replaces thymine as a base in RNA know tRNA structure

Supercoiling know difference between positive supercoiling (helix twists around its axis) and negative supercoiling (helix twists against its axis) topologic isomers same DNA sequence, different types of supercoiling type I topoisomerases (cleave only one strand, changes supercoiling one at a time) vs. type II topoisomerases (cleave both strands of DNA, changes supercoiling 2 at a time) o know drug interactions: camptothesin inhibits type I; quinalone inhibits DNA gyrase (~type II)

DNA packaging in euks: chromatin = DNA + histones histones 5 types = H1, H2a, H2b, H3, H4 nucleosome = 2H2a, 2H2b, 2H3, 2H4 1 H1 attaches to outside of nucleosome solenoids loops of 6 nucleosomes around an axis know packing ratios covalent modification of histones reduce its positive charge leads to unraveling e.g. phosphorylation, acetylation, methylation, ADP-ribosylation

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DNA Replication know properties of different DNA pol (prok and euk) DNA synthesis occurs 5 3 on both leading and lagging (~Okazaki fragments) strands within replication bubble (~bi-directional replication) helicases unwind and separate dsDNA via ATP hydrolysis o o rep is on leading strand; travels 3 5 (reference to template strand) hel II is on lagging strand; travels 5 3 (reference to template strand)

ssBP (single strand binding proteins) prevents reassociation of separated DNA after helicase unwinds it know topoisomerase function in DNA replication know that proks have 1 oriC that indicates origin of replication where dnaA of primosomes (dnaA + dnaB + primase) binds in euks, multiple origin sites are present understand how telomeres are added onto new strand using telomerases that add tandem repeats on 3 end of template strand using an RNA template strand KNOW DRUGS THAT INTERFERE WITH DNA REPLICATION o o o quinolones inhibit DNA gyrase (affects proks only) acyclovir antiviral drug against Herpes family; analogue of deoxyguanosine cytosine arabinoside arabinose is an epimer of ribose chain terminator

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TRANSCRIPTION
Know CENTRAL DOGMA o in proks and euks: DNA can be replicated or transcribed into RNA, which is then translated into proteins. o in viruses and telomerases: RNA is reverse transcribed into DNA o in viruses: RNA can be replicated prok mRNA: t1/2 = 20 min allows most bacterial proteins to be inducuble o inducible expressed when needed o constitutive always on euk mRNA: compartmentalization and functionally-specific cells o in nucleus replication and transcription o in cytosol translation RNA polymerases: o 2 types: DNA-dependent and RNA-dependent o NTPs source of energy; analogous to dNTPs in replication o new chain grows 53 o RNA/DNA template required o no primer required -4 -5 o high error rate (10 - 10 ) due to lack of proofreading o only non-sense strand is copied o in proks makes rRNA, mRNA, tRNA; has 4 subunits in holoenzyme (2) and 5 subunits in core enzyme (2) Initiation:

begins at promoters (slightly upstream of transcription site) which interact with -35 box = TTGACA Pribnow sequence (~-10) = TATAAT strong vs. weak promoters allows cell to control mRNA synthesis

subunit inhibited by rifamycin and rifampicin, while actinomycin D prevents strand separation Elongation: RNA holoenzyme has high processivity Positive supercoils removed by topoisomerases Termination: Rho-independent sites in transcript, GC-rich region followed by AT-rich region formation of hairpin loops RNA pol encounters hairpin loop and falls off

Rho-dependent sites GC-rich region that causes formation of hairpin loop in transcript RNA pol stalls Rho-factor moves down transcript (via ATP hydrolysis) until it sees stalled RNA pol Rho-factor catalyzes release of mRNA and RNA pol in euks similar features as prok transcription,but more complex 3 polymerases:

I in nucleolus, makes 5.8S, 18S, 28S rRNA not inhibited by -amanitin II in nucleoplasm, makes mRNA precursors, snRNA, hnRNA very high inhibition by -aminitin

III in nucleoplasm, makes 5S rRNA, tRNA, snRNA slight inhibition by -aminitin RNA pol II is the main transcription polymerase and has 2 promoters -25 box (~TATA box) = ATATAA -40 box (~CAAT box) = GGCCAATC

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POST-TRANSCRIPTIONAL MODIFICATION
6 types of modification: 1) 2) addition of CCA to 3-ends of tRNA by enzymes that dont need templates cleavage of tRNA primary transcripts a) 3) initially produced as one long mRNA chain that is cleaved into tRNA pieces (in both proks and euks)

base modification a) b) formation of minor bases in tRNA individuality mRNA editing conversion of bases into other bases i) in humans: 3 known cases (1) Wilms tumor susceptibility genes (2) Glutamate receptor in brain (3) **ApoB48 made from same transcript as ApoB100 C converted to U to create stop codon in ApoB48

4)

5-capping of euk mRNAs in nucleus (no capping in proks) a) b) c) d) 7-methyl guanosine attaches via 55 triphosphate linkage to beginning of mRNA transcript 2 stages: - guanyl transferase adds GTP to 5 end of mRNA - transmethylase methylates G know Cap 0 vs. Cap 1 vs. Cap 2 importance = identifies 5-end of transcript, protects from degradation, aids in transportation to cytoplasm from nucleus

5)

addition of polyA tail to 3-end of mRNA (~polyadenylation) a) b) c) is of varying length and determines length of transcript (~additional 150-200 bp) RNA pol II copies gene past 3-end; endoribonuclease cleaves transcript; polyA pol adds 3-AAA (via ATP hydrolysis) protects 3-end from degradation

6)

splicing in nucleus, removal of introns in mRNA (only in euks) a) b) c) d) exons coding region within gene introns spliced out of primary transcript know mechanism of splicing via SPLICEOSOMES alternative splicing different mature mRNAs produced from same precursor mRNA (depending on which exons are spliced together) [e.g. PFK-2/F2,6BP in liver and muscle differ]

GENETIC CODE
codons = 3 bases creates a reading frame that must be maintained degeneratecode 64 possible codons fdr 20 amino acids protects against mutational changes STOP CODONS = UAA, UAG, UGA START CODON = AUG (methionine in euks, f-methionine in proks) Code is non-overlapping and collinear

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PROTEIN SYNTHESIS / TRANSLATION

synthesized from N-terminal to C-terminal

mRNA is read from 53 (as opposed to replication and transcription) occurs in cytoplasm and is performed by ribosomes o o o o proks 70S = 50S + 30S euks 80S = 60S + 40S mitochondria has separate machinery, but makes few proteins (~13) constituents can be separated, but when mixed together, they all reassociate

tRNA adapter molecule that allows amino acids to interact with mRNA o o o ~80nt long with clover-leaf structure central single-stranded region contains ANTICODON that base pairs with codon on mRNA Wobble Hypothesis tRNA can recognize more than 1 codon first 2 bases at 5-end of codon interact with last 2 bases of anticodon via H-bonding last base of codon and first base of anticodon have less restrictive associations non-standard base pairing can occur o ATPAMP is needed to load amino acids onto tRNA by aminoacyl-tRNA synthetases (specific to each amino acid)

Translation more than 1 ribosome can simultaneously translate mRNA from 53 = efficiency (~polyribosomes) o Initiation: (similar in proks and euks, but proks is shown) starts at 5-AUG-3 codon with fmet-tRNAf in proks and met-tRNAf in euks

in proks: ribosomes recognize correct start point via Shine-Dalgarno sequence (7bp region upstream of AUG) in euks: no Shine-Dalgarno ribosomes start at first AUG encountered on mRNA eukaryotes = monocistronic (1 protein per mRNA), but prokaryotes = polycistronic (multiple genes per mRNA)

mRNA + 30S + fmet-tRNAf + GTP (+ IF-1, IF-2, IF-3) 30S initiation complex

fmet-tRNAf binds to P-site in 30S ribosome (only free aminoacyl-tRNA that binds to P-site; all others bind to A-site) determines reading frame A-site = amino acid P-site = peptidyl

30S initiation complex + 50S + GTP (- IF-1, IF-2, IF-3) 70S initiation complex o Elongation: fmet-tRNAf is in P-site aminoacyl-tRNA enters empty A-site on ribosome via Ef-Tu and GTP
NB: Ef-Tu does not interact with fmet so fmet is not incorporated into internal AUG sites

ester bond between fmet and terminal ribose of tRNAf is broken by peptidyl transferase (within 50S) energy drives formation of peptide bond tRNAf falls off P-site growing peptide-tRNA in A-site moves into P-site translocation mRNA moves a distance of 3nt relative to ribsome so next codon is in A-site EF-G and GTP needed

in euks, EF-G = EF-2 diphtheria toxin inactivates EF-2 and can be reversed by nicotinamide

Termination: at UAA, UAG, UGA no aminoacyl-tRNAs recognize these codons in proks: RF-1 recognizes UAA and UAG, RF-2 recognizes UAA and UGA, and RF-3 stimulates RF-1 and RF-2 binding in euks: RF recognizes all 3 stop codons that has ribosome-dependent GTPase activity for it to bind peptidyl transferase now catalyzes hydrolysis of ester bond between tRNA and completed polypeptide chain

o o

ribosome dissociates into subunits NB: **proteins fold as they are made

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ANTIBIOTICS o puromycin looks like 3-end of tRNA binds to A-site causes premature chain termination effective in euks and proks no clinical use o tetracyclines binds with smaller ribosomal unit prevents entry of aminoacyl-tRNA into A-site effective in euks and proks but more potent in some bacteria o streptomycin prevents binding of fmet-tRNAf to P-sites only affects proks o chloramphenicol interferes with peptidyl transferase activity of 50S only affects proks; toxic for all if it enters mitochondria o o o o o o cycloheximide inhibits peptidyl transferase in euk ribosomes (no clinical use) erythromycin blocks translocatn by binding to 50S prok ribosomes CBR703 prevents nucleotide addition by RNA pol rifamycin and rifampicin inhibits subunit in RNA pol and sequester Mg++ from RNA pol aminoglycosides binds to 30S subunit (e.g kanamycin, neomycin) fluoroquinolones inhibits bacterial topoisomerases (impedes replication, transcription, repair)

POST-TRANSLATIONAL MODIFICATION OF PROTEINS

1) 2) 3) 4) 5) 6) 7) folding of chain (proteins fold as translation occurs) removal of formyl goup or met disulfide bridges are formed glycosylation sugar moieties are added addition of prosthetic groups (e.g. heme) modification of amino acid side chains (e.g. hydroxylation or phosphorylation) removal of redundant sequences (e.g. activation of zymogens, **processing of insulin)

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MUTATIONS
I. Spontaneous mutations a. Tautomerism i. ii. iii. v. b. c. d. change to rare tautomeric forms causes illegitimate base pairs O: keto enol; N: amino imino shift must occur during replication other nucleases can also recognize and excise non-base-paired nucleotides excision occurs from nearest methylated GATC site to mismatch DNA pol then recopies

iv. is usually prevented by 35 exonuclease proofreading activity of DNA pol

5-bromouracil (5BU) analogue of thymine; mutagen (b/c induces mutations) nitrous acid (HNO3) induces deamination OH keto causes mismatches depurination and depyrimidation creates apurinic or apyrimidic sites i. ii. iii. can cause deletions if DNA pol skips missing base can cause substitution mutations any nucleotide is inserted into gap repair: (common to many repair processes after mismatched nucleotide is removed) 1. 2. 3. AP-endonucleases cleave DNA strand on 5-side leaving free 3-OH 53 exonuclease of DNA pol removes deoxyribose-P and replaces missing base DNA ligase seals nick

e.

deamination (of C and G) bacteria and higher cells have corrective mechanism i. ii. uracil-DNA glycosylase recognizes Us in DNA and breaks glycosidic bond between uracil and deoxyribose hypoxanthine-DNA glycosylase some As hypoxanthine which binds with C 1. removes hypoxanthine

II.

Spontaneous mutations caused by environment a. UV light can cause cross-linking of adjacent pyrimidines (e.g. thymidine dimers) i. b. c. UV endonucleases recognize backbone distortion 1. in E. coli: UvrABC cuts 8bp on 5-side and 4-5bp on 3-side; DNA pol fills gap; DNA ligase seals nick

Xeroderma pigmentosa genetic disorder; extreme skin sensitivity to UV light X-rays and ionizing radiation can create free radicals point mutations i. also causes DNA strand breaks

III. Chemical mutagens a. alkylating agents add alkyl groups to bases i. ii. b. i. ii. iii. DMS methylates Gs and As Nitrosoguanadine carcinogen polycyclic hydrocarbons intercalate between bases and promote frameshift mutations hycanthone intercalating agent actinomycin D

frameshift mutations deletions/insertions that change reading frames

iv. EtBr intercalating agent carcinogenic IV. All carcinogens are mutagens KNOW definitions: mutagen, point mutation, silent mutation, spontaneous mutation, back mutation, somatic mutation, transition mutation (purine to purine), transversion mutation (purine to pyrimidine, vice versa), frameshift mutation

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PROKARYOTE GENE REGULATION

transcription is main regulation step in prokaryotes regulate gene expression to maintain proper amounts of proteins and to respond to changes in environment constitutive genes always on (i.e. continually expressed) inducible genes turned on in response to environmental stimuli or presence of particular nutrient repressible genes products only required in certain quantitative amounts positive control mechanisms product(s) of regulatory genes are required for gene expression negative control mechanisms product(s) of regulatory genes are required to turn off or reduce gene expression ** regulated genes have promoters and operators o o promoters upstream of transcription start site that binds RNA pol operators located on 3-side of promoter (between promoter and transcription start site) where other proteins bind repressors bind to operator to prevent RNA pol from starting transcription activators bind to operator and either allow RNA pol to initiate transcription or stabilizes RNA pol in promoter region LAC OPERON o o allolactose lactose (-gal) glucose + galactose pieces: lacI = repressor (without inducer, LacI binds to operator)

lacI gene is not near promoter or operator

lacP = promoter where RNA pol binds to initiate transcription lacO = operator; if LacI binds here, prevents RNA pol from transcribing structural/functional genes = lacZ, lacY, lacA o o o with no inducer: (i.e. no lactose, allolactose, IPTG) LacI binds operator NO TRANSCRIPTION with inducer: inducer binds allosteric site on LacI LacI cant bind operator TRANSCRIPTION mutations lacI- = non-functional repressor Lac operon is always on lacZ-, lacY-, lacA- = non-functional enzymes/products lacP- = non-functional promoter, so RNA pol cant bind NO TRANSCRIPTION lacO- = non-functional operator, so repressor cant bind TRANSCRIPTION always on o if glucose is present: lac operon is repressed low cAMP levels (cAMP acts as hunger signal for when glucose is low) lac operon is only induced in absence of glucose and presence of lactose o if no glucose: AMP cAMP binds CRP (cAMP receptor protein) cAMP-CRP binds lacP and increases affinity of RNA pol transcription initiated
(assuming no repressor is bound to operator)

NB: if glucose, lactose cAMP-CRP not formed; cAMP-CRP only formed when glucose is low

repressor = sensitive to lactose levels; activator = sensitive to glucose levels + glucose - glucose no repressor, activator bound ON repressor bound, activator bound OFF
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+ lactose - lactose

no repressor, no activator OFF repressor bound, no activator OFF

TRP OPERON always on, except when [trp] is high o pieces: trpR = TrpR = repressor/regulatory protein that binds to trpO when trp is present

trp is co-repressor

trpP = where RNA pol binds trpO = where TrpR + trp binds prevents RNA pol binding and transcription trpA, trpB, trpC, trpD, trpE = molecules needed for trp synthesis o o without co-repressor/trp: TrpR is inactive cant bind TrpO TRANSCRIPTION with co-repressor/trp: trp binds TrpR active TrpR binds trpO prevents RNA pol binding NO TRANSCRIPTION as [trp] decreases, trp diffuses off TrpR and inactivates repressor o in proks: attenuation is a second level of control based on transcription and translation occurring at same time trpL (between trpO and transcription start site) produces leader peptide when trp-tRNA is present

[trp-tRNA] fast ribosome through trpL creates 3-4 hairpin loop RNA pol falls off terminates transcription [trp-tRNA] slow ribosome through trpL creates 2-3 hairpin loop blocks 3-4 hairpin loop RNA pol stalls while waiting for trp-tRNA transcription continues

EUKARYOTE GENE REGULATION

spatial expression tissue-specific genes temporal expression embryonic development (time/order-dependent)

occurs at: transcriptional control, mRNA processing, mRNA transport, mRNA stability, translation, protein stability/processing DIFFERENTIAL GENE TRANSCRIPTION: (2 mechanisms) 1) epigenetics changes in phenotypic effects (heritable) without changes in DNA sequence a) via modification of chromatin (histone modifications) i) ii) b) i) ii) euchromatin acetylation of histones heterochromatin deacetylation of histones, DNA methylation, phosphorylation, ubiquitination know Dnmt3, MeCP2, Dnmt1 DNA methylation diseases (1) Immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) defective Dnmt3b (a) chromosomes 1, 9, 16 are unstable (b) facial dysmorphism, mental retardation, recurrent/prolonged infections, variable immunodeficiency (2) Retts Syndrome mostly affects females; mutation in X-linked MeCP2 (a) loss of speech and hand skills; develops ~6-18 months (3) Prader-Willi Syndrome (in males) (4) Angelman Syndrome (in females) abnormal DNA methylation of specific DNA sequences (5) Cancers 2) differential expression and activation of transcription factors that bind to cis-regulator sequences a) basal transcription machinery initiate low levels of transcription i) ii) b) also needs activator, coactivator, chromatin remodeling protein to initiate efficient transcription interaction between basal transcriptional machinery located at a genes promoter and trans-acting proteins bound to cisregulatory elements TRANSCRIPTION repressor proteins reduce transcription levels through competition or quenching multiple sets of gene regulatory proteins can work together to influence transcriptional initiation

via DNA methylation can replicate but not transcribe

trans-acting factors repressors/activators on different chromosomes stimulate transcription i) ii)

c)

cis-regulatory elements conserved DNA (promoter/enhancers) where trans-acting factors bind; same chromosome
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i) d) e)

genes involved in same processes have similar cis-regulatory elements so have similar activators/repressors

DNA:protein and protein:protein interactions are important to transcription factors folding of DNA allows interactions btw. enhancers/repressors bound to distant cis-elements and basal transcription machinery (e.g. zinc fingers, helix-turn-helix, leucine zipper)

REGULATION OF GENE EXPRESSION BY REGULATING mRNA STABILITY AND TRANSLATION: mRNA needs to be stable in order for it to be transcribed important cell functions can be regulated by modifying o o mRNA stability (polyadenylation or targeted degradation) ex.: iron transport longer poly-A tails on mRNAs promotes bind proteins that activate translation factors mRNA translational efficiency (mRNA packaging, et al) ex.: iron storage (ferritin stores iron)

iron: ferritin mRNA blocked by iron regulatory proteins (IRPs) (to increase free iron) transferrin receptor expression is ON iron: ferritin protein made; excess iron is stored IRP1, IRP2, IRE is inactivated by iron transferrin receptor expression is OFF ferritin expression is ON ferritin expression is OFF

during embryo development, mother gives mRNA to unfertilized egg, that is protected by ribonucleoproteins (RNPs)

ionic changes in Ca++, Na+, H+ after fertilization cause RNPs to dissociate TRANSCRIPTION

GENES IN EMBRYONIC DEVELOPMENT

I. bcd and caudal mRNA: anterior/posterior polarity of fruitflies a. b. II. bcd gradient: bcd inhibits translation of caudal mRNA anterior caudal levels stays constant: caudal > bcd posterior

Hox expression: define final segmentation of embryos highly conserved areas a. functions: i. ii. iii. b. anterior/posterior patterning of limb bud ([Shh] pinky; [Shh] thumb) dorsal/ventral patterning of neural tube and floor plate separation of eye field in anterior CNS during embryonic development (Shh shuts off Pax6 in middle of optic field)

III. Sonic hedgehog: paracrine signaling between cells

Shh binds Patched within Patched/Smoothened complex conformational change causes Smoothened to phosphorylate GLI proteins allows transcription of Shh response genes

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MOLECULAR GENETICS
1) Tools: a) b) c) restriction enzymes cut DNA at precisely defined palindromic base sequences (sticky-ends vs. blunt-ends) i) recognition sequences can vary considerably in length but probability of occurrence of sequence can be calculated DNA ligases rejoin pieces of DNA vectors DNA or RNA used to insert fragments of foreign DNA for insertion into living cells i) ii) d) 2) usually contains oriC site, gene for antibiotic resistance and lacZ gene with other restriction enzyme sites size-limited to be able to enter bacteria

CsCl centrifugation DNA separation based on size within cesium chloride gradient

Assays / Methods: a) b) c) d) DNA gel electrophoresis separates DNA pieces based on size i) @ alkaline pH, DNA has a negative charge moves towards + in agarose or polyacrylamide gel Southern blotting transfers DNA from electrophoresis gel to nitrocellulose membrane that covalently binds DNA Northern blotting transfers mRNA to nitrocellulose membrane Western blotting transfers proteins to nitrocellulose membrane immunochemical technique i) blotting allows location within gel to be preserved for further analyses labeled ssDNA, cDNA, mRNA binds DNA/RNA of interest; labeled protein/antibody binds protein of interest > ii) e) i) radioactive or fluorescent labels high sensitivity of fluorescent probes allows detection of specific genes on chromosomes (ex.: FISH) isolated genomic DNA is digested with a restriction enzyme resulting cut DNA is separated by gel electrophoresis transferred to a membrane (Southern blotting) membrane is hybridized with a labeled DNA fragment corresponding to chromosomal region containing polymorphism for example: normal HbA -globin gene has restriction site for DdeI which disappears in HbS mutated genes changes pattern of RFLPs need: (1) primers (free 5 -OH) are required to initiate DNA synthesis; short (~20-25nts) ssDNA molecules that bind to specific DNA sequences; initiation sites for DNA polymerization (2) Deoxynucleotide tri-phosphates (dNTPs): dATP, dCTP, dGTP, dTTP for growing DNA strand (3) DNA polymerase: to synthesize new DNA strand using a complementary DNA strand as template (4) magnesium chloride (cofactor required for DNA pol), buffer (stable reaction conditions), template DNA ii) steps: (1) denaturation heat separates template DNA strands (2) annealing primers bind to complementary regions in template DNA (3) extension DNA pol replicates template strands iii) g) i) synthesis of a DNA strand from template DNA results in an exponential increase in number of DNA molecules (2n) insert gene into bacterial plasmid (1) using restriction enzymes and DNA ligases, insert DNA of interest into vector hybrid plasmid vector (2) transform hybrid vector into bacterial cell host (3) culture bacteria in presence of antibiotic isolate copies of plasmid (via selection) (a) cells that grow in presence of antibiotic have vector (b) cells cultured in presence of IPTG: cells with hybrid vector (contains lacZ gene disrupted by DNA of interest) do not form blue colonies ii) h) DNA of interest can be extracted from bacterial host by: lysing cells separating plasmids and bacterial chromosomes using size exclusion techniques restriction enzyme digest of plasmid gel electrophoresis similar principle as PCR: denaturation, annealing, extension
- 15 -

Restriction fragment length polymorphism (RFLPs) result from different patterns of distribution of restriction sites

ii) f)

Polymerase Chain Reaction (PCR) DNA amplification from a small amount template DNA i)

Vector cloning

Sanger Sequencing method sequence DNA i)

ii)

but , reaction mixture also contains ddNTPs (dideoxynucleotides, no 3-OH) that are radioactively or fluorescently labeled > DNA pol needs free 3-OH to be able to polymerize additional dNTPs to strand (1) chain termination occurs (2) fluorescent labeled: 1 reaction gel electrophoresis to visualize fluorescence (each ddNTP has own color) radioactive labeled: 4 separate reactions (1 for each ddNTP)

i)

Chromosome walking: end sequence of DNA is known i) ii) iii) primer is developed using known end sequence DNA at ends are sequenced/amplified using primer new DNA products are used to develop the next set of primers DNA strand of interest is sequenced from outside-in DNA is injected into mouse oocyte, 3 possibilities: (1) foreign DNA is hydrolyzed and gone (negatively selected by neomycin) (2) foreign DNA integrates itself randomly into a gene (negatively selected by gancyclovir) (3) **intended outcome: foreign DNA is slightly complementary and can bind to mice DNA during replication replaces normal functioning mouse gene KNOCKOUT (a) ES cells will be resistance to neomycin and will survive in the presence of gancyclovir

j)

Knockout mice: production of mice with genetic diseases that mimic human disease i)

3)

5 gene therapy strategies: (KNOW) a) b) c) d) e) gene augmentation replaces non-functional gene with functional copy targeted killing of disease cells for diseases that can be cured by eliminating certain populations of cells (~cancer) assisted killing of disease cells by immune system introduction of a gene into disease cells that will express a protein that makes the cells vulnerable to attack by the immune system targeted inhibition of gene expression introduce a gene whose product inhibits expression of pathogenic gene or interferes with activity of its product targeted gene mutation correction homologous recombination techniques used to replace or correct defective genes

4)

RNA Interference shuts defective genes OFF a) b) some human diseases are caused by overactive genes use complementary RNA to make double-stranded or triple-stranded mRNA ds or ts mRNA is naturally degraded by cell

- 16 -

NITROGEN METABOLISM
nitrogen enters through diet and leaves body as urea and ammonia need amino acids/proteins in diet since we only recapture 75% of proteins that we degrade protein turnover constant synthesis/degradation based on need o 2 systems of protein degradation: ubiquitin-proteasome proteolytic pathway

proteins destined for degradation are tagged with poly-ubiquitin chain which are recognized by proteasomes ubiquitin feeds protein through barrel of proteasome protein is cleaved non-specifically into small fragments

t1/2 is determined by nature of N-terminus residue e.g. asp: t1/2 = 3 minutes; ser: t1/2 = 3 hours PEST (pro, gln, ser, thr) are rapidly degraded by Lysosomes

DIGESTION OF DIETARY PROTEINS necessary since proteins are too large to be absorbed by intestines o digestive enzymes that degrade proteins are produced in stomach, pancreas, small intestines

1)

Stomach: a) b) HCl denatures proteins to increase susceptibility to hydrolysis pepsin secreted as pepsinogen by serous cells of stomach i) ii) activated by HCl or autocatalytically by other pepsins releases peptides and few free amino acids

2)

Pancreas a) specificity for R-group on amino acids i) ii) iii) trypsin cleaves at arg, lys chymotrypsin cleaves at phe, tyr mainly; also at trp, met, leu elastase cleaves at ala, gly, ser

iv) carboxypeptidase A cleaves at ala, ile, leu, val v) b) carboxypeptidase A cleaves at arg, lys

release of zymogens mediated by chylecystokinin (CCK) and secretin i) ii) CCK inhibits gastric motility; activates pancreatic and bile secretions secretin activates bicarbonate secretions (to pH)

c)

activation occurs in duodenum: trypsinogen (enteropeptidase) trypsin i) trypsin is common activator of pancreatic zymogens and phospholipase A2

d) 3)

abnormalities result in undigested fats and proteins in feces

Small intestine a) on luminal surface aminopeptidase cleaves oligopeptides at N-terminus

4)

Absorption of amino acids and dipeptides by intestinal epithelial cells a) Free amino acids enter hepatic portal system where its metabolized or released into circulation

- 17 -

TRANSPORT OF AMINO ACIDS INTO CELLS presence of -amino group prevents oxidative breakdown need to remove amino group to produce energy 1) transamination amino groups are transferred to glutamate a) b) amino acid + -ketoglutarate (aminotransferase/transaminase) -keto acid + glutamate
cofactor = vitB6(pyridoxal phosphate)

glutamate i) ii) is an amino group donor in synthesis of nonessential amino acids or is oxidatively deaminated

c)

aminotransferase (found in liver, kidney, muscle, intestine) i) ii) all amino acids are transaminated, except lys and thr (which are only deaminated) substrate specificity each aminotransferase is specific to a particular amino acid (1) e.g. alanine aminotransferase (ALT) and aspartate aminotransferase (AST) key liver enzymes

d) e) 2)

Keq = 1: balances degradation and biosynthesis diagnostic value: plasma levels normally; if levels increase, sign of cell death/damage

oxidative deamination of amino acids in liver and kidneys a) b) liberates amino group as free ammonia provides -keto acids and ammonia i) glutamate dehydrogenase (reversible and rapid) (1) glutamate + NAD+ (glutamate DH) -ketoglutarate + NADH + NH3 (2) -ketoglutarate + NADPH + NH3 (glutamate DH, ~reductive amination) glutamate + NADP+ (3) direction depends on concentrations of glutamate, -ketoglutarate, ammonia ii) iii) deamination of his, ser, thr, gln, asn irreversible other deamination enzymes: (1) glutaminase, asparaginase, histidase, serine dehydratase, c) d) ATP, GTP: allosteric inhibitors ADP, GDP: activators

GLUCOGENIC carbon skeletons of these amino acids enter gluconeogenesis (~pyr or TCA intermediates) ala, arg, asn, asp, cys, glu, gln, gly, his, pro, ser KETOGENIC carbon skeletons of these amino acids lead to acetyl-CoA or acetoacetate lys, leu GLUCOGENIC and KETOGENIC tyr, ile, phe, trp NB: to make urea free ammonia:aspartate = 1:1 ammonia toxicity free ammonia is trapped by glu and gln energy metabolism -ketoglutarate + NH3 glutamate glutamate + NH3 glutamine

- 18 -

Kidney excretes nitrogen in the form of: o creatine and creatine phosphate creatinine o purine nucleotides uric acid o ammonium ions (dependent on acid-base homeostasis) Positive nitrogen balance = nitrogen intake > nitrogen excretion growth, pregnancy, recovery from illness Negative nitrogen balane = nitrogen intake < nitrogen excretion protein malnutrition, physiological stress

TRANSPORT OF AMMONIA TO LIVER

2 mechanisms: 1) in most tissues: 2) mainly muscle:

glutamate + ammonia + ATP (glutamine synthase) glutamine + ADP + Pi glutamine (blood) glutamine (liver) (glutaminase) glutamate + ammonia

non-toxic transport

pyruvate + glutamate (alanine transaminase) alanine + -ketoglutarate alanine (muscle) alanine (blood) alanine (liver) (~aka glucose-alanine cycle) alanine (liver) + -ketoglutarate (alanine transaminase) pyruvate + glutamate pyruvate glucose

UREA CYCLE
urea: major disposal form of amino groups from amino acids (source of 90% N in urine) o carries 2Ns one from free ammonia, one from aspartate [NB: glutamate is the immediate precursor for both] o carries 1C, 1O from CO2 o produced in liver, transported in blood, excreted in kidneys into urine o 1 urea = free ammonia:aspartate = 1:1

Summary: 2 NH3 + CO2 + 4ATP + aspartate urea + fumarate + 4ADP + 4 Pi Preliminary reactions: 1) when [glutamate]: glutamate + acetyl-CoA N-acetyl glutamate + CoA N-acetyl glutamate glutamate + acetate N-acetyl glutamate activates carbamoyl phosphate synthetase I stimulated by: arginine Note central role of glutamate in bringing free ammonia and forming aspartate to participate in urea cycle.

2)

Reactions of cycle: 1) 2ATP + HCO3- + NH3 (carbamoyl phosphate synthetase I, CPSI, mitochondria) carbamyl phosphate + 2ADP + Pi RATE-LIMITING STEP; 2 ATPs hydrolyzed stimulated by: N-acetyl glutamate carbamoyl phosphate + ornithine (ornithine transcarbomylase, OTC, mitochondria) citrulline + Pi release of phosphate drives reaction citrulline is transported into cytosol from mitochondria citrulline + aspartate + ATP (arginosuccinate synthetase, cytosol) argininosuccinate + AMP + PPi arginosuccinate contains all components of urea utilizes 2 ATP essentially (since ATP AMP) argininosuccinate (arginosuccinate lyase, cytosol) Arg + fumarate [fumarate] is low in cytosol: fumarate (hydrolyzed) malate malate in cytosol (via malate shuttle) malate into mitochondria TCA malate (oxidation) oxaloacetate re-used to form aspartate

2)

3)

4)

[~urea-TCA bicycle]

5)

Arg + H2O (arginase, cytosol) ornithine + urea this isoform is only found in the liver; kidneys, small intestines have other isoforms where urea can not made ornithine enters mitochondria
- 19 -

- 20 -

AMINO ACID CATABOLISM

involves removal of -amino groups and break down of remaining carbon skeleton few things to memorize: o o o o ketogenic amino acids (Ls): leucine and lysine note both are essential glucogenic and ketogenic amino acids (TPIT): glucogenic amino acids: non-essential = tyrosine essential = phenylalanine, isoleucine, tryptophan

essential = MTV = methionine, threonine, caline non-essential = all others

essential amino acids: PVT TIM HALL Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Lysine, Leucine

break down into acetyl-CoA or acetoacetate ketogenic acetoacetate phenylalanine, leucine, lysine, tryptophan, tyrosine acetyl-CoA isoleucine break down into pyruvate, OAA, fumarate, succinyl-CoA, -ketoglutarate glucogenic pyruvate alanine, cysteine, glycine, serine, threonine, tryptophan OAA aspartate, asparagine fumarate phenylalanine, tyrosine (rings) succinyl-CoA threonine, isoleucine, methionine, valine -ketoglutarate proline, histidine, arginine, glutamate, glutamine [success = Tim V] [PLLAATT] AA=acetoacetate

some clinically relevant amino acids: methionine source of methyl groups in metabolism (via SAM)** glutamine storage and transport of ammonia; precursor of purines and pyrimidines phenylalanine precursor of tyrosine; elevated in PKU histidine precursoe of histamine; elevated in histidinemia alanine transport form of ammonia from muscle

**KNOW Fig. 20.14 in LIPPINCOTT! know key enzymes that play a role in disease processes know co-factors and which reactions they influence

tetrahydrobiopterin (BH4) is formed from GTP

- 21 -

AMINO ACID BIOSYNTHESIS

Non-essential amino acids can be synthesized within the body from intermediates of metabolism or from essential amino acids. o transamination from -keto acids: pyruvate + amino acid (alanine transaminase) alanine + -keto acid OAA + amino acid (aspartate transaminase) aspartate + -keto acid -ketoglutarate + amino acid (aminotransferase) glutamate + -keto acid -ketoglutarate + NADPH+ (glutamate DH) glutamate + NADP+ o by amidation: glutamate + ATP + NH3 (glutamate synthetase) glutamine + ADP + Pi (1) driven by ATP hydrolysis; major mechanism for ammonia detoxification in liver aspartate + ATP + glutamine (asparagine synthetase) asparagine + glutamate + AMP + PPi o glutamate (cyclization)(reduction) proline o phenylalanine (phenylalanine hydroxylase) tyrosine o glycine + methylene-THF (serine hydroxyl-methyl transferase) serine + THF OR 3-phosphoglycerate (oxidized) 3-phosphopyruvate (transaminated) 3-phosphoserine (hydrolysis) serine (1) de novo snthesis of serine is active in kidneys o methionine homocysteine homocysteine + seirne (cystathionine synthase) cystathionine cysteine + -keto butyrate (1) cystathionine synthase is inhibited by cysteine during growth: arginine demand is high becomes dietary essential o backup synthesis process in kidneys aspartate + citrulline arginine into blood to the liver normally in kidneys: arginine is made to produce creatine for the muscle, brain and heart or to produce NO biologically active amines: o histidine (decarboxylation, PLP) histamine: allergic/inflammatory responses; gastric secretion; neurotransmission o glutamate (decarboxylation) GABA: neurotransmitter o tryptophan (hydroxylated, BH4) (decarboxylated, PLP) seratonin: pain perception, temp. regulation, sleep/wake cycles o phenylalanine (hydroxylated) tyrosine (hydroxylated) DOPA (decarboxylated) dopamine: neurotransmitter tyrosine (1) DOPA (2) dopamine (3) norepinephrine (4) epinephrine (1) tyrosine hydroxylase/tyrosinase deficiency can cause albinism; cofactor = BH4 (2) DOPA decarboxylase; cofactor = PLP, B6 (3) dopamine -hydroxylase; cofactor = vitC (4) methylation with SAM THF deficiency SAM deficiency (methionine cant be recycled from homocysteine) less epinephrine

- 22 -

DISEASES TO KNOW
1) pancreatitis can occur as a result of accidental activation of trypsinogen within pancreas a) trypsin activates other pancreatic zymogens and phospholipase A2 degrades pancreas cystinuria defective transport mechanism for resorption of cystine in kidney tubules a) cystine is not reabsorbed gets excreted in the urine b) cystine can crystallize within kidney forms kidney stones NB: [aa]extracellular << [aa]intracellular 7 different transport systems are present to transport amino acids into cells ammonia toxicity hyperammonemia result of diseased liver or deficiency of urea cycle enzymes a) b) c) 4) if body cant get rid of ammonia through urea cycle more ammonia is trapped in glutamate and glutamine energy metabolism (since there is ketoglutarate and glutamate) glutamate affects CNS dramatically since glutamate is a precursor for GABA (key CNS neurotransmitter) treatment: phenylbutyrate nitrogen is excreted as phenylacetyl glutamine

2)

3)

ornithine transcarbamoylase deficiency X-linked dominant (most common urea cycle deficiency) a) results in free ammonia, glutamate b) hyperammonemia brain impairment coma death (if untreated) c) modify with protein diet containing only essential amino acids histidinemia histidase deficiency mental retardation folic acid deficiency FIGlu in urine since there is no THF, homocysteine ** homocystinuria elevated homocysteine levels associated with artherosclerotic vascular diseaseMI; dislocation of lens; osteoporosis; mental retardation a) caused by deficiency of cystathionine synthase elevated levels of methionine and its metabolites in blood b) byproduct of folic acid and B12 deficiency additionally, homocysteine is a source of methionine when [met]

5) 6) 7)

8)

maple syrup urine disease cant do oxidative decarboxylation of branched chain amino acids (i.e. leu, ile, val) a) b) common neurologic problems high mortality rate treat with altered diet (minimal intake of branched chain amino acids since they are dietary essential) and vitB1, B2, B3

9)

methylmalonyl-CoA mutase deficiency elevated levels of methylmalonyl-CoA in blood a) cant -oxidate odd-chains b) can cause metabolic acidosis and developmental problems c) cant degrade valine, isoleucine at all; partial degradation of methionine d) pernicious anemia vitB12 deficiency also can present with similar problems; B12 = cofactor for methylmalonyl-CoA mutase

10) phenylketonuria (PKU) deficiency of phenylalanine hydroxylase a) b) c) d) results in phenylalanine, phenylacetate and phenylpyruvate musty urine odor hypopigmentation tyrosine (product of phenylalanine breakdown) is a precursor for melanin i) tyrosine becomes a dietary essential amino acid CNS disorders: mental retardation, failure to walk/talk, seizures treat by avoiding phenylalanine in diet and tyrosine supplements

11) alkoptonuria causes urine to blacken when standing a) deficiency of homegentisate dehydroxygenase an enzyme involved in tyrosine breakdown b) presents with arthritis c) treat: low protein diet

- 23 -

12) type I tyrosinemia deficiency of fumaryl acetate hydrolase enzyme in last step of trosine breakdown
st 13) type II tyrosinemia tyrosinosis deficiency in tyrosine transaminase 1 enzyme in tyrosine breakdown

14) albinism deficiency in tyrosinase that produces melanin a) absence of pigment from hair, eyes, skin b) possible vision defects and photophobia 15) administration of methotrexate leads to THF depletion since DHF reductase is inhibited a) strong effect on dividing cells, since there are no purine and pyrimidines available for DNA synthesis b) treatment with 5-formyl THF (~leucovorin) modulates effects so methyl-THF can be used to make methionine 16) pernicious anemia vitB12 deficiency THF deficiency a) all THF enters cells through methyl-THF b) in order for homocysteine methyltransferase to use it to make THF, need vitB12 as a cofactor

ONE CARBON METABOLISM

some pathways require addition of single carbon groups which can exist in variety of oxidation states (e.g. methane, methanol, formaldehyde, formic acid, carbonic acid) these one carbon molecules are transferred from carrier compounds: o tetrahydrofolic acid (THF) folate (dehydrofolate reductase, 2 steps) THF

methotraxate inhibits dehydrofolate reductase

know names in pathway illustrated in Lippincott, fig. 20.11 understand THF pool: know which molecules contribute to each population and what theyre metabolized into tryptophan formate histidine formimino-THF formyl-THF de novo purine nucleotide synthesis methenyl-THF serine OR glycine + THF methylene-THF (reductase) dUMP (thymidine synthase) dTMP methyl-THF homocysteine methionine + THF (cofactor = B12) o S-adenosyl methionine (SAM) methyl-THF + homocysteine (cofactor B12) methionine + THF (-adenosine) (methyl donation) involved in methyl (-CH3) transfers o biotin carries carbonic acid (hydrated CO2) cofactor in pyruvate carboxylase, propionyl-CoA carboxylase, ACCase (+ATP) S-adenosyl homocysteine S-adenosyl methionine

- 24 -

PURINES AND PYRIMIDINES

ATP universal energy currency GTP G-proteins and translation PURINE SYNTHESIS [Lippincott, fig. 22.7] ribose 5-phosphate (from pentose phosphate pathway) + ATP (PRPP synthetase) PRPP o o o stimulated by Pi inhibited by IMP, AMP, GMP PRPP (glutamine:phosphoribosyl pyrophosphate amidotransferase) inosine-MP (IMP)
st 1 reaction is main site of regulation

CTP lipid activation UTP carbohydrate activation

10 step process (dont need to know the names of all the intermediates)

o o o

stimulated by PRPP inhibited by AMP, GMP, IMP

additional inputs: 2 glutamine, glycine, aspartate, 4 ATP, 2 formyl-THF, CO2, H2O NB: methotrexate reduces THF generation inhibits purine synthesis halts any dividing cells (effective for cancers) NB: sulfa drugs inhibit bacterias ability to generate folic acid; since humans dont synthesize their own folic acid, administration of sulfa drugs dont affect humans, while damaging bacteria [makes 1 NADH, uses 1 ATP] [uses 1 GTP]

IMP (inhibited by GMP) xanthosine-MP (GMP synthase) GMP IMP (inhibited by AMP) adenylosuccinate adenylosuccinate AMP + fumarate o o o NB: synthesis of AMP requires 1 GTP; synthesis of 1 GMP requires 1 ATP

de novo purine synthesis is costly alternative: salvage pathway utilizing purines from normal turnover and diet PRPP + hypoxanthine (hypoxanthine:guanine phosphoribosyltransferase, -PPi) IMP PRPP + guanine (hypoxanthine:guanine phosphoribosyltransferase, -PPi) GMP deficiency of hypoxanthine:guanine phosphoribosyltransferase LESCH-NYHAN SYNDROME o X-linked recessive disorder where hypoxanthine and guanine cant be salvaged results in: PRPP, IMP, GMP; de novo purine synthesis; uric acid hypouricemia presents with chorea (irregular, involuntary movements) and self-mutilation

PRPP + adenine (adenine phosphoribosyltransferase, -PPi) AMP

PYRIMIDINE SYNTHESIS glutamine + 2 ATP + CO2 (carbamoyl-phosphate synthetase II) carbamoyl phosphate (UMP synthase) UMP o o o o total inputs: 2 ATP, CO2, aspartate, H2O, NAD+, PRPP 1 NADH is generated CPS2 inhibited by UTP and stimulated by ATP and PRPP UMP synthase deficiency OROTIC ACIDURIA treat with uridine to shut down de novo pyrimidine synthesis

minimal use of salvage pathways, since its not that costly done by kinases conversion of NDPs to dNDPs (can only occur via diphosphates) acts on both purines and pyrimidines o o NDP + thioredoxin (reduced, 2SH) (ribonucleotide reductase) dNDP + thioredoxin (oxidized, S-S) inhibited by dATP thioredoxin (oxidized) + NADPH + H+ (thioredoxin reductase) thioredoxin (reduced) + NADP+

- 25 -

DEGRADATION OF PYRIMIDINES AND PURINES occurs in small intestines pancreatic enzymes hydrolyze nucleotides to nucleosides and free bases purine nucleotides uric acid dietary purines/pyrimidines usually not used in tissues degraded purine degradation: AMP (AMP deaminase) IMP GMP guanosine (pur. nuc. phosphorylase) guanine (pur. nuc. phosphorylase) hypoxanthine (xanthine oxidase) uric acid (xanthine oxidase) xanthine (guanase) o o o xanthine oxidase is inhibited by allopurinol adenosine deaminase deficiency (ADA) SCID; dATP in RBC inhibits DNA synthesis; die <2yo because of infection gout occurs as a result of hyperuricemia (uric acid in body that crystallizes in joints inflammation); treat with allopurinol since xanthine is much more soluble than uric acid

adenosine (adenosine deaminase) inosine

pyrimidine degradation: rings are opened and degraded very easily to precursors of acetyl-CoA and succinyl-CoA

- 26 -

HEME METABOLISM
Porphyrins cyclic compounds that readily bind metal ions (Fe most prevalent in humans: metalloporphyrin = HEME Heme Fe
++ ++

and Fe

+3

usually)

at center of tetrapyrrole ring of protoporphyrin IX; (in liver, heme is in in p450; in RBCs, heme is in hemoglobin)

Know configuration of porphyrin ring in humans, type III is significant porphyrin is colored; porphyrinogens (precursors) are colorless o Congenital erythropoietic porphyria type I porphyrins synthesized in high quantities

HEME SYNTHESIS in mitochondria: 2 glycine + 2 succinyl-CoA (-CoA, -CO2) 2 aminolevulinic acid in cytosol: 2 aminolevulinic acid (-2 H2O) porphobilinogen [inhibited by hemin, heme] [inhibited by lead]

4 porphobilinogen (1) hydrozymethylbilane (2,4) uroporphyrinogen III (cyt) (3,5,6) coporphyrinogen III (cyt) protoporphyrin IX (mit) (ferrochelatase) heme (mit, cyt) o 1) 2) 3) 4) 5) 6) ferrochelatase inhibited by lead acute intermittent porphyria enzyme deficiency; ring is not photosensitive; results in ALA, porphobilinogen inblood and urine; stimulation of p450 with barbiturates death; treat with heme (to inhibit synthesis) all defects after cyclization at this step will make faulty rings causes photosensitivity a) photosensitivity = skin condition, manifested in rashes or swelling, that results from exposure to sunlight porphyria cutanea tarda autosomal somatic dominant congenital erythropoietic porphyria varigate porphyria autosomal somatic dominant hereditary coproporphyria autosomal somatic dominant

HEME DEGRADATION heme (RBC) heme (macrophage) (heme oxygenase) biliverdin (biliverdin reductase) bilirubin bilirubin (blood) o o heme oxygenase uses O2 and NADPH to cleave heme ring; releases Fe+3 and Cu biliverdin has a green color

bilirubin (blood) bilirubin-albumin complex bilirubin (liver) (bilirubin glucuronyl transferase, ~conjugation) bilirubin diglucuronide bile (gut) bile (gut) (hydrolyzed and reduced by bacteria) urobilinogen (colorless) (oxidized by bacteria) stercobilin (feces) urobilinogen (colorless) can be reabsorbed into blood (enterohepatic urobilinogen cycle) liver re-excreted as bile urobilinogen (colorless) can be reabsorbed into blood (enterohepatic urobilinogen cycle) kidneys urobilin (yellow in urine) jaundice accumulation of bilirubin o o o hemolytic jaundice caused massive lysis of RBCs obstructive jaundice obstruction of bile duct hepatocellular jaundice from damage to liver cells

Crigler-Najjar syndrome genetic deficiency of bilirubin glucoronyl transferase o treat with fluorescent light that converts bilirubin to more water-soluble isomers

- 27 -

GAGs, PROTEOGLYCANS, GLYCOPROTEINS

found on proteins and lipids at cell surfaces allows for cell-cell interactions GAGs (~glycosaminoglycans) long, unbranched polysaccharide chains with repetitive disaccharides of amino sugar-acid sugar (we were responsible to know names, and needed to know the general structure; i.e. where carboxyl and amino groups were found on sugars) o o amino sugar: N-acetyl glucosamine and N-acetyl galactosamine (both D-sugars) acid sugars: D-glucuronic acid and L- iduronic acids

negatively charged bind lots of water (very wet/hydrated) high concentrations in extracellular matrix good for CT, mucous, sinovial fluid o o o o o know general details of heparin and hyaluronic acid (can be found in lecture notes) glucose and fructose-6-phosphate amino-sugars (N-acetyl glucosamine, N-acetyl galactosamine, N-acetyl neuraminic acid) diet D-glucuronic acid activated by UDP D-glucuronic acid (epimerization) L-glucuronic acid UDP-activated sugars are added to the reducing end synthesis occurs in ER and Golgi and involves specific transferases (for sulphates)

HYALURONIC ACID synthesis (exception) assembled in extracellular matrix, through plasma membrane (not in ER and Golgi) Degradation occurs within phagolysosomes PROTEOGLYCANS has a higher proportion of carbohydrates than proteins structure: basically, a core protein with many GAGs attached to it ** GAGs attach to protein via sugar link (xyl-gal-gal residues attached to GAG) at serine-OH appreciate 3D structure know that proteoglycan monomers can amass around hyaluronic acid through non-covalent interactions highly hydrated LYSOSOMAL STORAGE DISEASES (~mucopolysaccharidoses) know common features especially childhood onset (i.e. undetectable at birth gradual degeneration occurs due to GAG accumulation) know difference between Hurler, Hunter, and Sanfilippo (only affects CNS) GLYCOPROTEINS part of ECM, plasma membranes, lysosomes more proteins than carbohydrates (small chain, branched) glycoforms are identical protein backbones that have different CHO compositions attached to them connection to proteins via N-link (-OH in serine, threonine, hydroxylysine ) or O-link (amide N of asparagine) glycosylation offers protections for protein backbones from degradation know mannose triad and core pentasaccharide O-linked glycoproteins: synthesis occurs in RER first sugar added in ER lumen exported to Golgi where glycosylation continues (know general process outlined in biosynthesis figure) N-linked glycoproteins: o o o o o protein backbone synthesis occurs in RER in cytosolic side of ER membrane, oligosaccharides are assembled internalized into ER transferred to asn in protein sugar components are modified (e.g. phosphorylation) in ER or Golgi Lectins in liver recognize glycoproteins without sialic acids on ends of oligosaccharides bound and internalized hydrolyzed by lysosomal enzymes defective synthesis can impede degradation glycoproteinoses (disease states that result from accumulation of inclusion bodies, i.e. lysosomes)

Degradation: sialic acid residues cap ends of oligosaccharide; with time, sialic residues are degraded are removed

- 28 -

BLOOD COAGULATION
bleeding minimized by vasoconstriction, platelets, clotting with fibrin after cellular repair, process must be reversed thrombosis = excessive clotting thrombolysis = removal and stoppage of clotting intrinsic clotting = pathways requiring only factors in blood (~misnomer since you need negative charges extrinsic clotting requires thromboplastin (factor III) released during heavy tissue damage NB: negative charges simulate tissue damage KNOW INTRINSIC AND EXTRINSIC PATHWAY AS ILLUSTRATED IN NOTES PACKET; highlights: o o o o o o o o serine proteases kallikrein, XIIa, XIa, IXa, Xa, VIIa, IIa protein C inhibits factor VIIIa and Va both pathways create a cascade effet to amplify signal rapid clotting response without VIIIa hemophilia A without IX hemophilia B VIIIa and III (~thromboplastin) are not enzymes; they are cofactors that allow clotting factors to stick to platelet membrane damaged endothelial layers bind XII which is cleaved by kallikrein localization GLA proteins contain -carboxyglutamate; involved in Factors II, VII, IX and X KNOW how -carboxy is added using vitK-dependent carboxylase KNOW how vitKH2 is regenerated dicumarol inhibits vitK reductase chelating agents (e.g. EDTA, citrate) retard clotting by sequestering necessary metals fibrinogen (Factor I) does not aggregate because of electric-repulsion between charged goups at free amino terminals; once removed by thrombin, fibrin spontaneously aggregates soft clot clot HARDENS by covalent linkage of fibrin molecules by XIIIa (~glutaminidase)

to stop clotting process: know pathway illustrated in notes o o excess thrombin activates protein C active protein C inactivates Va and VIIIa

clot lysis: plasminogen (tissue plasmin activator, ~serine protease) plasmin responsible for fibrinolysis o plasmin is inhibited by 2-plasmin inhibitor

Serine proteases (e.g. chymotrypsin, trypsin) know they have catalytic triads (aspartate, histidine, serine) within hydrophobic pockets histidine is acts in both acid and base catalysis o o base catalysis ionizes -OH of serine by attacking carbonyl C of protein acid catalysis cleaves peptide bond, releasing one side while other side is covalently bound to serine

aspartate negative charges increase pKa of histidine to ~7.0 near physiological conditions

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OXIDASES AND OXYGENASES

80% of O2 consumed is used by cyt oxidase other 20% used in following types of reactions o o o oxidases: e.g. xanthine oxidase substrate is oxidized by O2 and O2 is not incorporated into substrate products of oxidases = H2O, H2O2, ROS (reactive oxygen species) latter two can cause oxidative damage to cells monooxygenases: substrates is hydroxylated; water is formed dioxygenases: S + O2 SO2

monoamine oxidase (MAO)inactivates hormones and neurotransmitters (e.g. epinephrine, norepinephrine, dopamine, seratonin) o used to treat depression and Parkinsons disease

oxygenases incorporate O2 into product pros of using O2 o o o release of lots of energy use of oxyradicals to kill bacteria, tumor cells neurotransmitters (~nitric oxide)

cons of using O2 o o o tissue damage by oxygen radicals damage by hyperbaric O2, septic shock, aging degenerative disease (e.g. artherosclerosis, type I diabetes, Parkinsons, etc.)

KNOW LAST PAGE OF OXIDASES AND OXYGENASES packet (has 5 slides of information)

MITOCHONDIRAL DISEASES
almost all problems with mitochondria manifestas as lactic acidosis (when pyruvate cant enter or be used in mitochondria) mitochondria life span = 7 days mitochondria have own circular DNA (~1600bp) with its own transcription and translation machinery o o o o codes for 13 polypeptides (components of e transport chain, ATP synthase and its own tRNA) no introns most mitochondrial genes are encoded in nucleus, but these 13 remain in mitochondria essential for proper functioning NB: at least 75% of mitochondria have to be damaged for diseased phenotype

non-Mendelian inheritance all mitochondria inherited from mother heteroplasmy each cell contains many mitochondria (cells in some tissues have more mitochondria per cell than other tissues) survival of slowest damage mitochondria persist over time since they have less oxidative damage to deal with Diseases: KSS (Kearns-Sayre syndrome) due to deletion; paralysis of eye muscles, retinal degeneration; cardiac conduction abnormalities MELAS (mit. Encephalomyopathy, lactic acidosis, stroke-like symptoms) point mutation in tRNA for leucine; affects CNS mitochondrial proteins synthesized in cytosol with mit-aminoacyl tRNA synthases, mit DNA-replication enzymes, mit ribosomal proteins, mit RNA polymerase 2-5 copies of mitochondrial genome per mitochondria allows competent proteins to be made even if point mutation arises genes are present on both strands of DNA (unlike in nuclear genes) mitochondrial genes encoded by mit genome: complex I, complex II, complex IV, ATP synthase, tRNAs, RNA
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DIABETES MELLITUS
2 groups: ~low insulin:glucagon double hit (insulin, glucagon) liver thinks your fasting o Type 1 IDDM insulin-dependent diabetes mellitus
1) 2) 3) 4) 5) 6)

insulin:glucagon ratio is low juvenile onset, often after bout with infectious disease due to progressive, auto-immune destruction of -cells in pancreas symptoms: polyuria (excessive urinating), polydipsia (excessive thirst), polyphagia (excessive eating) complication: ketoacidosis patients need insulin to maintain normal blood glucose levels

Type 2 NIDDM non-insulin-dependent diabetes mellitus

1) 2) 3) 4) 5) 6)

tissue lacks sensitivity to insulin and behaves as though the insulin:glucagon ratio is low patients are usually middle-aged and obese; genetic predisposition causes disturbance in downstream signal transduction pathways after meals, skeletal muscle and adipocytes fail to take up glucose effectively after many years of overproducing, -cells burn out may need intramuscular insulin complications: hyperosmolar coma (due to loss of Na+/K+)

in both cases elevated blood glucose sensed by -cells and causes:

o
o

glycolysis pyruvate and glycerol-3-phosphate oxid. phosphorylation ATP/ADP ratio ATP blocks ATP-sensitive K+-channels in plasma membrane depolarization Ca++ enters cells exocytosis of insulin
1)

Key: need functional mitochondria that uptake Ca++ in order for insulin to be secreted

IDDM ~simulation of starvation with insulin and glucagon o hyperglycemia increased hepatic glucose production and decreased peripheral utilization
1)

vicious cycle liver releases glucose, stays in blood and not taken up into cells, negative feedback from cells to liver to make more glucose liver cant oxidize all FAs VLDL decreased synthesis of lipoprotein lipases (due to insulin) hypertriglyceridemia

hypertriglyceridemia FA mobilization from adipose because of hormone-sensitive lipase activation (via glucagon)
1)

o o

ketosis -oxidation in liver exceeding TCA cycle capabilities long-term complications: damage to vasculature, blindness, ischemia in extremities, renal + cardiac failure

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CONNECTIVE TISSUE DISEASES

connective tissue: fibrous material forms framework and supports structure of body tissue; surrounds many organs; has lots of extracellular matrix COLLAGEN major component of skin and bone; most abundant protein = 25-30% of total body proteins o o o long, stiff triple-stranded helical structure with 3 collagen polypeptide chains wound around each other pro-collagen
1)

heteromeric with 3 strands; assembles itself and forms crosslinks to make large fibril

proline provides stability and fibril crosslinks glycine every third residue; facilitates tight packin of 3 helical polypeptide chains collagen assembly: 1) 3 precursor chains assemble to form pro-collagen; disulfide bonds form in C-terminus of propeptide hold ends together so peptide can wind around each other 2) proline and lysine (hydroxylases) hydroxyproline and hydroxylysine; form interchain hydrogen bonds that stabilize structure; require vitC as a cofactor w/o vitC, get scurvy fragile blood vessels, loose teeth fue to high collagen turnover in tissues oxidative deamination by lysyl oxidase makes aldehyde groups that form covalent crosslinks between collagen molecules

3) o

location of collagen assembly:

1) 2) 3) 4) 5) 6) 7)

synthesis of mRNA nucleus translation of pro- chain cytoplasm hydroxylation of prline and lysine ER assembly of triple-helix pro-collagen ER secretion into extra-cellular matrix (ECM) secretory vesicle cleavage by collagen peptidase ECM covalent crosslinking by lysyl oxidase ECM

o o

altered coding sequence ot substitution for glycine would change netire collagen molecule weak fibrils mutations in proteins that work as dimers or multimers can produce dominant negative loss of function

mutant peptide loses its function AND interferes with product of normal allele in heterozygote more severe effects than deletions or nonsense mutations wrecks triple helix reduced amount of functional collagen

hotspots for mutations middle (mature collagen) region OR C-terminus (responsible for twisting during assembly)

KERATIN intermediate filament (~cytoskeleton) o o o o o most abundant in epithelium (in hemidesmosomes and desmosomes) gives cell strength and shape + distributes tensile strength and shearing forces across tissues heterodimers = 1 type1 + 1 type2 polypeptide filament = staggered tetramers of dimers mutationi n keratin5 or keratin14 causes keratin filaments to aggregate into clumps within cells or make mechanically weak, frayed filaments found in gene sequences at N-terminus or C-terminus

DISEASES Ehler-Danlos results from mutation in collagen peptidase or lysyl hydroxylase deficiency o o o o o elastic skin, may have heart valve problems; loose joints brittle, fragile bones frequent bone fractures as fetuses, newborns, infants, adolescents type 1 mildest and most common; brittle bones type 2 most severe; soft, fragile bones short broken ribs; usually stillborn types 3 and 4 moderate severity Osteogenesis imperfecta (OI) mutation of codons for glycine residues in COL1A1 or COL1A2 genes

Epidermolysis bullosa mutation in keratin5 or keratin14; skin blisters in response to mild mechanical stress (~rupturing of cells in basal layers)
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***************************************************************************** Thanks to M. Ambati for her time and dedication towards putting this material together for her DES sessions. Please use this material as a supplement to the primary material/notes given to you in the course, and not as a sole authoritative source for studying. *****************************************************************************

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