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Mol Cell Biochem (2011) 350:127–134 DOI 10.1007/s11010-010-0690-4

Characterizing the novel protein p33MONOX

Manisha Mishra Noriko Inoue Klaus Heese

Received: 4 June 2010 / Accepted: 18 September 2010 / Published online: 14 December 2010 Springer Science+Business Media, LLC. 2010

Abstract The novel protein p33MONOX (p33Monoox- ygenase) was over-expressed in neuroblastoma cells dem- onstrating its inhibitory effect on the phosphorylation of the App (amyloid precursor protein) and Bcl2 (B-cell lymphoma 2) proteins but mediating higher activation of Mapk1/3 (mitogen-activated protein kinase 1/3). We employed a variety of cell biology techniques to show the localization of p33MONOX to the cytoplasm of pyramidal neurons in the mouse brain hippocampus. We also carried out a yeast-two-hybrid screening plus co-immunoprecipi- tation and bio-informatics to determine COBRA1 (cofactor of BRCA1 (breast cancer type 1)), NOL12 (nucleolar protein 12), and PRNP (prion protein) as p33MONOX- interacting proteins. Bio-computational analyses revealed a flavine-containing monooxygenase (FMO)-1 motif, thus linking p33MONOX to a group of previously character- ized proteins, the MICALs (molecule interacting with CasL). Concluding, p33MONOX might regulate pre- and

Electronic supplementary material

The online version of this

article (doi:10.1007/s11010-010-0690-4) contains supplementary material, which is available to authorized users.

M. Mishra K. Heese (&) Department of Molecular and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore, Singapore e-mail: kheese@ntu.edu.sg

N. Inoue Medical Center for Translational Research, Osaka University Hospital, Suita, Osaka, Japan

post-transcriptional control of dynamic processes related to growth cone guidance.


Alzheimer’s disease Apoptosis



Alzheimer’s Disease (AD) remains the most common cause of dementia in all age groups, characterized by progressive neurodegeneration and profound cognitive deficits [14]. Much of what is known about AD revolves around the amyloid precursor protein (APP) and presenilin-1 (PSEN1) and PSEN2 [5]. However, there remains a myriad of other proteins that could possibly play an equally crucial role in the development of AD. Although the etiology of sporadic AD is poorly understood, there is evidence that aberrant iron deposition, oxidative stress and mitochondria insufficiency play a role in the pathogenesis of sporadic AD and other aging-related neurodegenerative disorders. The excessive generation of free radicals may promote neurofibrillary tangle (NFT) formation as well as amyloid deposition in AD brains. Conversely, the neurotoxic effects of certain amyloid fragments may be mediated by free radical intermediates [68]. P33MONOX was discovered in our recent study on brain site-specific gene-expression analysis, when we com- pared the gene-expression pattern in the temporal and occipital lobe of early stage AD subjects with control patients [9]. In that study, p33MONOX, a novel gene with unknown functions as yet, was identified to be down-regu- lated in the occipital lobe of an early stage AD patient. In the current study, we characterized the potential biological sig- nificance of p33MONOX using molecular and cell biologi- cal as well as bio-computational analyses.



Mol Cell Biochem (2011) 350:127–134

Materials and methods


All reagents used for experiments were purchased from Sigma-Aldrich (Milwaukee, WI, USA) unless otherwise stated.

Cell culture and transfection

Rat B104 and human SHSY5Y neuroblastoma cells as well as rat PC12 cells (all from American Type Culture Col- lection (ATCC, Manassas, VA, USA)) were maintained in Dulbecco’s Modified Eagle Medium (D-MEM/F12(1:1)) plus 10% fetal bovine serum (FBS; Invitrogen, (Gibco), Carlsbad, CA, USA) at 37 C in humidified 5% CO 2 /95% air. A p33Monox expression construct was generated by inserting rat p33Monox cDNA in-frame with the red fluo- rescent protein (DsRed) (pDsRed-Express-N1; BD Bio- sciences Clontech, Palo Alto, CA, USA) at the C-terminus of p33Monox (p33-CT-DsRed). B104 cells were tran- siently transfected with the p33-CT-DsRed expression vector using the Lipofectamine 2000 (Invitrogen) trans- fection reagent (according to the manufacturer’s protocol) and maintained in D-MEM medium containing 10% FBS at 37 C. The transfected cells were then visualized by fluorescence microscopy (Nikon eclipse TE2000U, Nikon, Singapore). SHSY5Y, B104, and PC12 cells were stably transfected using a lentivirus expression system (p33 in EF.CMV.Gfp-Lenti-vector (elongation factor 1 alpha, cytomegalovirus promotors, green fluorescent protein; JHU-55, ATCC); co-expression of p33Monox and green Gfp) (control, mock-transfection) according to the manu- facturer’s protocol (Invitrogen) as briefly described in Supplementary materials and methods [10, 11].

Western blot analyses

Total protein cell lysates were subjected to western blot analyses as described previously (Supplementary materials and methods) [9, 10].

Animal material, immunohistochemistry (IHC) and immunocytochemistry (ICC)

Experimental methods, including the killing of animals, were performed in accordance with the International Guiding Principles for Animal Research (WHO) and approved by the local Institutional Animal Care & Use Committee (NTU-IACUC). Mouse tissues were isolated (C57BL/6J mice from the Animal Facility Centre at the National University (NUS) of Singapore) after humane killing of the animals using approved anaesthetic methods.

Mouse brain perfusion, IHC, and ICC were performed as described previously (Supplementary materials and methods) [11].

ProQuest TM two-hybrid-system with Gateway TM technology

The two-hybrid-system is an in vivo yeast-based system that identifies the interaction between two proteins (here X = p33MONOX and Y = human brain cDNA library or COBRA1) by reconstituting an active transcription factor. The analysis was performed according to the manufac- turer’s protocol (Invitrogen’s brain ProQuest TM two- hybrid-system, Singapore) using p33MONOX as bait. In the ProQuest TM two-hybrid-system, in comparison to standard two-hybrid-systems, false positives are reduced because three independent transcription events (from dis- tinct promoters) must occur at independent chromosomal loci. Positive clones were confirmed by retransformation assays and protein co-immunoprecipitation (Co-IP, Sup- plementary materials and methods) [10].


p33MONOX protein sequence analysis

Bio-computational analyses of the p33MONOX protein sequence among the species of human, mouse, and rat showed high sequence similarity (Fig. 1) pointing to the possibility that p33MONOX plays a crucial role that is evolutionarily conserved. The most encouraging information about p33MONOX is the presence of a flavine-containing monooxygenase (FMO)-1 motif. The proteins comprising the FMO motif belong to a family of microsomal NADPH (nicotinamide adenine dinucleotide phosphate)- and oxygen-dependent flavoenzymes (with flavin adenine nucleotide (FAD) as a co-factor) that are distributed ubiquitously in mammalian species, and catalyze the oxidation of soft nucleophilic heteroatom centers in drugs, pesticides, and xenobiotics, using nucleotides as electron donors. FMO-1 catalyzes the N-oxygenation of secondary and tertiary amines. In some contexts, while performing the oxidation, they can generate reactive oxygen species (ROS) [1215]. Thus, p33MONOX protein is more likely to be a NADPH- dependent oxidoreductase.

Sub-cellular localization and neuronal expression of p33Monox in the mouse brain

We investigated the sub-cellular localization of p33Monox to obtain more information about its physiological role,


Mol Cell Biochem (2011) 350:127–134


Fig. 1 Characteristic features of the p33MONOX protein sequence. Aligned protein sequences of p33MONOX in the human (H, black), mouse (M, blue), and rat (R, green) species, with red alphabets denoting the varying amino acid. There is evidently a high degree of conservation in the protein sequences amongst the species, suggesting a pivotal functional significance of p33MONOX. Bio-informatical analyses of p33MONOX’s protein sequence revealed a potential flavine-containing monooxygenase (FMO)-1 motif and several Ser-/Thr- phosphorylation sites

Mol Cell Biochem (2011) 350:127–134 129 Fig. 1 Characteristic features of the p33MONOX protein sequence. Aligned

distribution, and site of activity in the cell. For this pur-

physiologically expressed in neuronal pyramidal cells of

pose, a p33Monox-DsRed fluorescent fusion protein was

the hippocampus


also in


neurons of the cortex

transiently expressed in B104 neuroblastoma cells. Using

(Fig. 3).

fluorescence microscopy, we show that p33Monox expression was confined to the neuronal cytoplasm thus

p33Monox-mediated neuronal signaling


confirming the bio-computational analysis data (Fig. 2a). Besides, ICC of nerve growth factor (Ngf)-differentiated PC12 cells also verified the localization of p33Monox in the cytoplasm with a substantial expression in the axonal growth cone (Fig. 2b). Furthermore, we conducted an IHC analysis of the mouse brain for the p33Monox protein expression and localization. This analysis revealed that p33Monox was

To further corroborate the physiological significance of p33Monox, we analyzed its effect on the activation of pivotal proteins involved in neuronal survival and differ- entiation. Interestingly, upon over-expression in neuronal cells, p33Monox inhibited the phosphorylation of App (reduced Ab formation [16]) and Bcl2 (the functional sig- nificance of the dynamic phosphorylation status (regulated



Mol Cell Biochem (2011) 350:127–134

Fig. 2 Sub-cellular localization of p33Monox. a B104 cells were transfected with a p33-CT-DsRed expression vector as described in Supplementary materials and methods. Microscopic picture of a representative B104 cell under bright-field, red fluorescence (revealing cytoplasmic localization of p33Monox), and UV light (indicating nuclear DAPI staining), respectively; scale bar = 20 lm. b Co-ICC of Ngf-differentiated PC12 cells with p33Monox (green) and Tuba1a (tubulin, red) or Syp (synaptophysin, red) as indicated. DAPI staining was used to indicate the nucleus. Representative pictures are shown. Pictures show the strong co-localization (merged, yellow) of p33Monox with tubulin in the cytoplasm and with Syp in the axonal growth cones (arrows). Scale bar = 50 lm

130 Mol Cell Biochem (2011) 350:127–134 Fig. 2 Sub-cellular localization of p33Monox. a B104 cells were

by various kinases and protein phosphatase PP2A) has been discussed conflictive [1722]) while Erk1/2 (Mapk1/3) was activated (sustained phosphorylation is required for neu- ronal differentiation [23]) (Fig. 4a).

p33MONOX interacts with COBRA1, NOL12, and PRNP

Additional clarification about p33Monox’s potential cel- lular function was obtained by the yeast-two-hybrid screen combined with bio-informatic analyses (NCBI (National Center for Biotechnology information), EMBL-EBI (European Bioinformatics Institute), and SIB (Swiss Insti- tute of Bioinformatics, (Swiss-Prot & Tremble, ExPASy, and Proteomics tools)) databases were used). The analyses revealed that p33MONOX interacts with several proteins


involved in the control of gene transcription, such as COBRA1 (the co-factor of BRCA1 is a newly charac- terized member of the negative elongation factor (NELF) complex; confirmed by Co-IP (Fig. 4b)) [2428], NOL12 (nucleolar protein 12, also known as ribosomal RNA pro- cessing protein 17) [29, 30], and the prion protein (PRNP or PrP with isoform 2 as a potential growth suppressor that arrests the cell cycle at the G0/G1 phase) [3133] (Supplementary Tables 1 and 2). NOL12 itself interacts with several other proteins known to be pivotal regulators of gene transcription and cell cycle progression such as: SAP18 (or Sin3A-associated protein), CDK4 (cyclin-dependent kinase 4), SF3B3 (sub- unit 3 of the splicing factor 3b protein complex), and SLC25A38 (solute carrier family 25, member 38), a mitochondrial carrier protein that is widely expressed

Mol Cell Biochem (2011) 350:127–134


Mol Cell Biochem (2011) 350:127–134 131 Fig. 3 Co-IHC analysis of p33Monox ( green ) and

Fig. 3 Co-IHC analysis of p33Monox (green) and Mtap2 (red) in the mouse brain revealed that p33Monox is expressed in the cytoplasm of pyramidal neurons in the hippocampus. Top left entire hippocampus formation including CA1, CA2, CA3 regions, and the dentate gyrus. Scale bar = 100 lm. Right enlarged CA1 regions.

Scale bar = 20 lm. Bottom/left further magnified picture of the CA1 region, clearly shows the cytoplasmic localization of p33Monox. Scale bar = 5 lm. Bottom/right representative picture taken form the cortex area thus indicating that p33Monox is generally expressed in neurons in the mouse brain. Scale bar = 20 lm

in the central nervous system [34]. Interestingly, by direct interaction NOL12 links p33Monox to SOD2 (superoxide dismutase 2). This protein is a member of the iron/ manganese superoxide dismutase family that binds to the superoxide byproducts of oxidative phosphorylation and converts them to hydrogen peroxide and diatomic oxygen. Mutations in this gene have been associated with premature aging and sporadic motor neuron disease [35, 36] (further details in Supplementary Table 2).


In the present study, we revealed the specific sites of p33Monox expression and its intensive localization in neural axonal growth cones. Apart from this, we observed

that p33Monox showed an interesting inhibition of the phosphorylation of App and Bcl2 as well as an enhanced activation of Mapk1/3 [16, 20, 23]. We also found that p33Monox forms a complex with Cobra1, Nol12, and Prnp. Cobra1, also known as Nelf-b that is involved in control- ling axonal growth [25, 26], associates with the product of the breast cancer susceptibility gene Brca1, thus hinting at a plausible role of p33Monox in sequestering Cobra1 and thereby leading to profound effects on gene transcription signals during dynamic neurite outgrowth processes. Consequently, p33Monox can be likened to a group of cytosolic proteins, the MICALs (molecule interacting with CasL), which are also oxidoreductases that utilize FAD as a co-factor and communicate via ROS [3739]. During the past decade, we have begun to recognize that controlled production of ROS and regulated redox modifications of



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132 Mol Cell Biochem (2011) 350:127–134 Fig. 4 a p33Monox inhibits the phosphorylation of pivotal signaling

Fig. 4 a p33Monox inhibits the phosphorylation of pivotal signaling molecules as indicated. Neuronal B104 cells were transfected with p33Monox as described in Supplementary materials and methods. Thereafter, the phosphorylation status was checked by western blotting. C control, non-transfected, GFP mock-transfection with GFP, p33Monox p33Monox-transfected. b p33MONOX Co-IP. Upon transfection of neuronal SHSY5Y cells, Co-IP was performed as described in Supplementary materials and methods to confirm the interaction between p33MONOX and COBRA1. Control Co-IP with unspecific serum

transcription factors or enzymes (such as kinases and phosphatases) are an essential part of signal transduction pathways [4042]. Similar to p33Monox, the MICALs are expressed in neuronal axons that associate with several cytoskeletal/-associated proteins, and are required for semaphorin-mediated repulsive axon guidance that is cru- cial for neuronal development. P33Monox may be another candidate for directly mediating the cytoskeletal alterations characteristic of semaphorin signaling and could be a novel target for the attenuation of axonal repulsion (Fig. 5). Given the presence of high amounts of ROS and other oxidants in the spinal cord after injury [43], and that aging and AD have previously been closely linked to the accu- mulation of oxidative stress [4446], regulation of redox signaling using antioxidants and specific enzyme inhibitors may be a powerful approach for encouraging neuronal regeneration [37]. The plausible link between MICAL, p33Monox, and SOD2 points to a role of p33Monox

132 Mol Cell Biochem (2011) 350:127–134 Fig. 4 a p33Monox inhibits the phosphorylation of pivotal signaling

Fig. 5 Schematic illustration of the potential action of p33MONOX in mediating neuronal survival, differentiation, and axonal outgrowth. In light of experimental and bio-informatical evidences it can be speculated that p33MONOX may act as platform to recruit down- stream effectors (e.g., COBRA1, NOL12, PRNP, SOD2, NF-jB, ROS, kinases, or phosphatases) to their site of action. The activity of these effectors could then be selectively modulated by redox modifications of key amino acid residues that could either be the direct effect of p33MONOX’s monooxygenase activity or be the indirect consequence of a local increase in ROS. P33MONOX- mediated de-phosphorylation of App and Bcl2 as well as sustained phosphorylation of Mapk1/3 are required for the control of neuronal survival, differentiation, and growth-cone extension

in controlling ROS that have been recently suggested to be a key factor in the cellular changes of an AD brain as several reports have suggested that mitochondrial abnormalities and oxidative stress play a role in sporadic AD [4749]. For instance, the heme-oxygenase-1 (HO-1), a member of the stress protein superfamily that operates with the NADPH cytochrome P450 reductase to oxidize heme, is widely accepted as a sensitive and fairly ubiquitous up-regulated marker of oxidative stress. It has also been shown to be consistently co-localized to NFTs and senile plaques in AD brains [5052]. In conclusion, our data has shed new insights on the possible involvement of the novel protein p33Monox in the regulation of neuronal survival, differentiation, and axonal outgrowth and the connection among p33Monox, oxidative stress and AD will be a gripping topic for future investiga- tions, especially in view of potential antioxidant therapies.

Acknowledgments This study was supported by an A*STAR grant (BMRC/04/1/22/19/360) to K.H. We thank Ms H.J. Tang and S. Yusof (both from the School of Biological Sciences, Nanyang Technical University) for technical assistance. We are particularly grateful to Prof. Dr. R. Li (Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center, 15355 Lambda Drive, San Antonio, TX, 78245-3207, USA) for providing us the anti-COBRA1 antibody.


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