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

post-transcriptional control of dynamic processes related to

growth cone guidance.

Keywords Alzheimers disease Apoptosis Neurodegeneration

Introduction

Alzheimers 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.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11010-010-0690-4) contains supplementarymaterial, 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

123

Mol Cell Biochem (2011) 350:127134

DOI 10.1007/s11010-010-0690-4

Materials and methods

Reagents

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

Dulbeccos Modified Eagle Medium (D-MEM/F12(1:1))

plus 10% fetal bovine serum (FBS; Invitrogen, (Gibco),

Carlsbad, CA, USA) at 37C in humidified 5% CO2/95%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 manufacturers protocol)

and maintained in D-MEM medium containing 10% FBS

at 37C. The transfected cells were then visualized byfluorescence 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-

facturers 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].

ProQuestTM two-hybrid-system with GatewayTM

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-

turers protocol (Invitrogens brain ProQuestTM two-

hybrid-system, Singapore) using p33MONOX as bait. In

the ProQuestTM 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].

Results

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,

128 Mol Cell Biochem (2011) 350:127134

123

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

pose, a p33Monox-DsRed fluorescent fusion protein was

transiently expressed in B104 neuroblastoma cells. Using

fluorescence microscopy, we show that p33Monox

expression was confined to the neuronal cytoplasm thus

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

physiologically expressed in neuronal pyramidal cells of

the hippocampus and also in the neurons of the cortex

(Fig. 3).

p33Monox-mediated neuronal signaling

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

Fig. 1 Characteristic featuresof the p33MONOX protein

sequence. Aligned protein

sequences of p33MONOX in

the human (H, black), mouse(M, blue), and rat (R, green)species, with red alphabetsdenoting 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 p33MONOXs

protein sequence revealed a

potential flavine-containing

monooxygenase (FMO)-1 motif

and several Ser-/Thr-

phosphorylation sites

Mol Cell Biochem (2011) 350:127134 129

123

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 p33Monoxs 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

Fig. 2 Sub-cellular localizationof p33Monox. a B104 cellswere 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-ICCof Ngf-differentiated PC12 cells

with p33Monox (green) andTuba1a (tubulin, red) or Syp(synaptophysin, red) asindicated. 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) 350:127134

123

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).

Discussion

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

Fig. 3 Co-IHC analysis of p33Monox (green) and Mtap2 (red) inthe mouse brain revealed that p33Monox is expressed in the

cytoplasm of pyramidal neurons in the hippocampus. Top left entirehippocampus 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 CA1region, clearly shows the cytoplasmic localization of p33Monox.

Scale bar = 5 lm. Bottom/right representative picture taken form thecortex area thus indicating that p33Monox is generally expressed in

neurons in the mouse brain. Scale bar = 20 lm

Mol Cell Biochem (2011) 350:127134 131

123

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

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.

Fig. 4 a p33Monox inhibits the phosphorylation of pivotal signalingmolecules 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 withGFP, p33Monox p33Monox-transfected. b p33MONOX Co-IP. Upontransfection 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 withunspecific serum

Fig. 5 Schematic illustration of the potential action of p33MONOXin 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 p33MONOXs 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

132 Mol Cell Biochem (2011) 350:127134

123

References

1. Welsh KA, Butters N, Hughes JP, Mohs RC, Heyman A (1992)

Detection and staging of dementia in Alzheimers disease. Use of

the neuropsychological measures developed for the Consortium

to Establish a Registry for Alzheimers Disease. Arch Neurol

49:448452

2. Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R,

Hansen LA, Katzman R (1991) Physical basis of cognitive

alterations in Alzheimers disease: synapse loss is the major

correlate of cognitive impairment. Ann Neurol 30:572580

3. Heese K, Akatsu H (2006) Alzheimers diseasean interactive

perspective. Curr Alzheimer Res 3:109121

4. Mattson MP (2004) Pathways towards and away from Alzhei-

mers disease. Nature 430:631639

5. Selkoe DJ, Schenk D (2003) Alzheimers disease: molecular

understanding predicts amyloid-based therapeutics. Annu Rev

Pharmacol Toxicol 43:545584

6. Butterfield DA (2002) Amyloid beta-peptide (1-42)-induced

oxidative stress and neurotoxicity: implications for neurodegen-

eration in Alzheimers disease brain. A review. Free Radic Res

36:13071313

7. Butterfield DA, Castegna A, Lauderback CM, Drake J (2002)

Evidence that amyloid beta-peptide-induced lipid peroxidation

and its sequelae in Alzheimers disease brain contribute to neu-

ronal death. Neurobiol Aging 23:655664

8. Pappolla MA, Chyan YJ, Omar RA, Hsiao K, Perry G, Smith

MA, Bozner P (1998) Evidence of oxidative stress and in vivo

neurotoxicity of beta-amyloid in a transgenic mouse model of

Alzheimers disease: a chronic oxidative paradigm for testing

antioxidant therapies in vivo. Am J Pathol 152:871877

9. Yokota T, Mishra M, Akatsu H, Tani Y, Miyauchi T, Yamamoto

T, Kosaka K, Nagai Y, Sawada T, Heese K (2006) Brain site-

specific gene expression analysis in Alzheimers disease patients.

Eur J Clin Invest 36:820830

10. Heese K, Yamada T, Akatsu H, Yamamoto T, Kosaka K, Nagai

Y, Sawada T (2004) Characterizing the new transcription regu-

lator protein p60TRP. J Cell Biochem 91:10301042

11. Nehar S, Mishra M, Heese K (2009) Identification and charac-

terisation of the novel amyloid-beta peptide-induced protein p17.

FEBS Lett 583:32473253

12. Massey V (1995) Introduction: flavoprotein structure and mech-

anism. FASEB J 9:473475

13. Yeung CK, Lang DH, Thummel KE, Rettie AE (2000) Immu-

noquantitation of FMO1 in human liver, kidney, and intestine.

Drug Metab Dispos 28:11071111

14. Ziegler DM (1990) Flavin-containing monooxygenases: enzymes

adapted for multisubstrate specificity. Trends Pharmacol Sci

11:321324

15. Massey V (1994) Activation of molecular oxygen by flavins and

flavoproteins. J Biol Chem 269:2245922462

16. Lee MS, Kao SC, Lemere CA, Xia W, Tseng HC, Zhou Y, Neve

R, Ahlijanian MK, Tsai LH (2003) APP processing is regulated

by cytoplasmic phosphorylation. J Cell Biol 163:8395

17. Ito T, Deng X, Carr B, May WS (1997) Bcl-2 phosphorylation

required for anti-apoptosis function. J Biol Chem 272:1167111673

18. Deng X, Ito T, Carr B, Mumby M, May WS Jr (1998) Reversible

phosphorylation of Bcl2 following interleukin 3 or bryostatin 1 is

mediated by direct interaction with protein phosphatase 2A.

J Biol Chem 273:3415734163

19. Deng X, Gao F, Flagg T, May WS Jr (2004) Mono- and multisite

phosphorylation enhances Bcl2s antiapoptotic function and

inhibition of cell cycle entry functions. Proc Natl Acad Sci USA

101:153158

20. Yamamoto K, Ichijo H, Korsmeyer SJ (1999) BCL-2 is phos-

phorylated and inactivated by an ASK1/Jun N-terminal protein

kinase pathway normally activated at G(2)/M. Mol Cell Biol

19:84698478

21. Chang BS, Minn AJ, Muchmore SW, Fesik SW, Thompson CB

(1997) Identification of a novel regulatory domain in Bcl-X(L)

and Bcl-2. EMBO J 16:968977

22. Liu XA, Liao K, Liu R, Wang HH, Zhang Y, Zhang Q, Wang Q,

Li HL, Tian Q, Wang JZ (2010) Tau dephosphorylation poten-

tiates apoptosis by mechanisms involving a failed depho-

sphorylation/activation of Bcl-2. J Alzheimers Dis 19:953962

23. Kholodenko BN (2007) Untangling the signalling wires. Nat Cell

Biol 9:247249

24. McChesney PA, Aiyar SE, Lee OJ, Zaika A, Moskaluk C, Li R,

El-Rifai W (2006) Cofactor of BRCA1: a novel transcription

factor regulator in upper gastrointestinal adenocarcinomas. Can-

cer Res 66:13461353

25. Kramer PR, Wray S (2001) Nasal embryonic LHRH factor

(NELF) expression within the CNS and PNS of the rodent. Brain

Res Gene Expr Patterns 1:2326

26. Kramer PR, Wray S (2000) Novel gene expressed in nasal region

influences outgrowth of olfactory axons and migration of

luteinizing hormone-releasing hormone (LHRH) neurons. Genes

Dev 14:18241834

27. Sun J, Blair AL, Aiyar SE, Li R (2007) Cofactor of BRCA1

modulates androgen-dependent transcription and alternative

splicing. J Steroid Biochem Mol Biol 107:131139

28. Narita T, Yamaguchi Y, Yano K, Sugimoto S, Chanarat S, Wada

T, Kim DK, Hasegawa J, Omori M, Inukai N, Endoh M, Yamada

T, Handa H (2003) Human transcription elongation factor NELF:

identification of novel subunits and reconstitution of the func-

tionally active complex. Mol Cell Biol 23:18631873

29. Suzuki S, Kanno M, Fujiwara T, Sugiyama H, Yokoyama A,

Takahashi H, Tanaka J (2006) Molecular cloning and character-

ization of Nop25, a novel nucleolar RNA binding protein, highly

conserved in vertebrate species. Exp Cell Res 312:10311041

30. Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH,

Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S,

Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde

A, Toksoz E, Droege A, Krobitsch S, Korn B, Birchmeier W,

Lehrach H, Wanker EE (2005) A human proteinprotein inter-

action network: a resource for annotating the proteome. Cell

122:957968

31. Juanes ME, Elvira G, Garcia-Grande A, Calero M, Gasset M (2009)

Biosynthesis of prion protein nucleocytoplasmic isoforms by

alternative initiation of translation. J Biol Chem 284:27872794

32. Satoh J, Obayashi S, Misawa T, Sumiyoshi K, Oosumi K, Tab-

unoki H (2009) Protein microarray analysis identifies human

cellular prion protein interactors. Neuropathol Appl Neurobiol

35:1635

33. Haigh CL, Lewis VA, Vella LJ, Masters CL, Hill AF, Lawson

VA, Collins SJ (2009) PrPC-related signal transduction is influ-

enced by copper, membrane integrity and the alpha cleavage site.

Cell Res 19:10621078

34. Haitina T, Lindblom J, Renstrom T, Fredriksson R (2006)

Fourteen novel human members of mitochondrial solute carrier

family 25 (SLC25) widely expressed in the central nervous sys-

tem. Genomics 88:779790

35. Miao L, St Clair DK (2009) Regulation of superoxide dismutase

genes: implications in disease. Free Radic Biol Med 47:344356

36. Lynn S, Huang EJ, Elchuri S, Naeemuddin M, Nishinaka Y,

Yodoi J, Ferriero DM, Epstein CJ, Huang TT (2005) Selective

neuronal vulnerability and inadequate stress response in super-

oxide dismutase mutant mice. Free Radic Biol Med 38:817828

Mol Cell Biochem (2011) 350:127134 133

123

37. Terman JR, Mao T, Pasterkamp RJ, Yu HH, Kolodkin AL (2002)

MICALs, a family of conserved flavoprotein oxidoreductases,

function in plexin-mediated axonal repulsion. Cell 109:887900

38. Massey V, Palmer G (1966) On the existence of spectrally dis-

tinct classes of flavoprotein semiquinones. A new method for the

quantitative production of flavoprotein semiquinones. Biochem-

istry 5:31813189

39. Ventura A, Pelicci PG (2002) Semaphorins: green light for redox

signaling? Sci STKE 2002: pe44

40. Kamata H, Hirata H (1999) Redox regulation of cellular signal-

ling. Cell Signal 11:114

41. Finkel T (1998) Oxygen radicals and signaling. Curr Opin Cell

Biol 10:248253

42. Meng TC, Fukada T, Tonks NK (2002) Reversible oxidation and

inactivation of protein tyrosine phosphatases in vivo. Mol Cell

9:387399

43. Juurlink BH, Paterson PG (1998) Review of oxidative stress in

brain and spinal cord injury: suggestions for pharmacological and

nutritional management strategies. J Spinal Cord Med 21:309334

44. Lovell MA, Ehmann WD, Butler SM, Markesbery WR (1995)

Elevated thiobarbituric acid-reactive substances and antioxidant

enzyme activity in the brain in Alzheimers disease. Neurology

45:15941601

45. Schuessel K, Leutner S, Cairns NJ, Muller WE, Eckert A (2004)

Impact of gender on upregulation of antioxidant defence mecha-

nisms in Alzheimers disease brain. J Neural Transm 111:11671182

46. Nunomura A, Chiba S, Lippa CF, Cras P, Kalaria RN, Takeda A,

Honda K, Smith MA, Perry G (2004) Neuronal RNA oxidation is

a prominent feature of familial Alzheimers disease. Neurobiol

Dis 17:108113

47. Hirai K, Aliev G, Nunomura A, Fujioka H, Russell RL, Atwood

CS, Johnson AB, Kress Y, Vinters HV, Tabaton M, Shimohama

S, Cash AD, Siedlak SL, Harris PL, Jones PK, Petersen RB, Perry

G, Smith MA (2001) Mitochondrial abnormalities in Alzheimers

disease. J Neurosci 21:30173023

48. Behl C (2005) Oxidative stress in Alzheimers disease: implica-

tions for prevention and therapy. Subcell Biochem 38:6578

49. Reddy PH (2006) Amyloid precursor protein-mediated free rad-

icals and oxidative damage: implications for the development and

progression of Alzheimers disease. J Neurochem 96:113

50. Premkumar DR, Smith MA, Richey PL, Petersen RB, Castellani

R, Kutty RK, Wiggert B, Perry G, Kalaria RN (1995) Induction of

heme oxygenase-1 mRNA and protein in neocortex and cerebral

vessels in Alzheimers disease. J Neurochem 65:13991402

51. Schipper HM, Cisse S, Stopa EG (1995) Expression of heme

oxygenase-1 in the senescent and Alzheimer-diseased brain. Ann

Neurol 37:758768

52. Smith MA, Kutty RK, Richey PL, Yan SD, Stern D, Chader GJ,

Wiggert B, Petersen RB, Perry G (1994) Heme oxygenase-1 is

associated with the neurofibrillary pathology of Alzheimers

disease. Am J Pathol 145:4247

134 Mol Cell Biochem (2011) 350:127134

123

Characterizing the novel protein p33MONOXAbstractIntroductionMaterials and methodsReagentsCell culture and transfectionWestern blot analysesAnimal material, immunohistochemistry (IHC) and immunocytochemistry (ICC)ProQuesttrade two-hybrid-system with Gatewaytrade technology

Resultsp33MONOX protein sequence analysisSub-cellular localization and neuronal expression of p33Monox in the mouse brainp33Monox-mediated neuronal signalingp33MONOX interacts with COBRA1, NOL12, and PRNP

DiscussionAcknowledgmentsReferences

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 149 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 150 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 599 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 600 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ]>> setdistillerparams> setpagedevice