The Thioredoxin-like Protein Rod-derived Cone Viability Factor (RdCVFL) Interacts with TAU and Inhibits Its Phosphorylation in the Retina
2009; Elsevier BV; Volume: 8; Issue: 6 Linguagem: Inglês
10.1074/mcp.m800406-mcp200
ISSN1535-9484
AutoresRam Fridlich, François Delalande, Céline Jaillard, Jun Lu, Laetitia Poidevin, Thérèse Cronin, Ludivine Perrocheau, Géraldine Millet-Puel, Marie-Laure Niepon, Olivier Poch, Arne Holmgren, Alain Van Dorsselaer, José‐Alain Sahel, Thierry Léveillard,
Tópico(s)S100 Proteins and Annexins
ResumoRod-derived cone viability factor (RdCVF) is produced by the Nxnl1 gene that codes for a second polypeptide, RdCVFL, by alternative splicing. Although the role of RdCVF in promoting cone survival has been described, the implication of RdCVFL, a putative thioredoxin enzyme, in the protection of photoreceptors is presently unknown. Using a proteomics approach we identified 90 proteins interacting with RdCVFL including the microtubule-binding protein TAU. We demonstrate that the level of phosphorylation of TAU is increased in the retina of the Nxnl1−/− mice as it is hyperphosphorylated in the brain of patients suffering from Alzheimer disease, presumably in some cases through oxidative stress. Using a cell-based assay, we show that RdCVFL inhibits TAU phosphorylation. In vitro, RdCVFL protects TAU from oxidative damage. Photooxidative stress is implicated in retinal degeneration, particularly in retinitis pigmentosa, where it is considered to be a contributor to secondary cone death. The functional interaction between RdCVFL and TAU described here is the first characterization of the RdCVFL signaling pathway involved in neuronal cell death mediated by oxidative stress. Rod-derived cone viability factor (RdCVF) is produced by the Nxnl1 gene that codes for a second polypeptide, RdCVFL, by alternative splicing. Although the role of RdCVF in promoting cone survival has been described, the implication of RdCVFL, a putative thioredoxin enzyme, in the protection of photoreceptors is presently unknown. Using a proteomics approach we identified 90 proteins interacting with RdCVFL including the microtubule-binding protein TAU. We demonstrate that the level of phosphorylation of TAU is increased in the retina of the Nxnl1−/− mice as it is hyperphosphorylated in the brain of patients suffering from Alzheimer disease, presumably in some cases through oxidative stress. Using a cell-based assay, we show that RdCVFL inhibits TAU phosphorylation. In vitro, RdCVFL protects TAU from oxidative damage. Photooxidative stress is implicated in retinal degeneration, particularly in retinitis pigmentosa, where it is considered to be a contributor to secondary cone death. The functional interaction between RdCVFL and TAU described here is the first characterization of the RdCVFL signaling pathway involved in neuronal cell death mediated by oxidative stress. Oxidative stress is suspected to play a major role in numerous age-related diseases (1Jones D.P. Extracellular redox state: refining the definition of oxidative stress in aging.Rejuvenation Res. 2006; 9: 169-181Crossref PubMed Scopus (200) Google Scholar) and neurodegenerative disorders (2Reynolds A. Laurie C. Mosley R.L. Gendelman H.E. Oxidative stress and the pathogenesis of neurodegenerative disorders.Int. Rev. Neurobiol. 2007; 82: 297-325Crossref PubMed Scopus (339) Google Scholar). High levels of oxygen result in generation of reactive oxygen species that interact with proteins, lipids, or DNA and lead to cell dysfunction and death. In the eye, several diseases including glaucoma (3Lundmark P.O. Pandi-Perumal S.R. Srinivasan V. Cardinali D.P. Rosenstein R.E. Melatonin in the eye: implications for glaucoma.Exp. Eye Res. 2007; 84: 1021-1030Crossref PubMed Scopus (52) Google Scholar) and age-related macular degeneration (4Beatty S. Koh H. Phil M. Henson D. Boulton M. The role of oxidative stress in the pathogenesis of age-related macular degeneration.Surv. Ophthalmol. 2000; 45: 115-134Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar) are associated with oxidative stress. Retinal degenerations are a prevalent cause of severe loss of vision. Retinitis pigmentosa is a monogenic disease in which mutations occur in genes that lead to the death of rod photoreceptors resulting in night blindness. After rods die, cone photoreceptors gradually die, resulting in blindness. Recent evidence has implicated oxidative damage as a contributor to death of cones in retinitis pigmentosa and to death of both photoreceptor cell types in age-related macular degeneration (5Cingolani C. Rogers B. Lu L. Kachi S. Shen J. Campochiaro P.A. Retinal degeneration from oxidative damage.Free Radic. Biol. Med. 2006; 40: 660-669Crossref PubMed Scopus (79) Google Scholar). After rods die, oxygen consumption in the outer retina is markedly reduced, and tissue oxygen levels become substantially elevated (6Yu D.Y. Cringle S. Valter K. Walsh N. Lee D. 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The protection of thioredoxin 1 (TRX1) 1The abbreviations used are:TRXthioredoxinADAlzheimer diseaseCDScoding DNA sequenceGSK-3βglycogen synthase kinase-3RdCVFrod-derived cone viability factorRdCVFLrod-derived cone viability factor long isoformNxnl1nucleoredoxin-like 1STRINGSearch Tool for the Retrieval of Interacting Genes/ProteinsGOgene ontologyaaamino acidsHEKhuman embryonic kidneyAPI5apoptosis inhibitor 5PARP1poly(ADP-ribose) polymerase 1HAhemagglutininIAF5-(iodoacetamido)fluorescein against retinal photooxidative damage (9Tanito M. Masutani H. Nakamura H. Oka S. Ohira A. Yodoi J. Attenuation of retinal photooxidative damage in thioredoxin transgenic mice.Neurosci. Lett. 2002; 326: 142-146Crossref PubMed Scopus (56) Google Scholar) and its role in neurodegenerative diseases like Alzheimer disease are known (10Akterin S. Cowburn R.F. Miranda-Vizuete A. Jimenez A. Bogdanovic N. Winblad B. Cedazo-Minguez A. Involvement of glutaredoxin-1 and thioredoxin-1 in β-amyloid toxicity and Alzheimer's disease.Cell Death Differ. 2006; 13: 1454-1465Crossref PubMed Scopus (148) Google Scholar, 11Lovell M.A. Xie C. Gabbita S.P. Markesbery W.R. Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer's disease brain.Free Radic. Biol. Med. 2000; 28: 418-427Crossref PubMed Scopus (180) Google Scholar). TRX1, the substrate of thioredoxin reductase, can protect neuronal cells by scavenging free radicals, binding and inhibiting apoptosis signal-regulating kinase 1 (12Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1.EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2092) Google Scholar), regulating transcription factors, and maintaining redox homeostasis (13Lillig C.H. Holmgren A. Thioredoxin and related molecules—from biology to health and disease.Antioxid. Redox Signal. 2007; 9: 25-47Crossref PubMed Scopus (594) Google Scholar). Rod-derived cone viability factor (RdCVF) belongs to the family of thioredoxins (14Leveillard T. Mohand-Said S. Lorentz O. Hicks D. Fintz A.C. Clerin E. Simonutti M. Forster V. Cavusoglu N. Chalmel F. Dolle P. Poch O. Lambrou G. Sahel J.A. Identification and characterization of rod-derived cone viability factor.Nat. Genet. 2004; 36: 755-759Crossref PubMed Scopus (349) Google Scholar). RdCVF is a trophic factor secreted by rods that promotes cone viability encoded by the nucleoredoxin-like 1 (Nxnl1) gene, also called thioredoxin-like 6. The loss of rods results in a decrease of RdCVF expression, which presumably leads to cone death due to a lack of trophic support (15Cronin T. Leveillard T. Sahel J.A. Retinal degenerations: from cell signaling to cell therapy; pre-clinical and clinical issues.Curr. Gene Ther. 2007; 7: 121-129Crossref PubMed Scopus (33) Google Scholar, 16Sahel J.A. Saving cone cells in hereditary rod diseases: a possible role for rod-derived cone viability factor (RdCVF) therapy.Retina. 2005; 25: S38-S39Crossref PubMed Scopus (35) Google Scholar). The TRX active site containing two cysteines (CXXC) catalyzes the reduction of disulfide bonds in targeted proteins (17Arner E.S. Holmgren A. Physiological functions of thioredoxin and thioredoxin reductase.Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2026) Google Scholar). The Nxnl1 gene encodes two products via alternative splicing that contain an active site: a full-length protein (RdCVFL) that might carry a thioredoxin activity (18Wang X.W. Liou Y.C. Ho B. Ding J.L. An evolutionarily conserved 16-kDa thioredoxin-related protein is an antioxidant which regulates the NF-κB signaling pathway.Free Radic. Biol. Med. 2007; 42: 247-259Crossref PubMed Scopus (29) Google Scholar, 19Wang X.W. Tan B.Z. Sun M. Ho B. Ding J.L. Thioredoxin-like 6 protects retinal cell line from photooxidative damage by upregulating NF-κB activity.Free Radic. Biol. Med. 2008; 45: 336-344Crossref PubMed Scopus (21) Google Scholar) and a C-terminally truncated protein (RdCVF) with trophic activity for cones but without any enzymatic activity. This latter form resembles the cytokine TRX80, a form of human TRX1 that has no thioredoxin activity (20Pekkari K. Avila-Carino J. Gurunath R. Bengtsson A. Scheynius A. Holmgren A. Truncated thioredoxin (Trx80) exerts unique mitogenic cytokine effects via a mechanism independent of thiol oxido-reductase activity.FEBS Lett. 2003; 539: 143-148Crossref PubMed Scopus (35) Google Scholar). The trophic effect is carried specifically by the short isoform RdCVF (14Leveillard T. Mohand-Said S. Lorentz O. Hicks D. Fintz A.C. Clerin E. Simonutti M. Forster V. Cavusoglu N. Chalmel F. Dolle P. Poch O. Lambrou G. Sahel J.A. Identification and characterization of rod-derived cone viability factor.Nat. Genet. 2004; 36: 755-759Crossref PubMed Scopus (349) Google Scholar). The thiol oxidoreductase activity of RdCVFL has to date only been observed in the arthropod Carcinoscorpius (18Wang X.W. Liou Y.C. Ho B. Ding J.L. An evolutionarily conserved 16-kDa thioredoxin-related protein is an antioxidant which regulates the NF-κB signaling pathway.Free Radic. Biol. Med. 2007; 42: 247-259Crossref PubMed Scopus (29) Google Scholar), and the participation of RdCVFL in the protection of photoreceptors is currently unknown. To clarify the role of RdCVFL in photoreceptor survival, we identified a specific functional interaction between RdCVFL and TAU using a proteomics approach. thioredoxin Alzheimer disease coding DNA sequence glycogen synthase kinase-3 rod-derived cone viability factor rod-derived cone viability factor long isoform nucleoredoxin-like 1 Search Tool for the Retrieval of Interacting Genes/Proteins gene ontology amino acids human embryonic kidney apoptosis inhibitor 5 poly(ADP-ribose) polymerase 1 hemagglutinin 5-(iodoacetamido)fluorescein TAU protein is a microtubule-associated protein that has a role in assembly and stabilization of microtubules (21Drubin D.G. Kirschner M.W. Tau protein function in living cells.J. Cell Biol. 1986; 103: 2739-2746Crossref PubMed Scopus (584) Google Scholar). In the brains of patients with Alzheimer disease (AD) TAU was found to be hyperphosphorylated (22Grundke-Iqbal I. Iqbal K. Tung Y.C. Quinlan M. Wisniewski H.M. Binder L.I. Abnormal phosphorylation of the microtubule-associated protein τ (tau) in Alzheimer cytoskeletal pathology.Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4913-4917Crossref PubMed Scopus (2896) Google Scholar), leading to aggregation of the protein and to a decrease in TAU binding to microtubules (23Buee L. Bussiere T. Buee-Scherrer V. Delacourte A. Hof P.R. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders.Brain Res. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1574) Google Scholar) resulting in cell death. Phosphorylated TAU is also toxic to neuronal cells (24Brandt R. Hundelt M. Shahani N. Tau alteration and neuronal degeneration in tauopathies: mechanisms and models.Biochim. Biophys. Acta. 2005; 1739: 331-354Crossref PubMed Scopus (202) Google Scholar). The interaction between RdCVFL and TAU is the first description of the participation of RdCVFL in the protection of photoreceptors and of a novel redox signaling pathway involved in regulating neuronal cell death. Care and handling of mice in these studies conformed to the rules set by the Association for Research on Vision and Ophthalmology Resolution. Chicken retinal extracts are described in Fintz et al. (25Fintz A.C. Audo I. Hicks D. Mohand-Said S. Leveillard T. Sahel J. Partial characterization of retina-derived cone neuroprotection in two culture models of photoreceptor degeneration.Investig. Ophthalmol. Vis. Sci. 2003; 44: 818-825Crossref PubMed Scopus (53) Google Scholar). The Nxnl1−/− mice were generated on a pure BALB/c background. Retinas were solubilized in Nonidet P-40 lysis buffer (25 mm Hepes, pH 7.8, 1 mm EDTA, 1 mm DTT, 0.05 m KCl, 1% Nonidet P-40, 1 mm PMSF, protease inhibitors) followed by sonication and centrifugation. The soluble fraction was subjected to further analysis. Unless otherwise specified, all chemicals were obtained from Sigma. RdCVFL was cloned into pGEX-2TK plasmid (GH Healthcare, catalogue number 27-4587-01), overexpressed, and purified as described previously (14Leveillard T. Mohand-Said S. Lorentz O. Hicks D. Fintz A.C. Clerin E. Simonutti M. Forster V. Cavusoglu N. Chalmel F. Dolle P. Poch O. Lambrou G. Sahel J.A. Identification and characterization of rod-derived cone viability factor.Nat. Genet. 2004; 36: 755-759Crossref PubMed Scopus (349) Google Scholar). GST and GST-RdCVFL were bound to Sepharose 4B beads (GE Healthcare), which were washed and packed in a column. An amount of GST and GST-RdCVFL equivalent in molarity was used. The columns were equilibrated with 25 mm Hepes, pH 7.8, 1 mm EDTA, 1 mm DTT, 0.05 m KCl. Chick retinas (postnatal day 17) were lysed in Nonidet P-40 lysis buffer followed by Polytron disruption and centrifugation. The soluble fraction was diluted to a concentration of 0.5 mg/ml and loaded on the equilibrated columns at a flow of 50 µl/min. After washing, elution was carried out with a KCl gradient (25 mm Hepes, pH 7.8, 1 mm EDTA, 1 mm DTT, 0.05–0.5 m KCl). Four fractions were collected from each column, desalted with Centricon (Amicon), and loaded on a gel followed by silver staining (ProteosilverPlus, Sigma). Lanes (except lane C) were cut out and subjected to MS analysis. In-gel digestion was performed with an automated protein digestion system, MassPREP Station (Waters). The gel slices were washed three times in a mixture containing 25 mm NH4HCO3, CH3CN (1:1, v/v). The cysteine residues were reduced by 50 µl of 10 mm DTT at 57 °C and alkylated by 50 µl of 55 mm iodoacetamide. After dehydration with acetonitrile, the proteins were cleaved in gel with 40 µl of 12.5 ng/µl modified porcine trypsin (Promega) in 25 mm NH4HCO3 at 37 °C for 4 h. The tryptic peptides were extracted with 60% acetonitrile in 5% formic acid followed by a second extraction with 100% (v/v) acetonitrile. The peptides extracted from the gel were directly analyzed by nano-LC-MS/MS. Nano-LC-MS/MS was performed on an Agilent 1100 Series HPLC-Chip/MS system (Agilent Technologies) coupled to an Ultra High-Capacity Trap mass spectrometer system (Bruker Daltonics). The voltage applied to the capillary cap was optimized to −1850 V. For tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The three most abundant peptides, preferring doubly charged ions, were selected on each MS spectrum for further isolation and fragmentation. The MS/MS scanning was performed in the ultrascan resolution mode at a scan rate of 26,000 m/z per second. A total of six scans were averaged to obtain a MS/MS spectrum. The complete system was fully controlled by ChemStation (Agilent Technologies) and EsquireControl (Bruker Daltonics) software. The mass data recorded during nano-LC-MS/MS analyses were processed and converted into .mgf peak list format. The MS and the MS/MS data were searched using a local Mascot server (version, Mascot 2.2.0; Matrix Science). The MS/MS data were analyzed against a composite target-decoy database including the National Center for Biotechnology Information (NCBI) protein sequences of human, Rattus norvegicus, Mus musculus, and Aves downloaded in February 2008 and reversed versions of these sequences (total 1,693,251 entries; see supplemental methods). Searches were performed with a mass tolerance of 250 ppm for MS mode and 0.4 Da in MS/MS mode for nano-LC-MS/MS analysis. One missed cleavage per peptide was allowed, and variable modifications were taken into account such as carbamidomethylation of cysteine, oxidation of methionine, and N-acetyl protein. Searches were performed without constraining protein molecular weight or isoelectric point and without any taxonomic restriction. To minimize false positive identifications, the results were subjected to very stringent filtering criteria. For the identification of a protein with two peptides or more, at least two unique peptides had to have a Mascot ion score above 25. In the case of single peptide hits, the score of the unique peptide must be greater (minimal "difference score" of 12) than the 95% significance Mascot threshold. The spectra of these single peptide hits are provided (supplemental data). For the estimation of the false positive rate, a target-decoy database search was performed (26Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2873) Google Scholar, 27Peng J. Elias J.E. Thoreen C.C. Licklider L.J. Gygi S.P. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome.J. Proteome Res. 2003; 2: 43-50Crossref PubMed Scopus (1388) Google Scholar). In this approach peptides are matched against a database consisting of the native protein sequences found in the database (target) and of the sequence-reversed entries (decoy). The evaluations were performed using the peptide validation software Scaffold (Proteome Software). This strategy was used to obtain a final catalogue of proteins with an estimated false positive rate below 1%. Protein-protein interactions were obtained from the STRING database (28von Mering C. Jensen L.J. Kuhn M. Chaffron S. Doerks T. Kruger B. Snel B. Bork P. STRING 7—recent developments in the integration and prediction of protein interactions.Nucleic Acids Res. 2007; 35: D358-D362Crossref PubMed Scopus (527) Google Scholar) containing known and predicted physical and functional protein-protein interactions. STRING in protein mode was used, and only interactions with high confidence levels (>0.7) were kept. Network visualization was done with the Cytoscape software (29Shannon P. Markiel A. Ozier O. Baliga N.S. Wang J.T. Ramage D. Amin N. Schwikowski B. Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks.Genome Res. 2003; 13: 2498-2504Crossref PubMed Scopus (26615) Google Scholar). We used the DAVID (Database for Annotation, Visualization and Integrated Discovery) functional annotation tool (30Sherman B.T. Huang D.W. Tan Q. Guo Y. Bour S. Liu D. Stephens R. Baseler M.W. Lane H.C. Lempicki R.A. DAVID Knowledgebase: a gene-centered database integrating heterogeneous gene annotation resources to facilitate high-throughput gene functional analysis.BMC Bioinformatics. 2007; 8: 426Crossref PubMed Scopus (418) Google Scholar) to identify the enriched gene ontology (GO) terms within the gene lists. We used p values after Benjamini correction and considered only those where the corrected p value was inferior to 10−7. Cone-enriched cultures were made as described previously (14Leveillard T. Mohand-Said S. Lorentz O. Hicks D. Fintz A.C. Clerin E. Simonutti M. Forster V. Cavusoglu N. Chalmel F. Dolle P. Poch O. Lambrou G. Sahel J.A. Identification and characterization of rod-derived cone viability factor.Nat. Genet. 2004; 36: 755-759Crossref PubMed Scopus (349) Google Scholar, 25Fintz A.C. Audo I. Hicks D. Mohand-Said S. Leveillard T. Sahel J. Partial characterization of retina-derived cone neuroprotection in two culture models of photoreceptor degeneration.Investig. Ophthalmol. Vis. Sci. 2003; 44: 818-825Crossref PubMed Scopus (53) Google Scholar). Total RNA from chicken cone-enriched cultures was purified by cesium gradient (14Leveillard T. Mohand-Said S. Lorentz O. Hicks D. Fintz A.C. Clerin E. Simonutti M. Forster V. Cavusoglu N. Chalmel F. Dolle P. Poch O. Lambrou G. Sahel J.A. Identification and characterization of rod-derived cone viability factor.Nat. Genet. 2004; 36: 755-759Crossref PubMed Scopus (349) Google Scholar) and subjected to RT analysis. PCR was performed with 100 ng of cDNA. Cycling conditions were as follow: 94 °C for 5 min followed by 35 cycles of amplification (94 °C denaturation for 30 s, annealing for 30 s at a temperature range of 52–58 °C, and 72 °C elongation for 1 min) with a final incubation at 72 °C for 3 min. Details of primers are provided in supplemental Table II. The PCR products of human TRX1 and chicken TAU were cloned into the pCMV-HA/DEST vector (a gift from R. Roepman) by Gateway system cloning (BP and LR reactions). pCMV4-GSK-3β and the cDNA of human TAU (383 aa) were kindly provided by C. Gespach and I. Ginzburg, respectively. Chicken genomic DNA was purified using standard methods. COS-1 and HEK 293 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The cells were transfected using the calcium phosphate co-precipitation technique (14Leveillard T. Mohand-Said S. Lorentz O. Hicks D. Fintz A.C. Clerin E. Simonutti M. Forster V. Cavusoglu N. Chalmel F. Dolle P. Poch O. Lambrou G. Sahel J.A. Identification and characterization of rod-derived cone viability factor.Nat. Genet. 2004; 36: 755-759Crossref PubMed Scopus (349) Google Scholar). 48 h after transfection, cells were resuspended in lysis buffer (50 mm Tris, pH 7.5, 1 mm EDTA, 1 mm DTT, 50 µg/ml Nα-tosyl-l-lysinyl-chloromethyl ketone, 1% Triton X-100, protease inhibitors), sonicated, and centrifuged, and the supernatant was kept at −80 °C. Diluted transfected cell extracts were incubated overnight with different antibodies. Immunocomplexes were immobilized on protein A-Sepharose beads. Beads were washed, and protein complexes were eluted by adding Laemmli buffer. Western blotting is described in Leveillard et al. (14Leveillard T. Mohand-Said S. Lorentz O. Hicks D. Fintz A.C. Clerin E. Simonutti M. Forster V. Cavusoglu N. Chalmel F. Dolle P. Poch O. Lambrou G. Sahel J.A. Identification and characterization of rod-derived cone viability factor.Nat. Genet. 2004; 36: 755-759Crossref PubMed Scopus (349) Google Scholar). The antibodies used were as follows: anti-HA (Covance, catalogue number MMS-101R-0500; 1:1000), anti-RdCVF (RdCVF-N, 1:500), anti-Tau5 (Calbiochem, catalogue number 577801; 1:500), anti-AT8 (Autogen Bioclear, catalogue number 90206; 1:100), anti-actin (Chemicon, catalogue number MAB1501; 1:7500), and anti-human TRX1 (IMCO Corp., catalogue number ATRX-03; 1:1000). Nxnl1+/+ and Nxnl1−/− mice aged 10 months were used. Retinas were removed from eyes and fixed in 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.2, for 2 h. Immunohistochemistry was performed on 5-µm sections obtained from paraffin-embedded tissue blocks. TAU was visualized using anti-AT8 and -Tau5 antibodies. Sections were subjected to microwave irradiation for antigen retrieval. Nonspecific binding sites were blocked by incubation of sections for 1 h at room temperature with normal goat serum in PBS. Sections were incubated overnight at 4 °C with AT8 antibody (1:250) in PBS. Immunostaining was visualized using the avidin-biotin system (Vectastain, Vector Laboratories) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as the chromogen. After washing, sections were mounted in Vecstatin and imaged with a microscope (Leica). GST and GST-RdCVFL were eluted from beads using a buffer containing 10 mm glutathione. GST, GST-RdCVFL (in PBS), and the recombinant human TAU protein samples (Sigma, catalogue number T9392) in 50 mm MOPS, pH 6.8, 100 mm NaCl, 1 mm EDTA, 5 mm DTT, 1 mm PMSF were dialyzed against 50 mm Tris-HCl, pH 7.5, 1 mm EDTA overnight with Dispo-Biodialyzer devices (molecular mass cutoff, 10 kDa) (Sigma). 1 µm TAU protein was mixed with 2 µm GST or GST-RdCVFL. The mixture or TAU protein alone was treated with 200 µm H2O2 for 2 h at room temperature. The oxidized TAU was incubated with the thioredoxin system (500 µm NADPH (Sigma), 100 nm recombinant thioredoxin reductase, 20 µm thioredoxin1 (IMCO Corp., catalogue number TRX-01)) for 1 h. The free cysteines of the proteins were labeled by 100 µm fluorescent 5-(iodoacetamido)fluorescein for 20 min. This labeling was stopped by addition of 50 mm iodoacetamide. The sample loading buffer with or without 50 mm DTT was incubated with the samples at 56 °C for 20 min, and the proteins were separated on a gel. The fluorescent bands were detected under a UV light, and subsequently the protein bands were detected by Coomassie Blue staining. Supplemental Table I displays the 44 and 121 proteins that were identified as GST- and GST-RdCVFL-interacting proteins, respectively. Supplemental Table II displays the sequence primers used in this study. Supplemental data display the spectra of all the proteins identified with one peptide. Recombinant protein sequences used in the study are provided in supplemental Table III. RdCVF was originally identified using cone-enriched cultures from chicken embryos. Bird retinas are cone-dominated, and in culture, cones represent 70% of the cell population (25Fintz A.C. Audo I. Hicks D. Mohand-Said S. Leveillard T. Sahel J. Partial characterization of retina-derived cone neuroprotection in two culture models of photoreceptor degeneration.Investig. Ophthalmol. Vis. Sci. 2003; 44: 818-825Crossref PubMed Scopus (53) Google Scholar). In the mouse retina RdCVF and RdCVFL are encoded by two alternative mRNA transcripts corresponding to exon 1 and to the spliced exons 1 and 2, respectively. In the chicken genome there is no in-frame stop codon immediately 3′ to exon 1 (31Chalmel F. Leveillard T. Jaillard C. Lardenois A. Berdugo N. Morel E. Koehl P. Lambrou G. Holmgren A. Sahel J.A. Poch O. Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential.BMC Mol. Biol. 2007; 8: 74Crossref PubMed Scopus (56) Google Scholar). We analyzed the expression of NXNL1 mRNAs by RT-PCR in the cone-enriched culture (Fig. 1A). In these cultures, visinin is selectively expressed by cone cells (25Fintz A.C. Audo I. Hicks D. Mohand-Said S. Leveillard T. Sahel J. Partial characterization of retina-derived cone neuroprotection in two culture models of photoreceptor degeneration.Investig. Ophthalmol. Vis. Sci. 2003; 44: 818-825Crossref PubMed Scopus (53) Google Scholar). We detected an amplification product of RdCVFL mRNA but not RdCVF (Fig. 1B). The efficiency of RdCVF primers was validated on genomic DNA. The data demonstrate that RdCVFL is expressed by the chick cone culture. To identify RdCVFL-interacting proteins, we prepared soluble protein extract from chicken retina at postnatal day 17, a developmental stage rich in mature cones (result not shown). This retinal extract was loaded on GST-RdCVFL or GST columns. After washing, the elution was carried out with a 50–500 mm KCl gradient. The eluted proteins collected in four fractions were loaded on a gel and silver-stained (Fig. 2). For both columns the amount of protein eluted decreased with increasing KCl concentration. Some differences in the eluted profile are indicated by an asterisk in Fig. 2. Each lane was cut into 2-mm slices corresponding to different molecular weights and subjected to proteomics analysis. To identify the proteins, the trypsin-digested peptides in the individual slices were analyzed by nano-LC-MS/MS. Mass data collected during an LC-MS/MS analysis were processed and converted into a .pkl file to be submitted to the search software Mascot. A composite target-decoy database strategy was used to generate score criteria that yielded an estimated false positive rate of 1% (26Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2873) Google Scholar). 44 and 121 proteins were identified as GST- and GST-RdCVFL-interacting proteins, respectively (supplemental Table I). After subtraction of the redundant proteins and the proteins eluted from the GST column, 90 proteins were found to interact specifically with RdCVFL (Table I). Among the proteins identified in this study, we found proteins related to apoptosis (apoptosis inhibitor 5 (API5) and poly(ADP-ribose) polymerase 1 (PARP1)) and linked to neurodegenerative diseases (TAU/MAPT (microtubule-associated protein Tau)). TAU was identified in several gel slices corresponding to molecular mass of 30–60 kDa by three different peptides (supplemental Table I).Table IRdCVFL-interacting proteins identified by nano-LC-MS/MSSymbolProtein nameGIkDaEluted fractionACTBβ-Actin2110559DaThe proteins that figure in the interactome scheme (Fig. 3).AMFRAutocrine motility factor receptor11809621688BDAPI5API55752997459AB
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