Novel application of S-nitrosoglutathione-Sepharose to identify proteins that are potential targets for S-nitrosoglutathione-induced mixed-disulphide formation.
Site-specific S-glutathionylation is emerging as a novel mechanism by which S-nitrosoglutathione (GSNO) may modify functionally important protein thiols. Here, we show that GSNO-Sepharose mimicks site-specific S-glutathionylation of the transcription factors c-Jun and p50 by free GSNO in vitro. Both c-Jun and p50 were found to bind to immobilized GSNO through the formation of a mixed disulphide, involving a conserved cysteine residue located in the DNA-binding domains of these transcription factors. Furthermore, we show that c-Jun, p50, glycogen phosphorylase b, glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, glutaredoxin and caspase-3 can be precipitated from a mixture of purified thiol-containing proteins by the formation of a mixed-disulphide bond with GSNO-Sepharose. With few exceptions, protein binding to this matrix correlated well with the susceptibility of the investigated proteins to undergo GSNO- but not diamide-induced mixed-disulphide formation in vitro. Finally, it is shown that covalent GSNO-Sepharose chromatography of HeLa cell nuclear extracts results in the enrichment of proteins which incorporate glutathione in response to GSNO treatment. As suggested by DNA-binding assays, this group of nuclear proteins include the transcription factors activator protein-1, nuclear factor-kappaB and cAMP-response-element-binding protein. In conclusion, we introduce GSNO-Sepharose as a probe for site-specific S-glutathionylation and as a novel and potentially useful tool to isolate and identify proteins which are candidate targets for GSNO-induced mixed-disulphide formation.
Klatt
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Department of Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, Instituto Reina Sofía de Investigaciones Nefrológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, E-28006 Madrid, Spain. pklatt@cnb.uam.es
Pineda Molina
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Pérez-Sala
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D
Lamas
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D001665:Binding Sites; D002457:Cell Extracts / Q000737:chemistry; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009603:Nitroso Compounds / Q000737:chemistry* / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000378:metabolism; D026422:S-Nitrosoglutathione; D012685:Sepharose / Q000737:chemistry; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism
D002457:Cell Extracts; D004220:Disulfides; D009603:Nitroso Compounds; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D026422:S-Nitrosoglutathione; D012685:Sepharose; D005978:Glutathione