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. 2000 Jul 15;349(Pt 2):567–578. doi: 10.1042/0264-6021:3490567

Novel application of S-nitrosoglutathione-Sepharose to identify proteins that are potential targets for S-nitrosoglutathione-induced mixed-disulphide formation.

P Klatt 1, E Pineda Molina 1, D Pérez-Sala 1, S Lamas 1
PMCID: PMC1221180  PMID: 10880356

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abate C., Patel L., Rauscher F. J., 3rd, Curran T. Redox regulation of fos and jun DNA-binding activity in vitro. Science. 1990 Sep 7;249(4973):1157–1161. doi: 10.1126/science.2118682. [DOI] [PubMed] [Google Scholar]
  2. Anderson M. E., Meister A. Glutathione monoesters. Anal Biochem. 1989 Nov 15;183(1):16–20. doi: 10.1016/0003-2697(89)90164-4. [DOI] [PubMed] [Google Scholar]
  3. Barrett W. C., DeGnore J. P., Keng Y. F., Zhang Z. Y., Yim M. B., Chock P. B. Roles of superoxide radical anion in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase 1B. J Biol Chem. 1999 Dec 3;274(49):34543–34546. doi: 10.1074/jbc.274.49.34543. [DOI] [PubMed] [Google Scholar]
  4. Beckman J. S., Koppenol W. H. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol. 1996 Nov;271(5 Pt 1):C1424–C1437. doi: 10.1152/ajpcell.1996.271.5.C1424. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Chai Y. C., Jung C. H., Lii C. K., Ashraf S. S., Hendrich S., Wolf B., Sies H., Thomas J. A. Identification of an abundant S-thiolated rat liver protein as carbonic anhydrase III; characterization of S-thiolation and dethiolation reactions. Arch Biochem Biophys. 1991 Feb 1;284(2):270–278. doi: 10.1016/0003-9861(91)90295-t. [DOI] [PubMed] [Google Scholar]
  7. Chandra A., Srivastava S., Petrash J. M., Bhatnagar A., Srivastava S. K. Modification of aldose reductase by S-nitrosoglutathione. Biochemistry. 1997 Dec 16;36(50):15801–15809. doi: 10.1021/bi9714722. [DOI] [PubMed] [Google Scholar]
  8. Cornell N. W., Veech R. L. Enzymatic measurement of ethanol or NAD in acid extracts of biological samples. Anal Biochem. 1983 Jul 15;132(2):418–423. doi: 10.1016/0003-2697(83)90029-5. [DOI] [PubMed] [Google Scholar]
  9. Dafré A. L., Reischl E. Oxidative stress causes intracellular reversible S-thiolation of chicken hemoglobin under diamide and xanthine oxidase treatment. Arch Biochem Biophys. 1998 Oct 15;358(2):291–296. doi: 10.1006/abbi.1998.0848. [DOI] [PubMed] [Google Scholar]
  10. De Bruin S. H., Joordens J. J., Rollema H. S. An isolation procedure for the native alpha chain of bovine hemoglobin. A study of the functional properties of this chain and its hybrid with the human beta chain. Eur J Biochem. 1977 May 2;75(1):211–215. doi: 10.1111/j.1432-1033.1977.tb11519.x. [DOI] [PubMed] [Google Scholar]
  11. DeMaster E. G., Quast B. J., Redfern B., Nagasawa H. T. Reaction of nitric oxide with the free sulfhydryl group of human serum albumin yields a sulfenic acid and nitrous oxide. Biochemistry. 1995 Sep 12;34(36):11494–11499. doi: 10.1021/bi00036a023. [DOI] [PubMed] [Google Scholar]
  12. Denninger J. W., Marletta M. A. Guanylate cyclase and the .NO/cGMP signaling pathway. Biochim Biophys Acta. 1999 May 5;1411(2-3):334–350. doi: 10.1016/s0005-2728(99)00024-9. [DOI] [PubMed] [Google Scholar]
  13. Egorov T. A., Svenson A., Rydén L., Carlsson J. A rapid and specific method for isolation of thiol-containing peptides from large proteins by thiol-disulfide exchange on a solid support. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3029–3033. doi: 10.1073/pnas.72.8.3029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galter D., Mihm S., Dröge W. Distinct effects of glutathione disulphide on the nuclear transcription factor kappa B and the activator protein-1. Eur J Biochem. 1994 Apr 15;221(2):639–648. doi: 10.1111/j.1432-1033.1994.tb18776.x. [DOI] [PubMed] [Google Scholar]
  15. Gaston B. Nitric oxide and thiol groups. Biochim Biophys Acta. 1999 May 5;1411(2-3):323–333. doi: 10.1016/s0005-2728(99)00023-7. [DOI] [PubMed] [Google Scholar]
  16. Gergel D., Cederbaum A. I. Inhibition of the catalytic activity of alcohol dehydrogenase by nitric oxide is associated with S nitrosylation and the release of zinc. Biochemistry. 1996 Dec 17;35(50):16186–16194. doi: 10.1021/bi962043r. [DOI] [PubMed] [Google Scholar]
  17. Ghosh G., van Duyne G., Ghosh S., Sigler P. B. Structure of NF-kappa B p50 homodimer bound to a kappa B site. Nature. 1995 Jan 26;373(6512):303–310. doi: 10.1038/373303a0. [DOI] [PubMed] [Google Scholar]
  18. Glover J. N., Harrison S. C. Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature. 1995 Jan 19;373(6511):257–261. doi: 10.1038/373257a0. [DOI] [PubMed] [Google Scholar]
  19. Ji Y., Akerboom T. P., Sies H., Thomas J. A. S-nitrosylation and S-glutathiolation of protein sulfhydryls by S-nitroso glutathione. Arch Biochem Biophys. 1999 Feb 1;362(1):67–78. doi: 10.1006/abbi.1998.1013. [DOI] [PubMed] [Google Scholar]
  20. Kadonaga J. T., Carner K. R., Masiarz F. R., Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. doi: 10.1016/0092-8674(87)90594-0. [DOI] [PubMed] [Google Scholar]
  21. Kahlenberg A. Preparative isolation of band 3, the predominant polypeptides of the human erythrocyte membrane. Anal Biochem. 1976 Aug;74(2):337–342. doi: 10.1016/0003-2697(76)90214-1. [DOI] [PubMed] [Google Scholar]
  22. Klatt P., Molina E. P., De Lacoba M. G., Padilla C. A., Martinez-Galesteo E., Barcena J. A., Lamas S. Redox regulation of c-Jun DNA binding by reversible S-glutathiolation. FASEB J. 1999 Sep;13(12):1481–1490. doi: 10.1096/fasebj.13.12.1481. [DOI] [PubMed] [Google Scholar]
  23. Klatt P., Molina E. P., Lamas S. Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation. J Biol Chem. 1999 May 28;274(22):15857–15864. doi: 10.1074/jbc.274.22.15857. [DOI] [PubMed] [Google Scholar]
  24. Kondo T., Taniguchi N., Hirano T., Kawakami Y. A novel low-activity form of carbonic anhydrase I in erythrocytes of patients with primary aldosteronism. Evidence for the presence of a mixed disulfide with glutathione. J Biol Chem. 1984 Dec 25;259(24):15517–15522. [PubMed] [Google Scholar]
  25. Koppenol W. H. The basic chemistry of nitrogen monoxide and peroxynitrite. Free Radic Biol Med. 1998 Sep;25(4-5):385–391. doi: 10.1016/s0891-5849(98)00093-8. [DOI] [PubMed] [Google Scholar]
  26. Liu Z., Rudd M. A., Freedman J. E., Loscalzo J. S-Transnitrosation reactions are involved in the metabolic fate and biological actions of nitric oxide. J Pharmacol Exp Ther. 1998 Feb;284(2):526–534. [PubMed] [Google Scholar]
  27. Marletta M. A. Nitric oxide synthase: aspects concerning structure and catalysis. Cell. 1994 Sep 23;78(6):927–930. doi: 10.1016/0092-8674(94)90268-2. [DOI] [PubMed] [Google Scholar]
  28. Matthews J. R., Botting C. H., Panico M., Morris H. R., Hay R. T. Inhibition of NF-kappaB DNA binding by nitric oxide. Nucleic Acids Res. 1996 Jun 15;24(12):2236–2242. doi: 10.1093/nar/24.12.2236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matthews J. R., Wakasugi N., Virelizier J. L., Yodoi J., Hay R. T. Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res. 1992 Aug 11;20(15):3821–3830. doi: 10.1093/nar/20.15.3821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mayer B., Hemmens B. Biosynthesis and action of nitric oxide in mammalian cells. Trends Biochem Sci. 1997 Dec;22(12):477–481. doi: 10.1016/s0968-0004(97)01147-x. [DOI] [PubMed] [Google Scholar]
  31. Mohr S., Hallak H., de Boitte A., Lapetina E. G., Brüne B. Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem. 1999 Apr 2;274(14):9427–9430. doi: 10.1074/jbc.274.14.9427. [DOI] [PubMed] [Google Scholar]
  32. Morel Y., Barouki R. Down-regulation of cytochrome P450 1A1 gene promoter by oxidative stress. Critical contribution of nuclear factor 1. J Biol Chem. 1998 Oct 9;273(41):26969–26976. doi: 10.1074/jbc.273.41.26969. [DOI] [PubMed] [Google Scholar]
  33. Müller C. W., Rey F. A., Sodeoka M., Verdine G. L., Harrison S. C. Structure of the NF-kappa B p50 homodimer bound to DNA. Nature. 1995 Jan 26;373(6512):311–317. doi: 10.1038/373311a0. [DOI] [PubMed] [Google Scholar]
  34. Nikitovic D., Holmgren A. S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide. J Biol Chem. 1996 Aug 9;271(32):19180–19185. doi: 10.1074/jbc.271.32.19180. [DOI] [PubMed] [Google Scholar]
  35. Nikitovic D., Holmgren A., Spyrou G. Inhibition of AP-1 DNA binding by nitric oxide involving conserved cysteine residues in Jun and Fos. Biochem Biophys Res Commun. 1998 Jan 6;242(1):109–112. doi: 10.1006/bbrc.1997.7930. [DOI] [PubMed] [Google Scholar]
  36. Padgett C. M., Whorton A. R. Cellular responses to nitric oxide: role of protein S-thiolation/dethiolation. Arch Biochem Biophys. 1998 Oct 15;358(2):232–242. doi: 10.1006/abbi.1998.0859. [DOI] [PubMed] [Google Scholar]
  37. Park E. M., Thomas J. A. S-thiolation of creatine kinase and glycogen phosphorylase b initiated by partially reduced oxygen species. Biochim Biophys Acta. 1988 Feb 17;964(2):151–160. doi: 10.1016/0304-4165(88)90161-4. [DOI] [PubMed] [Google Scholar]
  38. Park J. W. Reaction of S-nitrosoglutathione with sulfhydryl groups in protein. Biochem Biophys Res Commun. 1988 Apr 29;152(2):916–920. doi: 10.1016/s0006-291x(88)80127-x. [DOI] [PubMed] [Google Scholar]
  39. Patel R. P., McAndrew J., Sellak H., White C. R., Jo H., Freeman B. A., Darley-Usmar V. M. Biological aspects of reactive nitrogen species. Biochim Biophys Acta. 1999 May 5;1411(2-3):385–400. doi: 10.1016/s0005-2728(99)00028-6. [DOI] [PubMed] [Google Scholar]
  40. Percival M. D., Ouellet M., Campagnolo C., Claveau D., Li C. Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid. Biochemistry. 1999 Oct 12;38(41):13574–13583. doi: 10.1021/bi991028u. [DOI] [PubMed] [Google Scholar]
  41. Ravichandran V., Seres T., Moriguchi T., Thomas J. A., Johnston R. B., Jr S-thiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the phagocytosis-associated respiratory burst in blood monocytes. J Biol Chem. 1994 Oct 7;269(40):25010–25015. [PubMed] [Google Scholar]
  42. Schininà M. E., Carlini P., Polticelli F., Zappacosta F., Bossa F., Calabrese L. Amino acid sequence of chicken Cu, Zn-containing superoxide dismutase and identification of glutathionyl adducts at exposed cysteine residues. Eur J Biochem. 1996 Apr 15;237(2):433–439. doi: 10.1111/j.1432-1033.1996.0433k.x. [DOI] [PubMed] [Google Scholar]
  43. Schuppe-Koistinen I., Moldéus P., Bergman T., Cotgreave I. A. S-thiolation of human endothelial cell glyceraldehyde-3-phosphate dehydrogenase after hydrogen peroxide treatment. Eur J Biochem. 1994 May 1;221(3):1033–1037. doi: 10.1111/j.1432-1033.1994.tb18821.x. [DOI] [PubMed] [Google Scholar]
  44. Smith R. E., MacQuarrie R. The role of cysteine residues in the catalytic activity of glycerol-3-phosphate dehydrogenase. Biochim Biophys Acta. 1979 Apr 12;567(2):269–277. doi: 10.1016/0005-2744(79)90112-8. [DOI] [PubMed] [Google Scholar]
  45. Snyder G. H., Cennerazzo M. J., Karalis A. J., Field D. Electrostatic influence of local cysteine environments on disulfide exchange kinetics. Biochemistry. 1981 Nov 10;20(23):6509–6519. doi: 10.1021/bi00526a001. [DOI] [PubMed] [Google Scholar]
  46. Stamler J. S., Hausladen A. Oxidative modifications in nitrosative stress. Nat Struct Biol. 1998 Apr;5(4):247–249. doi: 10.1038/nsb0498-247. [DOI] [PubMed] [Google Scholar]
  47. Stamler J. S., Jia L., Eu J. P., McMahon T. J., Demchenko I. T., Bonaventura J., Gernert K., Piantadosi C. A. Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science. 1997 Jun 27;276(5321):2034–2037. doi: 10.1126/science.276.5321.2034. [DOI] [PubMed] [Google Scholar]
  48. Stamler J. S. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell. 1994 Sep 23;78(6):931–936. doi: 10.1016/0092-8674(94)90269-0. [DOI] [PubMed] [Google Scholar]
  49. Stamler J. S., Simon D. I., Osborne J. A., Mullins M. E., Jaraki O., Michel T., Singel D. J., Loscalzo J. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):444–448. doi: 10.1073/pnas.89.1.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sykes B. C. The separation of two soft-tissue collagens by covalent chromatography. FEBS Lett. 1976 Jan 15;61(2):180–185. doi: 10.1016/0014-5793(76)81032-0. [DOI] [PubMed] [Google Scholar]
  51. Sáez G. T., Romero F. J., Viña J. Effects of glutathione depletion on gluconeogenesis in isolated hepatocytes. Arch Biochem Biophys. 1985 Aug 15;241(1):75–80. doi: 10.1016/0003-9861(85)90363-7. [DOI] [PubMed] [Google Scholar]
  52. Thomas J. A., Poland B., Honzatko R. Protein sulfhydryls and their role in the antioxidant function of protein S-thiolation. Arch Biochem Biophys. 1995 May 10;319(1):1–9. doi: 10.1006/abbi.1995.1261. [DOI] [PubMed] [Google Scholar]
  53. Tien M., Berlett B. S., Levine R. L., Chock P. B., Stadtman E. R. Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation. Proc Natl Acad Sci U S A. 1999 Jul 6;96(14):7809–7814. doi: 10.1073/pnas.96.14.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Viner R. I., Williams T. D., Schöneich C. Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase. Biochemistry. 1999 Sep 21;38(38):12408–12415. doi: 10.1021/bi9909445. [DOI] [PubMed] [Google Scholar]
  55. Wink D. A., Mitchell J. B. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med. 1998 Sep;25(4-5):434–456. doi: 10.1016/s0891-5849(98)00092-6. [DOI] [PubMed] [Google Scholar]
  56. Xanthoudakis S., Curran T. Analysis of c-Fos and c-Jun redox-dependent DNA binding activity. Methods Enzymol. 1994;234:163–174. doi: 10.1016/0076-6879(94)34086-2. [DOI] [PubMed] [Google Scholar]
  57. Yang Y., Jao S. c., Nanduri S., Starke D. W., Mieyal J. J., Qin J. Reactivity of the human thioltransferase (glutaredoxin) C7S, C25S, C78S, C82S mutant and NMR solution structure of its glutathionyl mixed disulfide intermediate reflect catalytic specificity. Biochemistry. 1998 Dec 8;37(49):17145–17156. doi: 10.1021/bi9806504. [DOI] [PubMed] [Google Scholar]
  58. Zech B., Wilm M., van Eldik R., Brüne B. Mass spectrometric analysis of nitric oxide-modified caspase-3. J Biol Chem. 1999 Jul 23;274(30):20931–20936. doi: 10.1074/jbc.274.30.20931. [DOI] [PubMed] [Google Scholar]
  59. Zweier J. L., Samouilov A., Kuppusamy P. Non-enzymatic nitric oxide synthesis in biological systems. Biochim Biophys Acta. 1999 May 5;1411(2-3):250–262. doi: 10.1016/s0005-2728(99)00018-3. [DOI] [PubMed] [Google Scholar]

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