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W2009953261 abstract "S-Nitrosoglutathione (GSNO) undergoes spontaneous degradation that generates several nitrogen-containing compounds and oxidized glutathione derivatives. We identified glutathione sulfonic acid, glutathione disulfideS-oxide (GS(O)SG), glutathione disulfideS-dioxide, and GSSG as the major decomposition products of GSNO. Each of these compounds and GSNO were tested for their efficacies to modify rat brain neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm). Among them, GS(O)SG was found to be the most potent in causing glutathiolation of both proteins; four glutathiones were incorporated into the four Cys residues of Ng, and two were incorporated into the two Cys residues of Nm. Ng and Nm are two in vivo substrates of protein kinase C; their phosphorylations by protein kinase C attenuate the binding affinities of both proteins for calmodulin. When compared with their respective unmodified forms, the glutathiolated Ng was a poorer substrate and glutathiolated Nm a better substrate for protein kinase C. Glutathiolation of these two proteins caused no change in their binding affinities for calmodulin. Treatment of [35S]cysteine-labeled rat brain slices with xanthine/xanthine oxidase or a combination of xanthine/xanthine oxidase with sodium nitroprusside resulted in an increase in cellular level of GS(O)SG. These treatments, as well as those by other oxidants, all resulted in an increase in thiolation of proteins; among them, thiolation of Ng was positively identified by immunoprecipitation. These results show that GS(O)SG is one of the most potent glutathiolating agents generated upon oxidative stress. S-Nitrosoglutathione (GSNO) undergoes spontaneous degradation that generates several nitrogen-containing compounds and oxidized glutathione derivatives. We identified glutathione sulfonic acid, glutathione disulfideS-oxide (GS(O)SG), glutathione disulfideS-dioxide, and GSSG as the major decomposition products of GSNO. Each of these compounds and GSNO were tested for their efficacies to modify rat brain neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm). Among them, GS(O)SG was found to be the most potent in causing glutathiolation of both proteins; four glutathiones were incorporated into the four Cys residues of Ng, and two were incorporated into the two Cys residues of Nm. Ng and Nm are two in vivo substrates of protein kinase C; their phosphorylations by protein kinase C attenuate the binding affinities of both proteins for calmodulin. When compared with their respective unmodified forms, the glutathiolated Ng was a poorer substrate and glutathiolated Nm a better substrate for protein kinase C. Glutathiolation of these two proteins caused no change in their binding affinities for calmodulin. Treatment of [35S]cysteine-labeled rat brain slices with xanthine/xanthine oxidase or a combination of xanthine/xanthine oxidase with sodium nitroprusside resulted in an increase in cellular level of GS(O)SG. These treatments, as well as those by other oxidants, all resulted in an increase in thiolation of proteins; among them, thiolation of Ng was positively identified by immunoprecipitation. These results show that GS(O)SG is one of the most potent glutathiolating agents generated upon oxidative stress. protein kinase C neurogranin/RC3 neuromodulin/GAP-43 calmodulin S-nitrosoglutathione sodium nitroprusside iodoacetamide acetamide xanthine xanthine oxidase electrospray ionization mass spectrometry artificial cerebrospinal fluid reduced oxidized glutathiolated residue nitric oxide glutathione sulfonic acid glutathione disulfide S-oxide glutathione disulfide S-dioxide high pressure liquid chromatography dithiothreitol polyacrylamide gel electrophoresis Protein S-glutathiolation can be induced in cells by mild oxidative stress (1Thomas J.A. Poland B. Honzatko R. Arch. Biochem. Biophys. 1995; 319: 1-9Crossref PubMed Scopus (363) Google Scholar). GSSG has been shown to oxidatively regulate the activity of several purified enzymes including carbonic anhydrase III (2Cabiscol E. Levine R.L. J. Biol. Chem. 1995; 270: 14742-14747Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 3Cabiscol E. Levine R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4170-4174Crossref PubMed Scopus (132) Google Scholar), protein kinase C (PKC)1 (4Ward N.E. Pierce D.S. Chung S.E. Gravitt K.R. O'Brian C.A. J. Biol. Chem. 1998; 273: 12558-12566Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), human aldose reductase (5Cappiello M. Voltarelli M. Cecconi I. Vilardo P.G. Dal Monte M. Marini I. Del Corso A. Wilson D.K. Quiocho F.A. Petrash J.M. Mura U. J. Biol. Chem. 1996; 271: 33539-33544Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and human immunodeficiency virus, type I protease (6Davis D.A. Dorsey K. Wingfield P.T. Stahl S.J. Kaufman J. Fales H.M. Levine R.L. Biochemistry. 1996; 35: 2482-2488Crossref PubMed Scopus (96) Google Scholar), and in each case the effects of glutathiolation can be reversed by reducing agents. As the concentration of reduced GSH in the mammalian cells is in the millimolar range and that of GSSG is less than 5% of GSH, glutathiolation of proteins by GSSG in vivo is not likely an efficient mechanism. More recently, the superoxide-induced glutathiolation of protein (7Barrett W.C. DeGnore J.P. Keng Y.-F. Zhang Z.-Y. Yim M.B. Chock P.B. J. Biol. Chem. 1999; 274: 34543-34546Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) and that induced by peroxynitrite, nitric oxide (NO), and nitrosothiol, in particular,S-nitrosoglutathione (GSNO), are thought to be the main avenues leading to protein S-thiolation (8Viner R.I. Williams T.D. Schöneich C. Biochemistry. 1999; 38: 12408-12415Crossref PubMed Scopus (213) Google Scholar, 9Padgett C.M. Whorton A.R. Arch. Biochem. Biophys. 1998; 358: 232-242Crossref PubMed Scopus (84) Google Scholar, 10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar, 11Mohr S. Hallak H. de Boitte A. Lapetina E.G. Brüne B. J. Biol. Chem. 1999; 274: 9427-9430Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 12Percival M.D. Ouellet M. Campagnolo C. Claveau D. Li C. Biochemistry. 1999; 38: 13574-13583Crossref PubMed Scopus (123) Google Scholar). In mammalian cells, a relatively high concentration of GSH (0.5–10 mm) serves as an NO sink to form GSNO (13Kharitonov V.G. Sundquist A.R. Skarma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 14Clancy R.M. Levartovsky D. Leszczynska-Piziak J. Yegudin J. Abramson S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3680-3684Crossref PubMed Scopus (305) Google Scholar, 15Mayer B. Pfeiffer S. Schrammel A. Koesling D. Schmidt K. Brunner F. J. Biol. Chem. 1998; 273: 3264-3270Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 16Gow A.J. Buerk D.G. Ischiropoulos H. J. Biol. Chem. 1997; 272: 2841-2845Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), which can undergo transnitrosylation with protein sulfhydryl group to formS-nitrosoprotein and GSH or to form protein-GSH mixed disulfide and nitroxyl (17Park J.W. Biochem. Biophys. Res. Commun. 1988; 52: 916-920Crossref Scopus (110) Google Scholar, 18Scharfstein J.S. Keaney Jr., J.F. Slivka A. Welch G.N. Vita J.A. Stamler J.S. Loscalzo J. J. Clin. Invest. 1994; 94: 1432-1439Crossref PubMed Scopus (255) Google Scholar, 19Meyer D.J. Kramer H. Ozer N. Coles B. Ketterer B. FEBS Lett. 1994; 345: 177-180Crossref PubMed Scopus (146) Google Scholar, 20Wong P.S.-Y. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (327) Google Scholar). GSNO can also release NO in the presence of cuprous ion (21Singh R.J. Hogg N. Joseph J. Kalyanaraman B. J. Biol. Chem. 1996; 271: 18596-18603Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar), ascorbate (22Kashiba-Iwatsuki M. Yamaguchi M. Inoue M. FEBS Lett. 1996; 389: 149-152Crossref PubMed Scopus (67) Google Scholar), or thiols (20Wong P.S.-Y. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (327) Google Scholar, 23Singh S.P. Wishnok J.S. Keshive M. Deen W.M. Tannenbaum S.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14428-14433Crossref PubMed Scopus (298) Google Scholar) and serves as a possible source of nitrsonium or nitroxyl ions (24Arnelle D.R. Stamler J.S. Arch. Biochem. Biophys. 1995; 318: 279-285Crossref PubMed Scopus (538) Google Scholar). In addition, GSNO is unstable in aqueous solution and undergoes decomposition, which is believed to be homolytic cleavage of the S–N bond to give NO and a thiyl radical (25Josephy P.D. Rehorek D. Junzen E.G. Tetrahedron Lett. 1984; 25: 1685-1688Crossref Scopus (66) Google Scholar, 26Sheu F.-S. Zhu W. Fung P.C. Biophys. J. 2000; 78: 1216-1226Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Indeed, the reactions involving GSNO are fairly complex and generate many potential products including ammonia, NO, nitrous oxide, nitrite, sulfinamide, hydroxylamine, and several oxidized forms of glutathione (20Wong P.S.-Y. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (327) Google Scholar, 23Singh S.P. Wishnok J.S. Keshive M. Deen W.M. Tannenbaum S.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14428-14433Crossref PubMed Scopus (298) Google Scholar). Recently, it was found that freshly prepared GSNO was effective inS-nitrosylation of proteins through transnitrosylation, whereas the decomposed GSNO was more effective inS-glutathiolation of proteins (10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar). It was suggested that glutathione sulfenic acid was the active component for glutathiolation of proteins.Neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm) are two prominent PKC substrates in the brain. Phoshorylations of both Ng and Nm reduce their binding affinities for calmodulin (CaM) (27Alexander K.A. Cimler B.M. Meier K.E. Storm D.R. J. Biol. Chem. 1987; 262: 6108-6113Abstract Full Text PDF PubMed Google Scholar, 28Houbre D. Duportail G. Deloulme J.-C. Baudier J. J. Biol. Chem. 1991; 266: 7121-7131Abstract Full Text PDF PubMed Google Scholar). Rat brain Ng contains four, and Nm contains two Cys residues; these Cys residues in Ng form two pairs of intramolecular disulfides upon oxidation by NO and other oxidants (29Sheu F.-S. Mahoney C.W. Seki K. Huang K.-P. J. Biol. Chem. 1996; 271: 22407-22413Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 30Li J. Pak J.H. Huang F.L. Huang K.-P. J. Biol. Chem. 1999; 274: 1294-1300Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and those in Nm undergo palmitoylation (31Skene J.H.P. Virág I. J. Cell Biol. 1989; 108: 613-624Crossref PubMed Scopus (313) Google Scholar). Intramolecular disulfide formation renders Ng a poorer substrate of PKC and also reduces its binding affinity for CaM (29Sheu F.-S. Mahoney C.W. Seki K. Huang K.-P. J. Biol. Chem. 1996; 271: 22407-22413Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The effect of oxidation of the two Cys residues of Nm has not been elucidated. However, it was shown that treatment of cultured dorsal root ganglion and PC-12 cells with 3-morpholino-sydononimine, which generates both NO and superoxide, inhibited palmitoylation of Nm (32Hess D.T. Patterson S.I. Smith D.S. Skene J.H.P. Nature. 1993; 366: 562-565Crossref PubMed Scopus (298) Google Scholar). Recently, we found that treatment of Ng with GSNO caused oxidation to form intramolecular disulfides, as well as glutathiolation; the extent of glutathiolation was greatly increased upon incubation of Ng with the decomposed GSNO (33Huang K.-P. Huang F.L. Li J. Schuck P. McPhie P. Biochemistry. 2000; 39: 7291-7299Crossref PubMed Scopus (39) Google Scholar). In this study, by using mass spectrometry, we have identified several glutathione derivatives as the degradation products of GSNO, including glutathione sulfonic acid (GSO3H), glutathione disulfide S-oxide (GS(O)SG), glutathione disulfideS-dioxide (GS(O)2SG), and GSSG. Among them, GS(O)SG was the most potent in causing glutathiolation of Ng and Nm. The level of this compound was found to increase upon treatment of rat brain slices with oxidants. Ng had been positively identified as a target of thiolation under oxidative stress.DISCUSSIONWhile testing the effect of GSNO on the oxidation of Ng to form intramolecular disulfide, we found that the freshly prepared GSNO was effective in this modification, but a partially decomposed GSNO was more effective in causing glutathiolation (33Huang K.-P. Huang F.L. Li J. Schuck P. McPhie P. Biochemistry. 2000; 39: 7291-7299Crossref PubMed Scopus (39) Google Scholar). Analysis of the decomposition products of GSNO led us to identify GS(O)SG as one of the most potent glutathiolating agents among the various glutathione derivatives tested, including GSO3H, GS(O)2SG, GSNO, and GSSG (Fig. 3). Although these latter compounds can oxidize Ng to form intramolecular disulfide bonds, they are not very effective for glutathiolation of this protein. Glutathiolation of protein by GS(O)SG likely proceeds by the following reactions,R1SH+GS(O)SG→R1SSG+GSOHREACTION1R2SH+GSOH→R2SSG+H2OREACTION2where R1 and R2 are either protein or any sulfhydryl-containing compound. Modification of proteins containing multiple sulfhydryl groups, such as Ng, by GS(O)SG is complicated by two competing reactions, namely, formation of intramolecular disulfide and glutathiolation. Partially glutathiolated Ng can be driven to form intramolecular disulfide, but the intramolecular disulfide form of Ng cannot be glutathiolated. Thus, at a low GS(O)SG concentration Ng forms intramolecular disulfide, and at a high concentration Ng is glutathiolated. When the ratio of GS(O)SG/–SH is equal or greater than one, both Ng and Nm are stoichiometrically glutathiolated.Protein S-thiolation, especiallyS-glutathiolation, has been recognized as one of the physiological responses to nitrosative and oxidative stresses. The mechanism by which these stresses induce proteinS-thiolation is poorly understood. Several mechanisms have been proposed for protein glutathiolation including the following: 1) thiol-disulfide exchange between protein thiols and GSSG (38Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar); 2) oxidation of protein thiols by oxy-radicals or H2O2 to form thiyl radicals or sulfenic acids and then to interact with GSH to produce mixed disulfide (39Park E.-M. Thomas J.A. Biochim. Biophys. Acta. 1988; 964: 151-160Crossref PubMed Scopus (71) Google Scholar); 3) nucleophilic attack of protein thiolate on GSNO to produce mixed disulfide (9Padgett C.M. Whorton A.R. Arch. Biochem. Biophys. 1998; 358: 232-242Crossref PubMed Scopus (84) Google Scholar, 10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar, 11Mohr S. Hallak H. de Boitte A. Lapetina E.G. Brüne B. J. Biol. Chem. 1999; 274: 9427-9430Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 12Percival M.D. Ouellet M. Campagnolo C. Claveau D. Li C. Biochemistry. 1999; 38: 13574-13583Crossref PubMed Scopus (123) Google Scholar); 4) oxidation of GSH to form sulfenic acid and then interact with protein thiols to form mixed disulfides (10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar); and 5) nitrosation of protein thiols followed by interaction with GSH to form mixed disulfides (9Padgett C.M. Whorton A.R. Arch. Biochem. Biophys. 1998; 358: 232-242Crossref PubMed Scopus (84) Google Scholar, 10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar). The present study suggests another mechanism that utilizes GS(O)SG as a potential GS– donor for protein glutathiolation. GS(O)SG is more potent than GSNO in glutathiolation of protein, and this degradation product of GSNO may account for some of the effects of GSNO. GS(O)SG is present at a low level in the control rat brain slices labeled with [35S]cysteine and is increased under oxidative stress. GS(O)SG and GS(O)2SG have been shown to be the major products generated by oxidation of GSH with H2O2 (40Finley J.W. Wheeler E.L. Witt S.C. J. Agric. Food Chem. 1981; 29: 404-407Crossref PubMed Scopus (96) Google Scholar). We also showed that GS(O)SG could be formed by oxidation of both GSH and GSSG with NO (data not shown). Although GSSG can be reduced by glutathione reductase to form GSH, it is unknown whether GS(O)SG and GS(O2)SG are also substrates of the reductase. Accumulation of GS(O)SG under basal physiological conditions could provide tonic glutathiolation of certain proteins that employ this modification mechanism for regulation of their activities or subcellular localization.Thiolation of Ng has been positively identified by immunoprecipitation of the [35S]cysteine-labeled rat brain cortical slices, whereas thiolation of Nm was not detected under the same conditions. The two Cys residues of Nm are the potential sites of palmitoylation, which is involved in the association of this protein with the membrane (31Skene J.H.P. Virág I. J. Cell Biol. 1989; 108: 613-624Crossref PubMed Scopus (313) Google Scholar). It is apparent that the acylated Nm cannot be thiolated. Under the basal conditions, in which the slices were incubated with ACSF without added oxidants, Ng was thiolated to a lower level as compared with those treated with oxidants. In addition, Ng also subjected to oxidation to form intramolecular disulfides in the presence of the oxidants. These findings indicate that Ng can undergo multiple oxidative modifications at its Cys residues. The nature of thiolation of Ng in the brain slices has not been determined; we do not know whether it is because of glutathiolation or mixed disulfides between Ng and Cys. It is interesting to note that both oxidation of Ng to form intramolecular disulfides and glutathiolation convert this protein to become a poorer substrate of PKC by reducing theV max value (see Table I and Ref. 29Sheu F.-S. Mahoney C.W. Seki K. Huang K.-P. J. Biol. Chem. 1996; 271: 22407-22413Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar); however, the former modification, but not the latter, also causes a reduction in the binding affinity of Ng toward CaM. Because the partially thiolated Ng can be converted into the intramolecular disulfide form of Ng, one may consider thiolation as an intermediate step toward intramolecular disulfide formation. Thus, oxidative stress may cause these two modifications of Ng and result in an attenuation of Ng phosphorylation by PKC.In summary, the current study provides evidence that GS(O)SG derived from GSNO is one of the most potent glutathiolating agents known so far. This compound can be easily prepared from the decomposed GSNO, by bubbling GSH or GSSG solution with NO gas, or by treatment of GSH with H2O2. GS(O)SG is relatively stable upon storage but is very reactive toward sulfhydryl group. GS(O)SG apparently is generated in situ following oxidative stress and may be responsible for the glutathiolation of proteins in vivo. Here, we have also demonstrated that Ng is thiolated in rat brain slices and that the extent of thiolation is enhanced under oxidative stress. Protein S-glutathiolation can be induced in cells by mild oxidative stress (1Thomas J.A. Poland B. Honzatko R. Arch. Biochem. Biophys. 1995; 319: 1-9Crossref PubMed Scopus (363) Google Scholar). GSSG has been shown to oxidatively regulate the activity of several purified enzymes including carbonic anhydrase III (2Cabiscol E. Levine R.L. J. Biol. Chem. 1995; 270: 14742-14747Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 3Cabiscol E. Levine R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4170-4174Crossref PubMed Scopus (132) Google Scholar), protein kinase C (PKC)1 (4Ward N.E. Pierce D.S. Chung S.E. Gravitt K.R. O'Brian C.A. J. Biol. Chem. 1998; 273: 12558-12566Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), human aldose reductase (5Cappiello M. Voltarelli M. Cecconi I. Vilardo P.G. Dal Monte M. Marini I. Del Corso A. Wilson D.K. Quiocho F.A. Petrash J.M. Mura U. J. Biol. Chem. 1996; 271: 33539-33544Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and human immunodeficiency virus, type I protease (6Davis D.A. Dorsey K. Wingfield P.T. Stahl S.J. Kaufman J. Fales H.M. Levine R.L. Biochemistry. 1996; 35: 2482-2488Crossref PubMed Scopus (96) Google Scholar), and in each case the effects of glutathiolation can be reversed by reducing agents. As the concentration of reduced GSH in the mammalian cells is in the millimolar range and that of GSSG is less than 5% of GSH, glutathiolation of proteins by GSSG in vivo is not likely an efficient mechanism. More recently, the superoxide-induced glutathiolation of protein (7Barrett W.C. DeGnore J.P. Keng Y.-F. Zhang Z.-Y. Yim M.B. Chock P.B. J. Biol. Chem. 1999; 274: 34543-34546Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) and that induced by peroxynitrite, nitric oxide (NO), and nitrosothiol, in particular,S-nitrosoglutathione (GSNO), are thought to be the main avenues leading to protein S-thiolation (8Viner R.I. Williams T.D. Schöneich C. Biochemistry. 1999; 38: 12408-12415Crossref PubMed Scopus (213) Google Scholar, 9Padgett C.M. Whorton A.R. Arch. Biochem. Biophys. 1998; 358: 232-242Crossref PubMed Scopus (84) Google Scholar, 10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar, 11Mohr S. Hallak H. de Boitte A. Lapetina E.G. Brüne B. J. Biol. Chem. 1999; 274: 9427-9430Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 12Percival M.D. Ouellet M. Campagnolo C. Claveau D. Li C. Biochemistry. 1999; 38: 13574-13583Crossref PubMed Scopus (123) Google Scholar). In mammalian cells, a relatively high concentration of GSH (0.5–10 mm) serves as an NO sink to form GSNO (13Kharitonov V.G. Sundquist A.R. Skarma V.S. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 14Clancy R.M. Levartovsky D. Leszczynska-Piziak J. Yegudin J. Abramson S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3680-3684Crossref PubMed Scopus (305) Google Scholar, 15Mayer B. Pfeiffer S. Schrammel A. Koesling D. Schmidt K. Brunner F. J. Biol. Chem. 1998; 273: 3264-3270Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 16Gow A.J. Buerk D.G. Ischiropoulos H. J. Biol. Chem. 1997; 272: 2841-2845Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), which can undergo transnitrosylation with protein sulfhydryl group to formS-nitrosoprotein and GSH or to form protein-GSH mixed disulfide and nitroxyl (17Park J.W. Biochem. Biophys. Res. Commun. 1988; 52: 916-920Crossref Scopus (110) Google Scholar, 18Scharfstein J.S. Keaney Jr., J.F. Slivka A. Welch G.N. Vita J.A. Stamler J.S. Loscalzo J. J. Clin. Invest. 1994; 94: 1432-1439Crossref PubMed Scopus (255) Google Scholar, 19Meyer D.J. Kramer H. Ozer N. Coles B. Ketterer B. FEBS Lett. 1994; 345: 177-180Crossref PubMed Scopus (146) Google Scholar, 20Wong P.S.-Y. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (327) Google Scholar). GSNO can also release NO in the presence of cuprous ion (21Singh R.J. Hogg N. Joseph J. Kalyanaraman B. J. Biol. Chem. 1996; 271: 18596-18603Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar), ascorbate (22Kashiba-Iwatsuki M. Yamaguchi M. Inoue M. FEBS Lett. 1996; 389: 149-152Crossref PubMed Scopus (67) Google Scholar), or thiols (20Wong P.S.-Y. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (327) Google Scholar, 23Singh S.P. Wishnok J.S. Keshive M. Deen W.M. Tannenbaum S.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14428-14433Crossref PubMed Scopus (298) Google Scholar) and serves as a possible source of nitrsonium or nitroxyl ions (24Arnelle D.R. Stamler J.S. Arch. Biochem. Biophys. 1995; 318: 279-285Crossref PubMed Scopus (538) Google Scholar). In addition, GSNO is unstable in aqueous solution and undergoes decomposition, which is believed to be homolytic cleavage of the S–N bond to give NO and a thiyl radical (25Josephy P.D. Rehorek D. Junzen E.G. Tetrahedron Lett. 1984; 25: 1685-1688Crossref Scopus (66) Google Scholar, 26Sheu F.-S. Zhu W. Fung P.C. Biophys. J. 2000; 78: 1216-1226Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Indeed, the reactions involving GSNO are fairly complex and generate many potential products including ammonia, NO, nitrous oxide, nitrite, sulfinamide, hydroxylamine, and several oxidized forms of glutathione (20Wong P.S.-Y. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (327) Google Scholar, 23Singh S.P. Wishnok J.S. Keshive M. Deen W.M. Tannenbaum S.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14428-14433Crossref PubMed Scopus (298) Google Scholar). Recently, it was found that freshly prepared GSNO was effective inS-nitrosylation of proteins through transnitrosylation, whereas the decomposed GSNO was more effective inS-glutathiolation of proteins (10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar). It was suggested that glutathione sulfenic acid was the active component for glutathiolation of proteins. Neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm) are two prominent PKC substrates in the brain. Phoshorylations of both Ng and Nm reduce their binding affinities for calmodulin (CaM) (27Alexander K.A. Cimler B.M. Meier K.E. Storm D.R. J. Biol. Chem. 1987; 262: 6108-6113Abstract Full Text PDF PubMed Google Scholar, 28Houbre D. Duportail G. Deloulme J.-C. Baudier J. J. Biol. Chem. 1991; 266: 7121-7131Abstract Full Text PDF PubMed Google Scholar). Rat brain Ng contains four, and Nm contains two Cys residues; these Cys residues in Ng form two pairs of intramolecular disulfides upon oxidation by NO and other oxidants (29Sheu F.-S. Mahoney C.W. Seki K. Huang K.-P. J. Biol. Chem. 1996; 271: 22407-22413Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 30Li J. Pak J.H. Huang F.L. Huang K.-P. J. Biol. Chem. 1999; 274: 1294-1300Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and those in Nm undergo palmitoylation (31Skene J.H.P. Virág I. J. Cell Biol. 1989; 108: 613-624Crossref PubMed Scopus (313) Google Scholar). Intramolecular disulfide formation renders Ng a poorer substrate of PKC and also reduces its binding affinity for CaM (29Sheu F.-S. Mahoney C.W. Seki K. Huang K.-P. J. Biol. Chem. 1996; 271: 22407-22413Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The effect of oxidation of the two Cys residues of Nm has not been elucidated. However, it was shown that treatment of cultured dorsal root ganglion and PC-12 cells with 3-morpholino-sydononimine, which generates both NO and superoxide, inhibited palmitoylation of Nm (32Hess D.T. Patterson S.I. Smith D.S. Skene J.H.P. Nature. 1993; 366: 562-565Crossref PubMed Scopus (298) Google Scholar). Recently, we found that treatment of Ng with GSNO caused oxidation to form intramolecular disulfides, as well as glutathiolation; the extent of glutathiolation was greatly increased upon incubation of Ng with the decomposed GSNO (33Huang K.-P. Huang F.L. Li J. Schuck P. McPhie P. Biochemistry. 2000; 39: 7291-7299Crossref PubMed Scopus (39) Google Scholar). In this study, by using mass spectrometry, we have identified several glutathione derivatives as the degradation products of GSNO, including glutathione sulfonic acid (GSO3H), glutathione disulfide S-oxide (GS(O)SG), glutathione disulfideS-dioxide (GS(O)2SG), and GSSG. Among them, GS(O)SG was the most potent in causing glutathiolation of Ng and Nm. The level of this compound was found to increase upon treatment of rat brain slices with oxidants. Ng had been positively identified as a target of thiolation under oxidative stress. DISCUSSIONWhile testing the effect of GSNO on the oxidation of Ng to form intramolecular disulfide, we found that the freshly prepared GSNO was effective in this modification, but a partially decomposed GSNO was more effective in causing glutathiolation (33Huang K.-P. Huang F.L. Li J. Schuck P. McPhie P. Biochemistry. 2000; 39: 7291-7299Crossref PubMed Scopus (39) Google Scholar). Analysis of the decomposition products of GSNO led us to identify GS(O)SG as one of the most potent glutathiolating agents among the various glutathione derivatives tested, including GSO3H, GS(O)2SG, GSNO, and GSSG (Fig. 3). Although these latter compounds can oxidize Ng to form intramolecular disulfide bonds, they are not very effective for glutathiolation of this protein. Glutathiolation of protein by GS(O)SG likely proceeds by the following reactions,R1SH+GS(O)SG→R1SSG+GSOHREACTION1R2SH+GSOH→R2SSG+H2OREACTION2where R1 and R2 are either protein or any sulfhydryl-containing compound. Modification of proteins containing multiple sulfhydryl groups, such as Ng, by GS(O)SG is complicated by two competing reactions, namely, formation of intramolecular disulfide and glutathiolation. Partially glutathiolated Ng can be driven to form intramolecular disulfide, but the intramolecular disulfide form of Ng cannot be glutathiolated. Thus, at a low GS(O)SG concentration Ng forms intramolecular disulfide, and at a high concentration Ng is glutathiolated. When the ratio of GS(O)SG/–SH is equal or greater than one, both Ng and Nm are stoichiometrically glutathiolated.Protein S-thiolation, especiallyS-glutathiolation, has been recognized as one of the physiological responses to nitrosative and oxidative stresses. The mechanism by which these stresses induce proteinS-thiolation is poorly understood. Several mechanisms have been proposed for protein glutathiolation including the following: 1) thiol-disulfide exchange between protein thiols and GSSG (38Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar); 2) oxidation of protein thiols by oxy-radicals or H2O2 to form thiyl radicals or sulfenic acids and then to interact with GSH to produce mixed disulfide (39Park E.-M. Thomas J.A. Biochim. Biophys. Acta. 1988; 964: 151-160Crossref PubMed Scopus (71) Google Scholar); 3) nucleophilic attack of protein thiolate on GSNO to produce mixed disulfide (9Padgett C.M. Whorton A.R. Arch. Biochem. Biophys. 1998; 358: 232-242Crossref PubMed Scopus (84) Google Scholar, 10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar, 11Mohr S. Hallak H. de Boitte A. Lapetina E.G. Brüne B. J. Biol. Chem. 1999; 274: 9427-9430Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 12Percival M.D. Ouellet M. Campagnolo C. Claveau D. Li C. Biochemistry. 1999; 38: 13574-13583Crossref PubMed Scopus (123) Google Scholar); 4) oxidation of GSH to form sulfenic acid and then interact with protein thiols to form mixed disulfides (10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar); and 5) nitrosation of protein thiols followed by interaction with GSH to form mixed disulfides (9Padgett C.M. Whorton A.R. Arch. Biochem. Biophys. 1998; 358: 232-242Crossref PubMed Scopus (84) Google Scholar, 10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar). The present study suggests another mechanism that utilizes GS(O)SG as a potential GS– donor for protein glutathiolation. GS(O)SG is more potent than GSNO in glutathiolation of protein, and this degradation product of GSNO may account for some of the effects of GSNO. GS(O)SG is present at a low level in the control rat brain slices labeled with [35S]cysteine and is increased under oxidative stress. GS(O)SG and GS(O)2SG have been shown to be the major products generated by oxidation of GSH with H2O2 (40Finley J.W. Wheeler E.L. Witt S.C. J. Agric. Food Chem. 1981; 29: 404-407Crossref PubMed Scopus (96) Google Scholar). We also showed that GS(O)SG could be formed by oxidation of both GSH and GSSG with NO (data not shown). Although GSSG can be reduced by glutathione reductase to form GSH, it is unknown whether GS(O)SG and GS(O2)SG are also substrates of the reductase. Accumulation of GS(O)SG under basal physiological conditions could provide tonic glutathiolation of certain proteins that employ this modification mechanism for regulation of their activities or subcellular localization.Thiolation of Ng has been positively identified by immunoprecipitation of the [35S]cysteine-labeled rat brain cortical slices, whereas thiolation of Nm was not detected under the same conditions. The two Cys residues of Nm are the potential sites of palmitoylation, which is involved in the association of this protein with the membrane (31Skene J.H.P. Virág I. J. Cell Biol. 1989; 108: 613-624Crossref PubMed Scopus (313) Google Scholar). It is apparent that the acylated Nm cannot be thiolated. Under the basal conditions, in which the slices were incubated with ACSF without added oxidants, Ng was thiolated to a lower level as compared with those treated with oxidants. In addition, Ng also subjected to oxidation to form intramolecular disulfides in the presence of the oxidants. These findings indicate that Ng can undergo multiple oxidative modifications at its Cys residues. The nature of thiolation of Ng in the brain slices has not been determined; we do not know whether it is because of glutathiolation or mixed disulfides between Ng and Cys. It is interesting to note that both oxidation of Ng to form intramolecular disulfides and glutathiolation convert this protein to become a poorer substrate of PKC by reducing theV max value (see Table I and Ref. 29Sheu F.-S. Mahoney C.W. Seki K. Huang K.-P. J. Biol. Chem. 1996; 271: 22407-22413Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar); however, the former modification, but not the latter, also causes a reduction in the binding affinity of Ng toward CaM. Because the partially thiolated Ng can be converted into the intramolecular disulfide form of Ng, one may consider thiolation as an intermediate step toward intramolecular disulfide formation. Thus, oxidative stress may cause these two modifications of Ng and result in an attenuation of Ng phosphorylation by PKC.In summary, the current study provides evidence that GS(O)SG derived from GSNO is one of the most potent glutathiolating agents known so far. This compound can be easily prepared from the decomposed GSNO, by bubbling GSH or GSSG solution with NO gas, or by treatment of GSH with H2O2. GS(O)SG is relatively stable upon storage but is very reactive toward sulfhydryl group. GS(O)SG apparently is generated in situ following oxidative stress and may be responsible for the glutathiolation of proteins in vivo. Here, we have also demonstrated that Ng is thiolated in rat brain slices and that the extent of thiolation is enhanced under oxidative stress. While testing the effect of GSNO on the oxidation of Ng to form intramolecular disulfide, we found that the freshly prepared GSNO was effective in this modification, but a partially decomposed GSNO was more effective in causing glutathiolation (33Huang K.-P. Huang F.L. Li J. Schuck P. McPhie P. Biochemistry. 2000; 39: 7291-7299Crossref PubMed Scopus (39) Google Scholar). Analysis of the decomposition products of GSNO led us to identify GS(O)SG as one of the most potent glutathiolating agents among the various glutathione derivatives tested, including GSO3H, GS(O)2SG, GSNO, and GSSG (Fig. 3). Although these latter compounds can oxidize Ng to form intramolecular disulfide bonds, they are not very effective for glutathiolation of this protein. Glutathiolation of protein by GS(O)SG likely proceeds by the following reactions,R1SH+GS(O)SG→R1SSG+GSOHREACTION1R2SH+GSOH→R2SSG+H2OREACTION2where R1 and R2 are either protein or any sulfhydryl-containing compound. Modification of proteins containing multiple sulfhydryl groups, such as Ng, by GS(O)SG is complicated by two competing reactions, namely, formation of intramolecular disulfide and glutathiolation. Partially glutathiolated Ng can be driven to form intramolecular disulfide, but the intramolecular disulfide form of Ng cannot be glutathiolated. Thus, at a low GS(O)SG concentration Ng forms intramolecular disulfide, and at a high concentration Ng is glutathiolated. When the ratio of GS(O)SG/–SH is equal or greater than one, both Ng and Nm are stoichiometrically glutathiolated. Protein S-thiolation, especiallyS-glutathiolation, has been recognized as one of the physiological responses to nitrosative and oxidative stresses. The mechanism by which these stresses induce proteinS-thiolation is poorly understood. Several mechanisms have been proposed for protein glutathiolation including the following: 1) thiol-disulfide exchange between protein thiols and GSSG (38Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar); 2) oxidation of protein thiols by oxy-radicals or H2O2 to form thiyl radicals or sulfenic acids and then to interact with GSH to produce mixed disulfide (39Park E.-M. Thomas J.A. Biochim. Biophys. Acta. 1988; 964: 151-160Crossref PubMed Scopus (71) Google Scholar); 3) nucleophilic attack of protein thiolate on GSNO to produce mixed disulfide (9Padgett C.M. Whorton A.R. Arch. Biochem. Biophys. 1998; 358: 232-242Crossref PubMed Scopus (84) Google Scholar, 10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar, 11Mohr S. Hallak H. de Boitte A. Lapetina E.G. Brüne B. J. Biol. Chem. 1999; 274: 9427-9430Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 12Percival M.D. Ouellet M. Campagnolo C. Claveau D. Li C. Biochemistry. 1999; 38: 13574-13583Crossref PubMed Scopus (123) Google Scholar); 4) oxidation of GSH to form sulfenic acid and then interact with protein thiols to form mixed disulfides (10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar); and 5) nitrosation of protein thiols followed by interaction with GSH to form mixed disulfides (9Padgett C.M. Whorton A.R. Arch. Biochem. Biophys. 1998; 358: 232-242Crossref PubMed Scopus (84) Google Scholar, 10Ji Y. Akerboom T.P.M. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar). The present study suggests another mechanism that utilizes GS(O)SG as a potential GS– donor for protein glutathiolation. GS(O)SG is more potent than GSNO in glutathiolation of protein, and this degradation product of GSNO may account for some of the effects of GSNO. GS(O)SG is present at a low level in the control rat brain slices labeled with [35S]cysteine and is increased under oxidative stress. GS(O)SG and GS(O)2SG have been shown to be the major products generated by oxidation of GSH with H2O2 (40Finley J.W. Wheeler E.L. Witt S.C. J. Agric. Food Chem. 1981; 29: 404-407Crossref PubMed Scopus (96) Google Scholar). We also showed that GS(O)SG could be formed by oxidation of both GSH and GSSG with NO (data not shown). Although GSSG can be reduced by glutathione reductase to form GSH, it is unknown whether GS(O)SG and GS(O2)SG are also substrates of the reductase. Accumulation of GS(O)SG under basal physiological conditions could provide tonic glutathiolation of certain proteins that employ this modification mechanism for regulation of their activities or subcellular localization. Thiolation of Ng has been positively identified by immunoprecipitation of the [35S]cysteine-labeled rat brain cortical slices, whereas thiolation of Nm was not detected under the same conditions. The two Cys residues of Nm are the potential sites of palmitoylation, which is involved in the association of this protein with the membrane (31Skene J.H.P. Virág I. J. Cell Biol. 1989; 108: 613-624Crossref PubMed Scopus (313) Google Scholar). It is apparent that the acylated Nm cannot be thiolated. Under the basal conditions, in which the slices were incubated with ACSF without added oxidants, Ng was thiolated to a lower level as compared with those treated with oxidants. In addition, Ng also subjected to oxidation to form intramolecular disulfides in the presence of the oxidants. These findings indicate that Ng can undergo multiple oxidative modifications at its Cys residues. The nature of thiolation of Ng in the brain slices has not been determined; we do not know whether it is because of glutathiolation or mixed disulfides between Ng and Cys. It is interesting to note that both oxidation of Ng to form intramolecular disulfides and glutathiolation convert this protein to become a poorer substrate of PKC by reducing theV max value (see Table I and Ref. 29Sheu F.-S. Mahoney C.W. Seki K. Huang K.-P. J. Biol. Chem. 1996; 271: 22407-22413Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar); however, the former modification, but not the latter, also causes a reduction in the binding affinity of Ng toward CaM. Because the partially thiolated Ng can be converted into the intramolecular disulfide form of Ng, one may consider thiolation as an intermediate step toward intramolecular disulfide formation. Thus, oxidative stress may cause these two modifications of Ng and result in an attenuation of Ng phosphorylation by PKC. In summary, the current study provides evidence that GS(O)SG derived from GSNO is one of the most potent glutathiolating agents known so far. This compound can be easily prepared from the decomposed GSNO, by bubbling GSH or GSSG solution with NO gas, or by treatment of GSH with H2O2. GS(O)SG is relatively stable upon storage but is very reactive toward sulfhydryl group. GS(O)SG apparently is generated in situ following oxidative stress and may be responsible for the glutathiolation of proteins in vivo. Here, we have also demonstrated that Ng is thiolated in rat brain slices and that the extent of thiolation is enhanced under oxidative stress." @default.
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W2009953261 title "Glutathiolation of Proteins by Glutathione DisulfideS-Oxide Derived from S-Nitrosoglutathione" @default.
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