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• W1993780555 abstract "The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys32-Gly-Pro-Cys35 thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys62–Cys69 disulfide as well as dimers and multimers. We have discovered that after incubation with H2O2 only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys69 and Cys73 were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys73, which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154–158). The reversible inhibition of human Trx1 activity by H2O2 and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control. The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys32-Gly-Pro-Cys35 thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys62–Cys69 disulfide as well as dimers and multimers. We have discovered that after incubation with H2O2 only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys69 and Cys73 were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys73, which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154–158). The reversible inhibition of human Trx1 activity by H2O2 and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control. Thioredoxin (Trx), 3The abbreviations used are: TrxthioredoxinTrxRthioredoxin reductaseDTNB5,5′-dithiobis-(2-nitrobenzoic acid)DTTdithiothreitolGSNOS-nitrosoglutathioneRNSreactive nitrogen speciesROSreactive oxygen speciesS-NOS-nitrosothiolwtwild type. a ubiquitous 12-kDa enzyme with the active site -Trp-CysN-Gly-Pro-CysC-, is evolutionary conserved from archaebacteria to man (1Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 2Lillig C.H. Holmgren A. Antioxid. Redox Signal. 2007; 9: 25-47Crossref PubMed Scopus (576) Google Scholar). The reduced or dithiol form Trx-(SH)2 utilizes its exposed nucleophilic CysN residue to attack a target protein disulfide and form a transient mixed disulfide, which is subsequently attacked by the CysC residue to generate oxidized Trx-S2 and the reduced protein (Reaction 1). Trx-S2 is a substrate for NADPH and thioredoxin reductase (TrxR) (Reaction 2). x0026;TRX-(SH)2+Protein-S2→Trx-S2+Protein-(SH)2x0026;Trx-S2+NADPH+H+→TrxRTrx-(SH)2+NADP+(Eq. 1) Escherichia coli Trx has only two Cys residues in the conserved active site (1Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar), but mammalian cytosolic thioredoxins (Trx1) have additional structural cysteines. For human Trx1, they are located in positions 62, 69, and 73 (see Fig. 1). Post-translational modifications of Trx1 via these cysteine residues, such as thiol oxidation, glutathionylation, and S-nitrosylation have been proposed to regulate function and are implicated in signal transduction pathways (3Casagrande S. Bonetto V. Fratelli M. Gianazza E. Eberini I. Massignan T. Salmona M. Chang G. Holmgren A. Ghezzi P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9745-9749Crossref PubMed Scopus (295) Google Scholar, 4Kuster G.M. Siwik D.A. Pimentel D.R. Colucci W.S. Antioxid. Redox Signal. 2006; 8: 2153-2159Crossref PubMed Scopus (35) Google Scholar). For instance, the loss of Trx activity and aggregation of the protein after oxidation was reported for the first time during purification (5Holmgren A. J. Biol. Chem. 1977; 252: 4600-4606Abstract Full Text PDF PubMed Google Scholar), and it was later shown to be due to the formation of a second disulfide motif (6Watson W.H. Pohl J. Montfort W.R. Stuchlik O. Reed M.S. Powis G. Jones D.P. J. Biol. Chem. 2003; 278: 33408-33415Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Watson et al. (6Watson W.H. Pohl J. Montfort W.R. Stuchlik O. Reed M.S. Powis G. Jones D.P. J. Biol. Chem. 2003; 278: 33408-33415Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) showed that under oxidizing conditions, besides the active site disulfide, a second disulfide may form between Cys62 and Cys69 with a considerable effect on Trx1 activity. These modifications are mediated by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are involved in numerous processes such as cell signaling (7Valko M. Leibfritz D. Moncol J. Cronin M.T.D. Mazur M. Telser J. Int. J. Biochem. Cell Biol. 2007; 39: 44-84Crossref PubMed Scopus (9567) Google Scholar, 8Keisari Y. Braun L. Flescher E. Immunobiology. 1983; 165: 78-89Crossref PubMed Scopus (58) Google Scholar). However, excessive ROS and RNS are also involved in pathogenesis of several diseases under oxidative or nitrosative stress (9Dhalla N.S. Temsah R.M. Netticadan T. J. Hypertens. 2000; 18: 655-673Crossref PubMed Scopus (1146) Google Scholar, 10Sayre L.M. Moreira P.I. Smith M.A. Perry G. Ann. Ist. Super. Sanita. 2005; 41: 143-164PubMed Google Scholar, 11Dalle-Donne I. Rossi R. Colombo R. Giustarini D. Milzani A. Clin. Chem. 2006; 52: 601-623Crossref PubMed Scopus (1280) Google Scholar, 12Valko M. Rhodes C.J. Moncol J. Izakovic M. Mazur M. Chem. Biol. Interact. 2006; 160: 1-40Crossref PubMed Scopus (4614) Google Scholar). thioredoxin thioredoxin reductase 5,5′-dithiobis-(2-nitrobenzoic acid) dithiothreitol S-nitrosoglutathione reactive nitrogen species reactive oxygen species S-nitrosothiol wild type. The role of ROS in cell signaling, specially via the effect on mitogen-activated protein kinases and activation of nuclear transcription factors, is well known (13Thannickal V.J. Fanburg B.L. Am. J. Physiol. 2000; 279: L1005-L1028Crossref PubMed Google Scholar, 14Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (999) Google Scholar). For example, H2O2 is involved in signaling pathways (15Veal E.A. Day A.M. Morgan B.A. Mol. Cell. 2007; 26: 1-14Abstract Full Text Full Text PDF PubMed Scopus (1201) Google Scholar, 16Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2285) Google Scholar); however, it is not precisely understood how H2O2 functions as a signaling molecule in vertebrates (17Stone J.R. Yang S. Antioxid. Redox Signal. 2006; 8: 243-270Crossref PubMed Scopus (895) Google Scholar). Proteins containing thiol groups are one of the molecular targets of H2O2 causing selective thiol oxidation (18Baty J.W. Hampton M.B. Winterbourn C.C. Biochem. J. 2005; 389: 785-795Crossref PubMed Scopus (126) Google Scholar). NO is also a reactive radical gas that is implicated in numerous physiological states (19Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2120) Google Scholar, 20Lowenstein C.J. Dinerman J.L. Snyder S.H. Ann. Intern. Med. 1994; 120: 227-237Crossref PubMed Scopus (842) Google Scholar, 21Rand M.J. Li C.G. Annu. Rev. Physiol. 1995; 57: 659-682Crossref PubMed Scopus (287) Google Scholar, 22Choi B.M. Pae H.O. Jang S.I. Kim Y.M. Chung H.T. J. Biochem. Mol. Biol. 2002; 35: 116-126Crossref PubMed Google Scholar) as well as pathophysiologic conditions (23Vincent J.L. Zhang H. Szabo C. Preiser J.C. Am. J. Respir. Crit. Care Med. 2000; 161: 1781-1785Crossref PubMed Scopus (266) Google Scholar, 24Boje K.M. Front Biosci. 2004; 9: 763-776Crossref PubMed Scopus (125) Google Scholar). NO exerts its effects via cGMP formation as well as cGMP-independent mechanisms such as nitration and nitrosylation of proteins (25Thippeswamy T. McKay J.S. Quinn J.P. Morris R. Histol. Histopathol. 2006; 21: 445-458PubMed Google Scholar). S-Nitrosylation is a post-translational modification, involved in the regulation of structure or activity of certain proteins (26Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1626) Google Scholar, 27Stamler J.S. Lamas S. Fang F.C. Cell. 2001; 106: 675-683Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar). Human Trx1 is such a protein, but the available data are controversial. In two studies, S-nitrosylation of hTrx1 at only one structural Cys residue was reported. Haendeler et al. (28Haendeler J. Hoffmann J. Tischler V. Berk B.C. Zeiher A.M. Dimmeler S. Nat. Cell Biol. 2002; 4: 743-749Crossref PubMed Scopus (339) Google Scholar) detected the S-nitrosylation of Cys69 under basal conditions, which was asserted to be essential for anti-apoptotic and redox regulatory function of the protein and to increase Trx catalytic activity. But, Mitchell and Marletta (29Mitchell D.A. Marletta M.A. Nat. Chem. Biol. 2005; 1: 154-158Crossref PubMed Scopus (224) Google Scholar) showed the oxidation of active site cysteines to a disulfide as well as the extensive nitrosylation of only Cys73 after treating human Trx1 with 50 molar equivalents of S-nitrosoglutathione (GSNO) followed by the biotin switch method and matrix-assisted laser desorption ionization mapping. They could not detect any significant S-nitrosylation of Cys62 and Cys69. S-Nitrosylation of Cys73 was implicated in the reversible and specific transfer of a nitrosothiol between Trx (Cys73) and caspase 3 (Cys163) in vitro (29Mitchell D.A. Marletta M.A. Nat. Chem. Biol. 2005; 1: 154-158Crossref PubMed Scopus (224) Google Scholar). This transnitrosation reaction was suggested to be involved in the regulation of apoptosis. On the other hand, in three other studies, the S-NO content of hTrx was increased after incubation with GSNO in a concentration-dependent manner to a maximum of about 2 mol of S-NO/mol of hTrx (30Sumbayev V.V. Arch. Biochem. Biophys. 2003; 415: 133-136Crossref PubMed Scopus (84) Google Scholar, 31Tao L. Gao E. Bryan N.S. Qu Y. Liu H.-R. Hu A. Christopher T.A. Lopez B.L. Yodoi J. Koch W.J. Feelisch M. Ma X.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11471-11476Crossref PubMed Scopus (168) Google Scholar, 32Yasinska I.M. Kozhukhar A.V. Sumbayev V.V. Arch. Biochem. Biophys. 2004; 428: 198-203Crossref PubMed Scopus (29) Google Scholar). Sumbayev (30Sumbayev V.V. Arch. Biochem. Biophys. 2003; 415: 133-136Crossref PubMed Scopus (84) Google Scholar) showed that S-nitrosylation of Trx correlated directly with apoptosis signal-regulating kinase 1 activation. In another study, comparable with the endogenous S-NO content in endothelial cells that had been utilized by Haendeler et al. (28Haendeler J. Hoffmann J. Tischler V. Berk B.C. Zeiher A.M. Dimmeler S. Nat. Cell Biol. 2002; 4: 743-749Crossref PubMed Scopus (339) Google Scholar), only 0.3 ± 0.09 mol of S-NO/mol of Trx was detected. However, under this condition, the cardio-protective effect of hTrx1 was markedly enhanced (31Tao L. Gao E. Bryan N.S. Qu Y. Liu H.-R. Hu A. Christopher T.A. Lopez B.L. Yodoi J. Koch W.J. Feelisch M. Ma X.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11471-11476Crossref PubMed Scopus (168) Google Scholar). Moreover, in a recent study regarding the crystal structure of human Trx1, only Cys62 of GSNO-treated sample became stably nitrosylated in the crystal at neutral pH; Cys69 could also be nitrosylated but required a more alkaline pH (33Weichsel A. Brailey J.L. Montfort W.R. Biochemistry. 2007; 46: 1219-1227Crossref PubMed Scopus (64) Google Scholar). In this paper we studied the reaction of human Trx1 with diamide and H2O2 as well as GSNO, which is a substrate for mammalian TrxR and was previously shown to inhibit the protein-disulfide reductase activity of the mammalian Trx system (34Nikitovic D. Holmgren A. J. Biol. Chem. 1996; 271: 19180-19185Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). We hypothesized that this effect could be due to the S-nitrosylation of Trx (28Haendeler J. Hoffmann J. Tischler V. Berk B.C. Zeiher A.M. Dimmeler S. Nat. Cell Biol. 2002; 4: 743-749Crossref PubMed Scopus (339) Google Scholar, 29Mitchell D.A. Marletta M.A. Nat. Chem. Biol. 2005; 1: 154-158Crossref PubMed Scopus (224) Google Scholar), inhibiting its activity rather than stimulating activity. Our results also suggest a mechanism for reversible inactivation of thioredoxin via H2O2 generated from NADPH oxidases in signaling. Chemicals and Enzyme—Bovine serum albumin, diamide, DTNB, DTT, NADPH, and insulin were purchased from Sigma. The NAP-5 Sephadex G-25 column was purchased from Amersham Biosciences. NuPAGE® Bis-Tris gels, NuPAGE® LDS sample buffer (4×), and running buffer were from Invitrogen. GSNO was prepared as previously reported (35Hart T.W. Tetrahedron Lett. 1985; 26: 2013-2016Crossref Scopus (377) Google Scholar) by the reaction of acidified sodium nitrite and reduced glutathione. The yield was calculated using the extinction coefficient of 920 m–1 cm–1 at 335 nm. E. coli Trx1 and TrxR (36Holmgren A. Björnstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (809) Google Scholar), wild type (wt) hTrx1, and its mutants C62S, C32S/C35S, and C62S/C73S were prepared as described (37Bizzarri C. Holmgren A. Pekkari K. Chang G. Colotta F. Ghezzi P. Bertini R. Antioxid. Redox Signal. 2005; 7: 1189-1194Crossref PubMed Scopus (22) Google Scholar, 38Ren X. Björnstedt M. Shen B. Ericson M.L. Holmgren A. Biochemistry. 1993; 32: 9701-9708Crossref PubMed Scopus (138) Google Scholar). E. coli P34H Trx was also utilized (39Krause G. Lundstrom J. Barea J.L. Pueyo de la Cuesta C. Holmgren A. J. Biol. Chem. 1991; 266: 9494-9500Abstract Full Text PDF PubMed Google Scholar). Rat recombinant TrxR1 was kindly provided by Olle Rengby and Dr. Elias Arner from the Department of Medical Biochemistry and Biophysics of the Karolinska Institutet (40Arner E.S. Sarioglu H. Lottspeich F. Holmgren A. Böck A. J. Mol. Biol. 1999; 292: 1003-1016Crossref PubMed Scopus (196) Google Scholar). Reduction of Human Trx1 and Treatment with Oxidants—The protein (100 μm in TE buffer (50 mm Tris-HCl, 1 mm EDTA, pH 7.4)) was incubated with 100 molar equivalents of DTT for 15 min at 37 °C for reduction. Thereafter, DTT was removed by chromatography using a NAP-5 column equilibrated with TE buffer. After confirming the complete reduction of Trx1 by measuring the number of free thiol groups (as described below), it was incubated with different concentrations of either diamide for less than 2 min at room temperature or H2O2 for 15 min at 37 °C. For the latter, DTT-treated Trx1 was desalted on a NAP-5 Sephadex G-25 column equilibrated with 50 mm Tris-HCl, pH 7.4, or phosphate-buffered saline, pH 7.4 with or without 1 mm EDTA. S-Nitrosylation Method—The fully reduced Trx1, prepared as described above, was incubated with 10 molar equivalents of GSNO at 37 °C for 60 min, which was followed by a desalting step on a column of NAP-5 Sephadex G-25 equilibrated with TE buffer. Spectrophotometric Analysis of GSNO-treated Thioredoxin—GSNO-treated Trx samples were analyzed spectrophotometrically to quantitate the number of nitrosothiols, using a molar extinction coefficient of 920 m–1 cm–1 at 335 nm. Localization of Nitrosothiols—Using wt and different mutants of hTrx1 including C32S/C35S, C62S/C73S, and C62S, the localization of nitrosothiols was investigated. E. coli Trx was also utilized to investigate whether active site cysteines can be nitrosylated. Determination of Total Thiol Groups—DTNB was used to determine the number of free thiols in reduced, oxidized, or nitrosylated thioredoxin samples (41Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (20873) Google Scholar). Briefly, 10 μm of reduced, oxidized, or nitrosylated protein was incubated with 6 m guanidine hydrochloride and 1 mm DTNB in a final volume of 500 μl of 0.2 m Tris-Cl, pH 8.0. A molar extinction coefficient of 13,600 m–1 cm–1 at 412 nm was used to calculate the number of free thiols. For diamide-treated Trx1, because diamide had not been removed from the sample, the reaction between diamide and free thionitrobenzoate, which is the product of the reaction between thiols and DTNB, was also studied to rule out any bleaching effect of diamide and subsequent interference with the determination of free thiol content. Guanidine hydrochloride 6 m in 0.1 m Tris-HCl, pH 8.0, and 1 mm DTNB were mixed with 25 μm DTT. DTT was excluded from the reference cuvette. After recording the initial absorbance at 412 nm, diamide with a final concentration of 100 μm was added to both the reference and sample cuvettes, and changes at 412 nm were recorded. The decrease of absorbance with a rate of 0.040 min–1 indicated the oxidation of thionitrobenzoate with diamide (data not shown); however, it did not interfere with the determination of free thiol content because of the slow rate of the reaction. Activity of Oxidized Thioredoxin as a Substrate for Thioredoxin Reductase—Oxidized or S-nitrosylated hTrx1 was tested as a substrate for TrxR by following the oxidation of NADPH (Reaction 2). The experiment was performed at 25 °C using 100 μm NADPH and either 20 μm oxidized or nitrosylated Trx1 in a final volume of 500 μl of TE buffer. Trx1 was excluded from the reference cuvette. The reaction was started by adding 10 nm rat TrxR, and the oxidation of NADPH was monitored by following A340 nm. Insulin Disulfide Bond Reduction Activity of Thioredoxin—Two assays were utilized to study the effects of oxidative post-translational modifications of Trx1 on the protein disulfide bond reductase activity of the Trx system. First, as described before (42Luthman M. Holmgren A. Biochemistry. 1982; 21: 6628-6633Crossref PubMed Scopus (503) Google Scholar), we mixed 200 μm NADPH and 160 μm insulin in a final volume of 500 μl of TE buffer. In 1-cm semi-micro quartz cuvettes, 2 μm of Trx sample was added to the mixture at 25 °C. Trx1 was excluded from the reference cuvette. After starting the reactions by the addition of 10 nm rat recombinant TrxR to all samples, A340 nm was recorded to calculate and compare the velocity of the reactions. Insulin precipitation assay was also utilized as described (43Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). Insulin disulfide bonds can be reduced by DTT leading to the precipitation of reduced insulin, and this reaction is catalyzed by Trx. This activity of Trx was compared between oxidized and nitrosylated molecules using 160 μm insulin, 2 μm of either oxidized or nitrosylated Trx, and 1 mm DTT in a final volume of 500 μl of 50 mm potassium phosphate buffer, pH 7.0, and 2 mm EDTA. Increase in turbidity was followed by recording A650 nm. TrxR Activity Assay—In a final volume of 500 μl of TE buffer, we mixed 5 mm DTNB, 0.2 mm NADPH, and varying concentrations of either H2O2 or GSNO. The reactions were started by adding 5 nm rat TrxR to all samples, and A412 nm was monitored for 10 min. TrxR was excluded from the reference cuvette. SDS-PAGE—NuPAGE® 12% Bis-Tris gels were utilized for SDS-PAGE electrophoresis to study the redox state of reduced and oxidized human Trx1 samples. Fifteen μg of reduced Trx1 as well as samples treated with different concentrations of either diamide or H2O2 were loaded on the gel, and electrophoresis was performed with 200 V for 45 min. DTT was excluded from the sample buffer, and a nonreducing gel was utilized. Oxidation of Human Thioredoxin 1 by Diamide—Chemically reduced human Trx1, with five thiols, was incubated with different concentrations of diamide for 2 min at room temperature; thereafter, the number of free thiols was determined. A decrease in the number of free thiols was recorded by increasing the ratio between diamide and Trx1 (Fig. 1B). With 10 molar equivalents of diamide, all of the cysteine residues were oxidized, and essentially no free thiol was detected in the Trx1. When the structure and redox state of diamide-treated human Trx1 was investigated using nonreducing SDS-PAGE electrophoresis, it was seen that oxidation by diamide led to a complex mixture of monomer, mainly dimers, but also oligomers. The reduced protein was as expected at 12 kDa. The effect of diamide on C62S/C73S Trx1, which has only one structural cysteine (Cys69), showed a progressive dimerization (data not shown). The effect of diamide on the activity of human Trx1 was measured using insulin disulfides as a substrate and recycling of Trx-S2 by NADPH and TrxR (Reactions 1 and 2). With one and three molar equivalents of diamide, the initial activity of Trx1 was decreased to 57 and 6% of the control sample, respectively (Fig. 2A). The inhibition of Trx catalysis was transient, and activity was regained after a lag phase as seen from the progress curves. The duration of the lag phase was a function of diamide concentrations. For the Trx1 C62S/C73S mutant protein (Fig. 2B), the activity was not affected by diamide, indicating that the dimers generated by Cys69 oxidation were active. No further studies of this protein were made. The results are in agreement with those of Watson et al. (6Watson W.H. Pohl J. Montfort W.R. Stuchlik O. Reed M.S. Powis G. Jones D.P. J. Biol. Chem. 2003; 278: 33408-33415Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), who, using His-tagged Trx1 showed that diamide generated a second structural disulfide Cys62–Cys69 with a loss of activity. Obviously, diamide is a strong oxidant inducing profound changes in thioredoxin. In particular, the generation of multimers as observed in this study was not evident from the native gel electrophoresis by Watson et al. (6Watson W.H. Pohl J. Montfort W.R. Stuchlik O. Reed M.S. Powis G. Jones D.P. J. Biol. Chem. 2003; 278: 33408-33415Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Oxidation of Human Thioredoxin 1 by H2O2—The oxidation of human Trx1 by H2O2 was studied using the same method as described for diamide, except excluding EDTA from the buffer and changing the incubation time to 15 min and the temperature to 37 °C. Similar to the effect of diamide, a progressive decrease in the number of sulfhydryl groups was observed (Fig. 3). However, the number of free thiols did not reach zero. Even with a 100-fold molar excess of H2O2, one free thiol was left in the structure of human Trx1. The redox state of Trx1 after treatment with H2O2 was investigated using nonreducing SDS-PAGE electrophoresis as described. Remarkably, H2O2-treated Trx1 even after treatment with 100 molar equivalents of H2O2 was still in the monomeric form. The same result was obtained with and without EDTA present; EDTA presence decreased the oxidation rate. Thus, H2O2 gives rise to clean two-disulfide monomers. To study the effect of H2O2 on Trx activity, 100 μm human Trx1 was also incubated with 10, 50, or 100 molar excess of H2O2 for 15 min at 37 °C and then desalted on a NAP-5 column, and the activity was measured. A progressive inhibition of Trx1 activity was recorded by increasing the concentration of H2O2 (Fig. 4), which was reversible, and Trx1 regained its activity after a lag phase. The duration of lag phase was again a function of the H2O2 concentration. S-Nitrosylation of Human Thioredoxin 1 by GSNO—Reduced Trx1 was treated with 10 molar equivalents of GSNO for 60 min at 37 °C. After chromatography on a NAP-5 column to remove excess GSNO, the number of thiols and nitrosothiols were measured in both reduced and GSNO-treated samples (Table 1). The spectrum of GSNO-treated Trx1 showed an extra peak of absorbance at 335 nm, indicating the S-nitrosylation of the protein (Fig. 5). The number of nitrosothiols was calculated using a molar extinction coefficient of 920 m–1 cm–1 at 335 nm (Table 1). There was close to two nitrosothiols in nitrosylated hTrx1. To find the localization of these nitrosothiols, different mutants of hTrx1 as well as the wt protein were utilized (Table 1). Furthermore, E. coli Trx was treated by GSNO to investigate whether cysteines in the active site can be nitrosylated. As shown in Table 1, nitrosylated human C32S/C35S and C62S mutants as well as wt Trx displayed close to two nitrosothiols, whereas there was only one nitrosothiol in the structure of nitrosylated C62S/C73S Trx, and E. coli Trx was not nitrosylated but oxidized. Therefore, cysteines 69 and 73 became modified by GSNO, and cysteines 32 and 35 in the active site were oxidized to a disulfide.TABLE 1The characteristics of reduced and GSNO-treated Trx1Human thioredoxinNumber of free SH groups in fully reduced formNumber of free SH groups in GSNO-treated formNumber of NO groups in GSNO-treated formWild type5.18 ± 0.161.07 ± 0.202.08 ± 0.19C62S/C73S2.89 ± 0.290.23 ± 0.120.78 ± 0.04C32S/C35S3.25 ± 0.301.37 ± 0.341.97 ± 0.19C62S3.40 ± 0.560.02 ± 0.041.71 ± 0.15E. coli Trx1.97 ± 0.150.03 ± 0.030.19 ± 0.17 Open table in a new tab The Effect of Nitrosylation of Trx1 on Its Catalytic Activity—The effect of nitrosylation on the activity of Trx as a substrate for TrxR was studied. Oxidation of NADPH by 20 μm of either oxidized or nitrosylated Trx1 in the presence of 10 nm rat TrxR was recorded (Fig. 6). As expected the Trx-S2 was rapidly reduced, oxidizing a stoichiometric amount of NADPH (Reaction 1). The rate of NADPH oxidation with nitrosylated hTrx1 was very slow; but interestingly, the rate was increased after a lag phase reaching finally up to 60 μm NADPH oxidized. The interpretation of this is that active Trx-(SH)2 molecules can reduce the nitrosothiol groups of Trx1. Therefore, nitrosothiols may be regarded as reduced by an autocatalytic reaction in which fully active Trx-(SH)2 was the catalyst. The NADPH oxidation result showing two nitrosothiols in nitrosylated Trx1 corroborated our spectral data regarding the number of nitrosothiols (Table 1). In addition, reduction of insulin disulfide bonds (Reactions 1 and 2) was used to investigate the effect of nitrosylation. Fig. 7 shows that the fully nitrosylated Trx was almost inactive, although it started to regain activity after a lag phase. When the experiments were done using nitrosylated double mutant of human Trx (C62S/C73S) with a single nitrosothiol on Cys 69 (Table 1), the initial protein-disulfide reductase activity of hTrx1 was not increased (data not shown), in contrast to what has been reported (28Haendeler J. Hoffmann J. Tischler V. Berk B.C. Zeiher A.M. Dimmeler S. Nat. Cell Biol. 2002; 4: 743-749Crossref PubMed Scopus (339) Google Scholar). Preincubation of reduced hTrx1 with varying concentrations of GSNO for 30 min at 37 °C was also used to study the effect of nitrosylation on the reductase activity. Fig. 8 shows a progressive inhibition of Trx activity with higher concentrations of GSNO and under no conditions an increase in activity. Also in assays of thioredoxin activity using DTT and insulin precipitation (43Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar), there was a lag phase consistent with the initial inhibition of activity. Protection of Active Site Cysteines from S-Nitrosylation by the Pro34 Residue—Protein-disulfide isomerase, a Trx fold protein, which has two active sites (-Trp-Cys-Gly-His-Cys-) with homology to the active site of Trx, is known to be nitrosylated on both thioredoxin domains (44Uehara T. Nakamura T. Yao D. Shi Z.-Q. Gu Z. Ma Y. Masliah E. Nomura Y. Lipton S.A. Nature. 2006; 441: 513-517Crossref PubMed Scopus (747) Google Scholar). The nitrosylation of the active site Cys residues in protein-disulfide isomerase inhibits catalytic activity (44Uehara T. Nakamura T. Yao D. Shi Z.-Q. Gu Z. Ma Y. Masliah E. Nomura Y. Lipton S.A. Nature. 2006; 441: 513-517Crossref PubMed Scopus (747) Google Scholar). We hypothesized that the Pro34 of Trx has a protective effect against the S-nitrosylation of the Cys residues in the active site. To test this hypothesis, we used E. coli Trx1 P34H, a mutant with increased isomerase activity (39Krause G. Lundstrom J. Barea J.L. Pueyo de la Cuesta C. Holmgren A. J. Biol. Chem. 1991; 266: 9494-9500Abstract Full Text PDF PubMed Google Scholar). P34H Trx1 wa" @default.
• W1993780555 created "2016-06-24" @default.
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• W1993780555 date "2008-08-01" @default.
• W1993780555 modified "2023-10-13" @default.
• W1993780555 title "Regulation of the Catalytic Activity and Structure of Human Thioredoxin 1 via Oxidation and S-Nitrosylation of Cysteine Residues" @default.
• W1993780555 cites W1269597682 @default.
• W1993780555 cites W1534880720 @default.
• W1993780555 cites W1583295309 @default.
• W1993780555 cites W1603513572 @default.
• W1993780555 cites W1788190869 @default.
• W1993780555 cites W1821918060 @default.
• W1993780555 cites W1974335367 @default.
• W1993780555 cites W1975186819 @default.
• W1993780555 cites W1985916621 @default.
• W1993780555 cites W1987194238 @default.
• W1993780555 cites W1992533564 @default.
• W1993780555 cites W1993408206 @default.
• W1993780555 cites W1995698822 @default.
• W1993780555 cites W2004494212 @default.
• W1993780555 cites W2004724154 @default.
• W1993780555 cites W2006370393 @default.
• W1993780555 cites W2011855358 @default.
• W1993780555 cites W2016051676 @default.
• W1993780555 cites W2019738475 @default.
• W1993780555 cites W2021727296 @default.
• W1993780555 cites W2022699060 @default.
• W1993780555 cites W2026207397 @default.
• W1993780555 cites W2029925670 @default.
• W1993780555 cites W2036140137 @default.
• W1993780555 cites W2048005287 @default.
• W1993780555 cites W2048553024 @default.
• W1993780555 cites W2048569323 @default.
• W1993780555 cites W2050083026 @default.
• W1993780555 cites W2054027791 @default.
• W1993780555 cites W2063078080 @default.
• W1993780555 cites W2065020384 @default.
• W1993780555 cites W2065546882 @default.
• W1993780555 cites W2065842157 @default.
• W1993780555 cites W2067593902 @default.
• W1993780555 cites W2071468228 @default.
• W1993780555 cites W2076102443 @default.
• W1993780555 cites W2079310705 @default.
• W1993780555 cites W2079912175 @default.
• W1993780555 cites W2080357351 @default.
• W1993780555 cites W2081748503 @default.
• W1993780555 cites W2081785725 @default.
• W1993780555 cites W2084398452 @default.
• W1993780555 cites W2085601435 @default.
• W1993780555 cites W2085664820 @default.
• W1993780555 cites W2091876246 @default.
• W1993780555 cites W2092685208 @default.
• W1993780555 cites W2100494125 @default.
• W1993780555 cites W2109719349 @default.
• W1993780555 cites W2118023300 @default.
• W1993780555 cites W2120705173 @default.
• W1993780555 cites W2134621793 @default.
• W1993780555 cites W2144229295 @default.
• W1993780555 cites W2165201727 @default.
• W1993780555 cites W2173112447 @default.
• W1993780555 cites W2179702494 @default.
• W1993780555 cites W4247896860 @default.
• W1993780555 doi "https://doi.org/10.1074/jbc.m801047200" @default.
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