Overview of Protein Glutathionylation

Aleksandra Filipovska1, Michael P. Murphy1

1 Medical Research Council, Dunn Human Nutrition Unit, Cambridge
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.10
DOI:  10.1002/0471140856.tx0610s28
Online Posting Date:  June, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Many proteins contain free thiols that can be modified by the reversible formation of mixed disulfides with glutathione. Protein glutathionylation is of significance for defense against oxidative damage and in redox signaling. Here we outline the mechanisms and possible significance of protein glutathionylation.

Keywords: mitochondria; glutathione; oxidative stress; protein thiols; redox regulation

PDF or HTML at Wiley Online Library

Table of Contents

  • Protein Thiols and Glutathione
  • Protein Glutathionylation
  • Fate of Glutathionylated Proteins
  • Glutaredoxin
  • Thioredoxin
  • Physiological Roles of Protein Glutathionylation
  • Conclusions
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Arner, E.S. and Holmgren, A. 2000. Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 26:6102‐6109.
   Axelsson, K. and Mannervik, B. 1980. General specificity of cytoplasmic thioltransferase (thiol:disulfide oxidoreducatse) from rat liver for thiol and disulfide substrates. Biochim. Biophys. Acta 613:324‐336.
   Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., and Freeman, B.A. 1990. Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U.S.A. 87:1620‐1624.
   Beer, S.M., Taylor, E.R., Brown, S.E., Dahm, C.C., Costa, N.J., Runswick, M.J., and Murphy, M.P. 2004. Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: Implications for mitochondrial redox regulation and antioxidant defense. J. Biol. Chem. 279:47939‐47951.
   Bolanos, J.P., Almeida, A., Stewart, V., Peuchen, S., Land, J.M., Clark, J.B., and Heales, S.J. 1997. Nitric oxide‐mediated mitochondrial damage in the brain: Mechanisms and implications for neurodegenerative diseases. J. Neurochem. 68:2227‐2240.
   Brennan, J.P., Wait, R., Begum, S., Bell, J.R., Dunn, M.J., and Eaton, P. 2004. Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis. J. Biol. Chem. 279:41352‐41360.
   Bushweller, J.H., Aslund, F., Wuthrich, K., and Holmgren, A. 1992. Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14—S) and its mixed disulfide with glutathione. Biochemistry 31:9288‐9293.
   Cabiscol, E. and Levine, R.L. 1996. The phosphatase activity of carbonic anhydrase III is reversibly regulated by glutathiolation. Proc. Natl. Acad. Sci. U.S.A. 93:4170‐4174.
   Costa, N.J., Dahm, C.C., Hurrell, F., Taylor, E.R., and Murphy, M.P. 2003. Interactions of mitochondrial thiols with nitric oxide. Antioxid. Redox Signal 5:291‐305.
   Cotgreave, I.A. and Gerdes, R.G. 1998. Recent trends in glutathione biochemistry—glutathione‐protein interactions: A molecular link between oxidative stress and cell proliferation? Biochem. Biophys. Res. Commun. 242:1‐9.
   Di Simplicio, P., Cacace, M.G., Lusini, L., Giannerini, F., Giustarini, D., and Rossi, R. 1998. Role of protein –SH groups in redox homeostasis: The erythrocyte as a model system. Arch. Biochem. Biophys. 355:145‐152.
   Esposito, L.A., Kokoszka, J.E., Waymire, K.G., Cottrell, B., MacGregor, G.R., and Wallace, D.C. 2000. Mitochondrial oxidative stress in mice lacking the glutathione peroxidase‐1 gene. Free Radic. Biol. Med. 28:754‐766.
   Fernandez‐Checa, J.C., Kaplowitz, N., Garcia‐Ruiz, C., and Colell, A. 1998. Mitochondrial glutathione: Importance and transport. Semin. Liver Dis. 18:389‐401.
   Foster, M.W. and Stamler, J.S. 2004. New insights into protein S‐nitrosylation: Mitochondria as a model system. J. Biol. Chem. 279:25891‐25897.
   Fratelli, M., Demol, H., Puype, M., Casagrande, S., Eberini, I., Salmona, M., Bonetto, V., Mengozzi, M., Duffieux, F., Miclet, E., Bachi, A., Vandekerckhove, J., Gianazza, E., and Ghezzi, P. 2002. Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 99:3505‐3510.
   Fratelli, M., Demol, H., Puype, M., Casagrande, S., Villa, P., Eberini, I., Vandekerckhove, J., Gi anazza, E., and Ghezzi, P. 2003. Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells. Proteomics 3:1154‐1161.
   Gardner, J.L. and Gallagher, E.P. 2001. Development of a peptide antibody specific to human glutathione S‐transferase alpha 4‐4 (hGSTA4‐4) reveals preferential localization in human liver mitochondria. Arch. Biochem. Biophys. 390:19‐27.
   Gilbert, H.F. 1984. Redox control of enzyme activities by thiol/disulfide exchange. Meth. Enzymol. 107:330‐351.
   Gilbert, H.F. 1990. Molecular and cellular aspects of thiol‐disulfide exchange. Adv. Enzymol. Relat. Areas Mol. Biol. 63:69‐172.
   Gilbert, H.F. 1995. Thiol/disulfide exchange equilibria and disulfide bond stability. Meth. Enzymol. 251:8‐28.
   Gitler, C., Zarmi, B., and Kalef, E. 1997. General methods to identify and enrich vicinal thiol proteins present in intact cells in the oxidised disulfide state. Anal. Biochem. 252:48‐55.
   Giustarini, D., Rossi, R., Milzani, A., Coombo, R., and Dalle‐Donne, R. 2004. S‐Glutathionylation: From redox regulation of protein functions to human diseases. J. Cell. Mol. Med. 8:201‐212.
   Gladyshev, V.N., Liu, A., Novoselov, S.V., Krysan, K., Sun, O.A., Kryukov, V.M., Kryukov, G.V., and Lou, M.F. 2001. Identification and characterization of a new mammalian glutaredoxin (thioltransferase), Grx2. J. Biol. Chem. 276:30374‐30380.
   Gravina, S.A. and Mieyal, J.J. 1993. Thioltransferase is a specific glutathionyl mixed disulfide oxidoreductase. Biochemistry 32:3368‐3376.
   Griffith, O.W. and Meister, A. 1985. Origin and turnover of mitochondrial glutathione. Proc. Natl. Acad. Sci. U.S.A. 82:4668‐4672.
   Holmgren, A. 1985. Thioredoxin. Annu. Rev. Biochem. 54:237‐271.
   Jacob, C., Giles, G.I., Giles, N.M., and Sies, H. 2003. Sulfur and selenium: The role of oxidation state in protein structure and function. Angew Chem. Int. Ed. Engl. 42:4742‐4758.
   Johansson, C., Lillig, C.H., and Holmgren, A. 2004. Human mitochondrial glutaredoxin reduces S‐glutathionylated proteins with high affinity accepting electrons from either glutathione or thioredoxin reductase. J. Biol. Chem. 279:7537‐7543.
   Jung, C.H. and Thomas, J.A. 1996. S‐glutathiolated hepatocyte proteins and insulin disulfides as substrates for reduction by glutaredoxin, thioredoxin, protein disulfide isomerase, and glutathione. Arch. Biochem. Biophys. 335:61‐72.
   Kamphuis, I.G., Drenth, J., and Baker, E.N. 1985. Thiol proteases: Comparative studies based on the high‐resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain. J. Mol. Biol. 182:317‐329.
   Kelner, M.J. and Montoya, M.A. 2000. Structural organization of the human glutathione reductase gene: Determination of correct cDNA sequence and identification of a mitochondrial leader sequence. Biochem. Biophys. Res. Commun. 269:366‐368.
   Klatt, P. and Lamas, S. 2000. Regulation of protein function by S‐glutathiolation in response to oxidative and nitrosative stress. Eur. J. Biochem. 267:4928‐4944.
   Klatt, P., Molina, E.P., De Lacoba, M.G., Padilla, C.A., Martinez‐Galesteo, E., Barcena, J.A., and Lamas, S. 1999. Redox regulation of c‐Jun DNA binding by reversible S‐glutathiolation. FASEB J. 13:1481‐1490.
   Lillig, C.H., Lonn, M.E., Enoksson, M., Fernandes, A.P., and Holmgren, A. 2004. Short interfering RNA‐mediated silencing of glutaredoxin 2 increases the sensitivity of HeLa cells toward doxorubicin and phenylarsine oxide. Proc. Natl. Acad. Sci. U.S.A. 101:13227‐13232.
   Lundberg, M., Johansson, C., Chandra, J., Enoks son, M., Jacobsson, G., Ljung, J., Joh ansson, M., and Holmgren, A. 2001. Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms. J. Biol. Chem. 276:26269‐26275.
   Mallis, R.J., Poland, B.W., Chatterjee, T.K., Fisher, R.A., Darmawan, S., Honkzatko, R.N., and Thomas, J.A. 2000. Crystal structure of S‐glutathiolated carbonic anhydrase III. FEBS Lett. 482:237‐241.
   Meister, A. 1995. Mitochondrial changes associated with glutathione deficiency. Biochim. Biophys. Acta 1271:35‐42.
   Musil, D., Zucic, D., Turk, D., Engh, R.A., Mayr, L., Huber, R., Popovik, T., Turk, V., Towartari, T., Katunuma, N., and Bodem, W. 1991. The refined 2.15 Å X‐ray crystal structure of human liver cathepsin B: The structural basis for its specificity. EMBO J. 10:2321‐2330.
   O'Donovan, D.J., Katkin, J.P., Tamura, T., Smith, C.V., and Welty, S.E. 2000. Attenuation of hyperoxia‐induced growth inhibition in H441 cells by gene transfer of mitochondrially targeted glutathione reductase. Am. J. Respir. Cell Mol. Biol. 22:732‐738.
   Padgett, C.M. and Whorton, A.R. 1995a. Regulation of cellular thiol redox status by nitric oxide. Cell Biochem. Biophys. 27:157‐177.
   Padgett, C.M. and Whorton, A.R. 1995b. S‐nitrosoglutathione reversibly inhibits GAPDH by S‐nitrosylation. Am. J. Physiol. 269:C739‐C749.
   Panfili, E., Sandri, G., and Ernster, L. 1991. Distribution of glutathione peroxidases and glutathione reductase in rat brain mitochondria. FEBS Lett. 290:35‐37.
   Park, E.‐M. and Thomas, J.A. 1989. Reduction of protein mixed disulfides (dethiolation) by E. coli thioredoxin: A study with glycogen phosphorylase b and creatine kinase. Arch. Biochem. Biophys. 272:25‐31.
   Pemble, S.E., Wardle, A.F., and Taylor, J.B. 1996. Glutathione S‐transferase class Kappa: Characterization by the cloning of rat mitochondrial GST and identification of a human homologue. Biochem. J. 319:749‐754.
   Radi, R., Beckman, J.S., Bush, K.M., and Free man, B.A. 1991. Peroxynitrite oxidation of sulfhydryls. J. Biol. Chem. 266:4244‐4250.
   Reed, D. 1990. Glutathione: Toxicological implications. Annu. Rev. Pharmacol. Toxicol. 30:603‐631.
   Ruoppolo, M., Lundstrom‐Ljung, J., Talamo, F., Pucci, P., and Marino, G. 1997. Effect of glutaredoxin and protein disulfide isomerase on the glutathione‐dependent folding of ribonuclease A. Biochemistry 36:12259‐12267.
   Seres, T., Ravichandran, V., Moriguchi, T., Roku tan, K., Thomas, J.A., and Johnston, R.B. Jr. 1996. Protein S‐thiolation and dethiolation during the respiratory burst in human monocytes: A reversible post‐translational modification with potential for buffering the effects of oxidant stress. J. Immunol. 156:1973‐1980.
   Spyrou, G., Enmark, E., Miranda‐Vizuete, A., and Gustafsson, J. 1997. Cloning and expression of a novel mammalian thioredoxin. J. Biol. Chem. 272:2936‐2941.
   Stamler, J.S. 1994. Redox signaling: Nitrosylation and related target interactions of nitric oxide. Cell 78:931‐936.
   Stamler, J.S. and Hausladen, A. 1998. Oxidative modifications in nitrosative stress. Nat. Struct. Biol. 5:247‐249.
   Starke, D.W., Chock, P.B., and Meiyal, J.J. 2003. Glutathione‐thiyl radical scavenging and transferase properties of human glutaredoxin (thioltransferase): Potential role in redox signal transduction. J. Biol. Chem. 278:14607‐14613.
   Steffen, M., Sarkela, T.M., Gybina, A.A., Steele, T.W., Trasseth, N.J., Kuehl, D., and Giulivi, C. 2001. Metabolism of S‐nitrosoglutathione in intact mitochondria. Biochem. J. 356:395‐402.
   Tamura, T., McMicken, H.W., Smith, C.V., and Hansen, T.N. 1996. Mitochondrial targeting of glutathione reductase requires a leader sequence. Biochem. Biophys. Res. Commun. 222:659‐663.
   Tamura, T., McMicken, H.W., Smith, C.V., and Hansen, T.N. 1997. Gene structure for mouse glutathione reductase, including a putative mitochondrial targeting signal. Biochem. Biophys. Res. Commun. 237:419‐422.
   Thomas, J.A., Poland, B., and Honkzatko, R. 1995. Protein sulfhydryls and their role in the antioxidant function of protein S‐thiolation. Arch. Biochem. Biophys. 319:1‐9.
   van den Dobbelsteen, D.J., Nobel, C.S., Schlegel, J., Cotgreave, I.A., Orrenius, S., and Slater, A.F. 1996. Rapid and specific efflux of reduced glutathione during apoptosis induced by anti‐Fas/APO‐1 antibody. J. Biol. Chem. 271:15420‐15427.
   Winterbourn, C.C. 1993. Superoxide as an intracellular radical sink. Free Rad. Biol. Med. 14:85‐90.
   Zheng, M., Aslund, F. and Storz, G. 1998. Activation of the OxyR transcription factor by reversible disulfide bond formation. Science 279:1718‐1721.
   Ziegler, D.M. 1985. Role of reversible oxidation‐reduction of enzyme thiols‐disulfides in metabolic regulation. Annu. Rev. Biochem. 54:305‐329.
PDF or HTML at Wiley Online Library