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Specifically Targeted Modification of Human Aldose Reductase by Physiological Disulfides

1996; Elsevier BV; Volume: 271; Issue: 52 Linguagem: Inglês

10.1074/jbc.271.52.33539

1083-351X

Mario Cappiello, M Voltarelli, I Cecconi, Pier Giuseppe Vilardo, Massimo Dal Monte, I Marini, Antonella Del Corso, David K. Wilson, Florante A. Quiocho, J. Mark Petrash, Umberto Mura,

Heme Oxygenase-1 and Carbon Monoxide

Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303. Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303. INTRODUCTIONAldose reductase (alditol:NADP oxidoreductase, EC 1.1.1.21) (ALR2) 1The abbreviations used are: ALR2aldose reductaseDTTdithiothreitolr.m.s.root mean square. catalyzes with a broad catalytic efficiency the NADPH-dependent reduction of aldo-sugars and a variety of aromatic and aliphatic aldehydes to their corresponding alcohols. This enzyme is the first in a pathway that results in the transformation of glucose to fructose using sorbitol as a metabolic intermediate. This so-called "polyol pathway" is not a "high flux" metabolic route except in hyperglycemic conditions such as diabetes mellitus and galactosemia, where elevated concentrations of glucose and galactose, respectively, result in enhanced accumulation of their corresponding polyols in various tissues such as the eye lens (1Varma S.D. Zadunaisky J.A. Davidson H. Current Topics in Eye Research. Vol. 3. Academic Press, New York1980: 91-153Google Scholar, 2Birlouez-Aragon I. Alloussi S. Morawiec M. Fevrier C. Curr. Eye Res. 1989; 8: 449-457Crossref PubMed Scopus (5) Google Scholar). Since these polyols do not readily permeate cell membranes, their intracellular accumulation is thought to create an osmotic imbalance, resulting ultimately in sugar cataract formation (3Kinoshita J.H. Invest. Ophthalmol. & Visual Sci. 1974; 13: 713-723Google Scholar, 4Kinoshita J.H. Am. J. Ophthalmol. 1986; 102: 685-692Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 5Dvornik D. Porte D. Aldose Reductase Inhibition: An Approach to the Prevention of Diabetic Complications. McGraw-Hill Book Co., New York1987: 69-151Google Scholar). Intensive effort has been mounted to identify inhibitors of aldose reductase for use as therapeutic tools against diabetic complications such as cataract and retinopathy (6Kador P.F. Kinoshita J.H. Sharpless N.E. J. Med. Chem. 1985; 28: 841-849Crossref PubMed Scopus (290) Google Scholar, 7Deck L.M. Vander Jagt D.L. Royer R.E. J. Med. Chem. 1991; 34: 3301-3305Crossref PubMed Scopus (43) Google Scholar, 8Smar M.W. Ares J.J. Nakayama T. Habe H. Kador P.F. Miller D.D. J. Med. Chem. 1992; 35: 1117-1120Crossref PubMed Scopus (18) Google Scholar, 9Petrash J.M. Tarle I. Wilson D.K. Quiocho F.A. Diabetes. 1994; 43: 955-959Crossref PubMed Scopus (47) Google Scholar).Aldose reductase is subject to modifications leading to enzyme forms with an altered sensitivity to various inhibitors. Thus, the so-called "activated" ALR2 generated through apparently different processes such as isomerization (10Grimshaw C.E. Shahbaz M. Jahangiri G. Putnay C.G. McKercher S.R. Mathur E.J. Biochemistry. 1989; 28: 5343-5353Crossref PubMed Scopus (53) Google Scholar), glycosylation (11Srivastava S.K. Ansari N.H. Bhatnagar A. Hair G. Liu S.-Q. Das B. Baynes J.W. Monnier V.M. The Maillard Reaction in Aging Diabetes and Nutrition. Alan R. Liss, Inc., New York1989: 171-184Google Scholar), and thiol-dependent oxidation (12Del Corso A. Camici M. Mura U. Biochem. Biophys. Res. Commun. 1987; 148: 369-375Crossref PubMed Scopus (26) Google Scholar, 13Del Corso A. Barsacchi D. Camici M. Garland D. Mura U. Arch. Biochem. Biophys. 1989; 270: 604-610Crossref PubMed Scopus (34) Google Scholar, 14Liu S.-Q. Bhatnagar A. Srivastava S.K. Biochim. Biophys. Acta. 1992; 1120: 329-336Crossref PubMed Scopus (25) Google Scholar), besides displaying differences in substrate specificity, has a greatly reduced sensitivity to different aldose reductase inhibitors. Indeed, others recently reported the purification of human ALR2 with kinetic properties consistent with those described for an oxidized form of the enzyme (15Grimshaw C.E. Lai C.-J. Arch. Biochem. Biophys. 1996; 327: 89-97Crossref PubMed Scopus (32) Google Scholar). The potential involvement of cysteine residues in catalysis and inhibition has been widely investigated (16Liu S.-Q. Bhatnagar A. Das B. Srivastava S.K. Arch. Biochem. Biophys. 1989; 275: 112-121Crossref PubMed Scopus (19) Google Scholar, 17Bhatnagar A. Liu S.-Q. Das B. Srivastava S.K. Mol. Pharmacol. 1989; 36: 825-830PubMed Google Scholar, 18Vander Jagt D.L. Robinson B. Taylor K.K. Hunsaker L.A. J. Biol. Chem. 1990; 265: 20982-20987Abstract Full Text PDF PubMed Google Scholar, 19Petrash J.M. Harter T.M. Devine C.S. Olins P.O. Bhatnagar A. Liu S. Srivastava S.K. J. Biol. Chem. 1992; 267: 24833-24840Abstract Full Text PDF PubMed Google Scholar, 20Bhatnagar A. Liu S.-Q. Petrash J.M. Srivastava S.K. Mol. Pharmacol. 1992; 42: 917-921PubMed Google Scholar, 21Liu S.-Q. Bhatnagar A. Ansari N.H. Srivastava S.K. Biochim. Biophys. Acta. 1993; 1164: 268-272Crossref PubMed Scopus (23) Google Scholar). Many studies indicate that Cys-298, an accessible residue located close to the active site, is a possible modulator of ALR2 susceptibility to inhibition. Carboxymethylation of ALR2 generates an enzyme form with differentially altered susceptibility to inhibition by Tolrestat (N-[[5-(trifluoromethyl)-6-methoxy-1-naphthalenyl]thioxomethyl]-N-methylglycine) and Sorbinil ((S)-6-fluorospiro[chroman-4,4′-imidazolidine]-2′,5′-dione), suggesting the existence of two distinct inhibitor-binding sites on ALR2 (14Liu S.-Q. Bhatnagar A. Srivastava S.K. Biochim. Biophys. Acta. 1992; 1120: 329-336Crossref PubMed Scopus (25) Google Scholar). Besides carboxymethylation, Cys-298 appears to be the target residue in the menadione-induced inactivation of human placental ALR2 (20Bhatnagar A. Liu S.-Q. Petrash J.M. Srivastava S.K. Mol. Pharmacol. 1992; 42: 917-921PubMed Google Scholar) as well as in the dithiodiethanol-induced activation of the enzyme (22Bohren K.M. Gabbay K.H. Weiner H. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 4. Plenum Press, New York1993: 267-277Google Scholar). Among significant differences in structural and kinetic properties (23Del Corso A. Barsacchi D. Giannessi M. Tozzi M.G. Camici M. Mura U. J. Biol. Chem. 1989; 264: 17653-17655Abstract Full Text PDF PubMed Google Scholar, 24Del Corso A. Barsacchi D. Giannessi M. Tozzi M.G. Houben J.L. Zandomeneghi M. Camici M. Mura U. Sakamoto N. Kinoshita J.H. Kador P.F. Hotta N. Current Concepts of Aldose Reductase and Its Inhibition. Elsevier Science Publishers B. V., Amsterdam1990: 195-198Google Scholar, 25Del Corso A. Barsacchi D. Giannessi M. Tozzi M.G. Camici M. Houben J.L. Zandomeneghi M. Mura U. Arch. Biochem. Biophys. 1990; 283: 512-518Crossref PubMed Scopus (41) Google Scholar, 26Giannessi M. Del Corso A. Cappiello M. Marini I. Barsacchi D. Garland D. Camici M. Mura U. Arch. Biochem. Biophys. 1993; 300: 423-429Crossref PubMed Scopus (31) Google Scholar), the enzyme form characterized by a mixed disulfide linkage between 2-mercaptoethanol and the cysteine residue appears completely insensitive to Sorbinil.Cystine and glutathione disulfide are both capable of inducing reversible inactivation of bovine lens ALR2 in vitro (27Cappiello M. Voltarelli M. Giannessi M. Cecconi I. Camici G. Manao G. Del Corso A. Mura U. Exp. Eye Res. 1994; 58: 491-501Crossref PubMed Scopus (58) Google Scholar). The enzyme form modified by GSSG, which displayed as much as 40% of the native enzyme activity and is insensitive to Sorbinil, has been demonstrated to occur in situ in cultured bovine lenses subjected to oxidative stress by hyperbaric oxygen treatment (28Cappiello M. Vilardo P.G. Cecconi I. Leverenz V. Giblin F.J. Del Corso A. Mura U. Biochem. Biophys. Res. Commun. 1995; 207: 775-782Crossref PubMed Scopus (35) Google Scholar). Thus, the possibility that ALR2 may undergo in vivo reversible thiolation must be considered in the design of new ALR2 inhibitors. In this study, we demonstrate that ALR2 is susceptible to glutathione disulfide- and cystine-dependent oxidation and that Cys-298 is the target residue for such post-translational modifications. INTRODUCTIONAldose reductase (alditol:NADP oxidoreductase, EC 1.1.1.21) (ALR2) 1The abbreviations used are: ALR2aldose reductaseDTTdithiothreitolr.m.s.root mean square. catalyzes with a broad catalytic efficiency the NADPH-dependent reduction of aldo-sugars and a variety of aromatic and aliphatic aldehydes to their corresponding alcohols. This enzyme is the first in a pathway that results in the transformation of glucose to fructose using sorbitol as a metabolic intermediate. This so-called "polyol pathway" is not a "high flux" metabolic route except in hyperglycemic conditions such as diabetes mellitus and galactosemia, where elevated concentrations of glucose and galactose, respectively, result in enhanced accumulation of their corresponding polyols in various tissues such as the eye lens (1Varma S.D. Zadunaisky J.A. Davidson H. Current Topics in Eye Research. Vol. 3. Academic Press, New York1980: 91-153Google Scholar, 2Birlouez-Aragon I. Alloussi S. Morawiec M. Fevrier C. Curr. Eye Res. 1989; 8: 449-457Crossref PubMed Scopus (5) Google Scholar). Since these polyols do not readily permeate cell membranes, their intracellular accumulation is thought to create an osmotic imbalance, resulting ultimately in sugar cataract formation (3Kinoshita J.H. Invest. Ophthalmol. & Visual Sci. 1974; 13: 713-723Google Scholar, 4Kinoshita J.H. Am. J. Ophthalmol. 1986; 102: 685-692Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 5Dvornik D. Porte D. Aldose Reductase Inhibition: An Approach to the Prevention of Diabetic Complications. McGraw-Hill Book Co., New York1987: 69-151Google Scholar). Intensive effort has been mounted to identify inhibitors of aldose reductase for use as therapeutic tools against diabetic complications such as cataract and retinopathy (6Kador P.F. Kinoshita J.H. Sharpless N.E. J. Med. Chem. 1985; 28: 841-849Crossref PubMed Scopus (290) Google Scholar, 7Deck L.M. Vander Jagt D.L. Royer R.E. J. Med. Chem. 1991; 34: 3301-3305Crossref PubMed Scopus (43) Google Scholar, 8Smar M.W. Ares J.J. Nakayama T. Habe H. Kador P.F. Miller D.D. J. Med. Chem. 1992; 35: 1117-1120Crossref PubMed Scopus (18) Google Scholar, 9Petrash J.M. Tarle I. Wilson D.K. Quiocho F.A. Diabetes. 1994; 43: 955-959Crossref PubMed Scopus (47) Google Scholar).Aldose reductase is subject to modifications leading to enzyme forms with an altered sensitivity to various inhibitors. Thus, the so-called "activated" ALR2 generated through apparently different processes such as isomerization (10Grimshaw C.E. Shahbaz M. Jahangiri G. Putnay C.G. McKercher S.R. Mathur E.J. Biochemistry. 1989; 28: 5343-5353Crossref PubMed Scopus (53) Google Scholar), glycosylation (11Srivastava S.K. Ansari N.H. Bhatnagar A. Hair G. Liu S.-Q. Das B. Baynes J.W. Monnier V.M. The Maillard Reaction in Aging Diabetes and Nutrition. Alan R. Liss, Inc., New York1989: 171-184Google Scholar), and thiol-dependent oxidation (12Del Corso A. Camici M. Mura U. Biochem. Biophys. Res. Commun. 1987; 148: 369-375Crossref PubMed Scopus (26) Google Scholar, 13Del Corso A. Barsacchi D. Camici M. Garland D. Mura U. Arch. Biochem. Biophys. 1989; 270: 604-610Crossref PubMed Scopus (34) Google Scholar, 14Liu S.-Q. Bhatnagar A. Srivastava S.K. Biochim. Biophys. Acta. 1992; 1120: 329-336Crossref PubMed Scopus (25) Google Scholar), besides displaying differences in substrate specificity, has a greatly reduced sensitivity to different aldose reductase inhibitors. Indeed, others recently reported the purification of human ALR2 with kinetic properties consistent with those described for an oxidized form of the enzyme (15Grimshaw C.E. Lai C.-J. Arch. Biochem. Biophys. 1996; 327: 89-97Crossref PubMed Scopus (32) Google Scholar). The potential involvement of cysteine residues in catalysis and inhibition has been widely investigated (16Liu S.-Q. Bhatnagar A. Das B. Srivastava S.K. Arch. Biochem. Biophys. 1989; 275: 112-121Crossref PubMed Scopus (19) Google Scholar, 17Bhatnagar A. Liu S.-Q. Das B. Srivastava S.K. Mol. Pharmacol. 1989; 36: 825-830PubMed Google Scholar, 18Vander Jagt D.L. Robinson B. Taylor K.K. Hunsaker L.A. J. Biol. Chem. 1990; 265: 20982-20987Abstract Full Text PDF PubMed Google Scholar, 19Petrash J.M. Harter T.M. Devine C.S. Olins P.O. Bhatnagar A. Liu S. Srivastava S.K. J. Biol. Chem. 1992; 267: 24833-24840Abstract Full Text PDF PubMed Google Scholar, 20Bhatnagar A. Liu S.-Q. Petrash J.M. Srivastava S.K. Mol. Pharmacol. 1992; 42: 917-921PubMed Google Scholar, 21Liu S.-Q. Bhatnagar A. Ansari N.H. Srivastava S.K. Biochim. Biophys. Acta. 1993; 1164: 268-272Crossref PubMed Scopus (23) Google Scholar). Many studies indicate that Cys-298, an accessible residue located close to the active site, is a possible modulator of ALR2 susceptibility to inhibition. Carboxymethylation of ALR2 generates an enzyme form with differentially altered susceptibility to inhibition by Tolrestat (N-[[5-(trifluoromethyl)-6-methoxy-1-naphthalenyl]thioxomethyl]-N-methylglycine) and Sorbinil ((S)-6-fluorospiro[chroman-4,4′-imidazolidine]-2′,5′-dione), suggesting the existence of two distinct inhibitor-binding sites on ALR2 (14Liu S.-Q. Bhatnagar A. Srivastava S.K. Biochim. Biophys. Acta. 1992; 1120: 329-336Crossref PubMed Scopus (25) Google Scholar). Besides carboxymethylation, Cys-298 appears to be the target residue in the menadione-induced inactivation of human placental ALR2 (20Bhatnagar A. Liu S.-Q. Petrash J.M. Srivastava S.K. Mol. Pharmacol. 1992; 42: 917-921PubMed Google Scholar) as well as in the dithiodiethanol-induced activation of the enzyme (22Bohren K.M. Gabbay K.H. Weiner H. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 4. Plenum Press, New York1993: 267-277Google Scholar). Among significant differences in structural and kinetic properties (23Del Corso A. Barsacchi D. Giannessi M. Tozzi M.G. Camici M. Mura U. J. Biol. Chem. 1989; 264: 17653-17655Abstract Full Text PDF PubMed Google Scholar, 24Del Corso A. Barsacchi D. Giannessi M. Tozzi M.G. Houben J.L. Zandomeneghi M. Camici M. Mura U. Sakamoto N. Kinoshita J.H. Kador P.F. Hotta N. Current Concepts of Aldose Reductase and Its Inhibition. Elsevier Science Publishers B. V., Amsterdam1990: 195-198Google Scholar, 25Del Corso A. Barsacchi D. Giannessi M. Tozzi M.G. Camici M. Houben J.L. Zandomeneghi M. Mura U. Arch. Biochem. Biophys. 1990; 283: 512-518Crossref PubMed Scopus (41) Google Scholar, 26Giannessi M. Del Corso A. Cappiello M. Marini I. Barsacchi D. Garland D. Camici M. Mura U. Arch. Biochem. Biophys. 1993; 300: 423-429Crossref PubMed Scopus (31) Google Scholar), the enzyme form characterized by a mixed disulfide linkage between 2-mercaptoethanol and the cysteine residue appears completely insensitive to Sorbinil.Cystine and glutathione disulfide are both capable of inducing reversible inactivation of bovine lens ALR2 in vitro (27Cappiello M. Voltarelli M. Giannessi M. Cecconi I. Camici G. Manao G. Del Corso A. Mura U. Exp. Eye Res. 1994; 58: 491-501Crossref PubMed Scopus (58) Google Scholar). The enzyme form modified by GSSG, which displayed as much as 40% of the native enzyme activity and is insensitive to Sorbinil, has been demonstrated to occur in situ in cultured bovine lenses subjected to oxidative stress by hyperbaric oxygen treatment (28Cappiello M. Vilardo P.G. Cecconi I. Leverenz V. Giblin F.J. Del Corso A. Mura U. Biochem. Biophys. Res. Commun. 1995; 207: 775-782Crossref PubMed Scopus (35) Google Scholar). Thus, the possibility that ALR2 may undergo in vivo reversible thiolation must be considered in the design of new ALR2 inhibitors. In this study, we demonstrate that ALR2 is susceptible to glutathione disulfide- and cystine-dependent oxidation and that Cys-298 is the target residue for such post-translational modifications.