A role for glutathione in reactions between oxidants and proteins

Mallis, Robert
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This dissertation examines the role of reduced glutathione (GSH) in the oxidation of proteins. Carbonic anhydrase III is a cytosolic protein which is S-glutathiolated in cells under oxidative stress. The pure protein was found here to be S-glutathiolated by two oxidants, 2,2'-Azobis(2-amidinopropane)dihydrochloride (AAPH) and hydrogen peroxide (H2O2). At physiologically relevant molar ratios of GSH to protein, GSH was found to mediate protection of carbonic anhydrase III from irreversible oxidation via S-glutathiolation. Both oxidants were found to react directly with the protein. S-glutathiolation. appears to result from the formation of an activated protein thiol that reacts with GSH;H-Ras, a low molecular weight G-protein that regulates proliferation and differentiation in cells, may be regulated by oxidative events. For this reason, oxidative modifications of the cysteine residues of H-Ras were studied. The pure protein was modified on multiple thiols when incubated with thiol oxidants. H2O2 + GSH was found to S-glutathiolate H-Ras on at least one cysteine, while either diamide + GSH or glutathione disulfide (GSSG) was found to S-glutathiolate at least two H-Ras cysteines. The NO donor S-nitrosoglutathione caused S-nitrosylation of H-Ras on four cysteine residues;Within NIH/3T3 cells overexpressing H-Ras, H-Ras was S-glutathiolated on multiple thiols by diamide. At least one of the cysteine residues modified in cells by diamide is normally lipidated, suggesting a role for oxidation in regulating the membrane association of H-Ras. In NIH/3T3 cells overexpressing H-Ras, S-nitrosocysteine was found to cause both S-nitrosylation and S-glutathiolation of H-Ras. Thus, oxidative modification of H-Ras can occur simultaneously on multiple thiols and by multiple mechanisms;The effect of S-nitrosocysteine on low molecular weight thiols and soluble proteins in NIH/3T3 cells was also studied. S-nitrosocysteine was found to be an effective S-nitrosating agent, causing S-nitrosylation of glutathione and proteins. S-nitrosocysteine was also found to be an effective oxidant, causing formation of glutathione disulfide, cysteine-glutathione disulfide, cystine, S-glutathiolated and S-cysteylated proteins. The biological effects of S-nitrosocysteine are likely to be mediated not only through nitrosative, but also oxidative events. Thus cellular GSH pools interact significantly with S-nitrosocysteine and may be a factor in cell-specific variations in responses to S-nitrosothiols.

Biochemistry, biophysics, and molecular biology, Biochemistry