REGULATION OF UBIQUITIN-CONJUGATING ENZYMES BY GLUTATHIONE FOLLOWING OXIDATIVE STRESS

Upon oxidative stress cells show an increase in the oxidized glutathio ne (GSSG) to reduced glutathione (GSH) ratio with a concomitant decrea se in activity of the ubiquitinylation pathway, Because most of the en zymes involved in the attachment of ubiquitin to substrate proteins co ntain active site sulfhydryls that might be covalently modified (thiol ated) upon enhancement of GSSG levels (glutathiolation), it appeared p lausible that glutathiolation might alter ubiquitinylation rates upon cellular oxidative stress. This hypothesis was explored using intact r etina and retinal pigment epithelial (RPE) cell models, Exposure of in tact bovine retina and RPE cells to H2O2 (0.1-1.7 mu mol/mg) resulted in a dose-dependent increase in the GSSG:GSH ratio and coincident dose -dependent reductions in the levels of endogenous ubiquitin-activating enzyme (E1)-ubiquitin thiol esters and endogenous protein-ubiquitin c onjugates and in the ability to form de novo retinal protein(125)I-lab eled ubiquitin conjugates. Oxidant-induced decrements in ubiquitin con jugates were associated with 60-80% reductions in El and ubiquitin-con jugating enzyme (E2) activities as measured by formation of ubiquitin thiol esters, When GSH levels in RPE cells recovered to preoxidation l evels following H2O2 removal, endogenous El activity and protein-ubiqu itin conjugates were restored, Evidence that S thiolation of El and E2 enzymes is the biochemical link between cellular redox state and E1/E 2 activities includes: (i) 5-fold increases in levels of immunoprecipi table, dithiothreitollabile S-35-E1 adducts in metabolically labeled, H2O2-treated, RPE cells; (ii) diminished formation of El-and E2-I-125- labeled ubiquitin thiol esters, oligomerization of E2(25K), and coinci dent reductions in protein-I-125-labeled ubiquitin conjugates in super natants from nonstressed retinas upon addition of levels of GSSG equiv alent to levels measured in oxidatively stressed retinas; and (iii) pa rtial restoration of El and E2 activities and levels of protein-I-125- labeled ubiquitin conjugates in supernatants from H2O2-treated retinas when GSSG:GSH ratios were restored to preoxidation levels by the addi tion of physiological levels of GSH. These data suggest that the cellu lar redox status modulates protein ubiquitinylation via reversible S t hiolation of El and E2 enzymes, presumably by glutathione.