Redox regulation of ubiquitin-conjugating enzymes: mechanistic insights using the thiol-specific oxidant diamide.
The ubiquitin-proteasome pathway (UPP) regulates critical cell processes, including the cell cycle, cytokine-induced gene expression, differentiation, and cell death. Recently we demonstrated that this pathway responds to oxidative stress in mammalian cells and proposed that activities of ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) are regulated by cellular redox status (i.e., GSSG:GSH ratio). To test this hypothesis, we altered the GSSG:GSH ratio in retinal pigment epithelial cells with the thiol-specific oxidant, diamide, and assessed activities of the UPP. Treatment of cells with diamide resulted in a dose-dependent increase in the GSSG:GSH ratio resulting from loss of GSH and a coincident increase in GSSG. Increases in the GSSG:GSH ratio from 0.02 in untreated cells to > or = 0.5 in diamide-treated cells were accompanied by dose-dependent reductions in the levels of endogenous Ub-protein conjugates, endogenous E1-ubiquitin thiol esters, and de novo ubiquitin-conjugating activity. As determined by the ability to form E1-ubiquitin and E2s-ubiquitin thiol esters, E1 and E2s were both inhibited by elevated GSSG:GSH ratios. Inhibition of E1 was associated with the formation of E1-protein mixed disulfides. Activities of E1 and E2s gradually recovered to preoxidation levels, coincident with gradual recovery of the GSSG:GSH ratio. These data support S-thiolation/dethiolation as a mechanism regulating E1 and E2 activities in response to oxidant insult. Ubiquitin-dependent proteolytic capacity was regulated by the GSSG:GSH ratio in a manner consistent with altered ubiquitin-conjugating activity. However, ubiquitin-independent proteolysis was unaffected by changes in the GSSG:GSH ratio. Potential adaptive and pathological consequences of redox regulation of UPP activities are discussed.
Obin
M
M
Laboratory for Nutrition and Vision Research, JMUSDA-HNRCA at Tufts University, Boston, Massachusetts 02111, USA.
Shang
F
F
Gong
X
X
Handelman
G
G
Blumberg
J
J
Taylor
A
A
D002461:Cell Line, Transformed; D003958:Diamide / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D016877:Oxidants; D010084:Oxidation-Reduction; D013539:Simian virus 40; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014452:Ubiquitins / Q000378:metabolism*
D016877:Oxidants; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D014452:Ubiquitins; D003958:Diamide; D008025:Ligases; D005978:Glutathione; D019803:Glutathione Disulfide