Redox regulation of ubiquitin-conjugating enzymes: mechanistic insights using the thiol-specific oxidant diamide

FASEB J. 1998 May;12(7):561-9. doi: 10.1096/fasebj.12.7.561.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line, Transformed
  • Diamide / pharmacology*
  • Glutathione / metabolism*
  • Glutathione Disulfide / metabolism
  • Humans
  • Kinetics
  • Ligases / metabolism*
  • Oxidants
  • Oxidation-Reduction
  • Simian virus 40
  • Sulfhydryl Compounds / metabolism
  • Sulfhydryl Reagents / pharmacology*
  • Ubiquitins / metabolism*


  • Oxidants
  • Sulfhydryl Compounds
  • Sulfhydryl Reagents
  • Ubiquitins
  • Diamide
  • Ligases
  • Glutathione
  • Glutathione Disulfide