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Redox regulation of ubiquitin‐conjugating enzymes: mechanistic insights using the thiol‐specific oxidant diamide
Author(s) -
Obin Martin,
Shang Fu,
Gong Xin,
Handelman Garry,
Blumberg Jeffrey,
Taylor Allen
Publication year - 1998
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.12.7.561
Subject(s) - ubiquitin , glutathione , oxidative stress , ubiquitin ligase , ubiquitin conjugating enzyme , biochemistry , thiol , chemistry , endogeny , proteasome , enzyme , gene
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 ≥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.