Figure 3: The catalytic cysteines in DUBs are prone to oxidation in vitro and in vivo.

(a) Reversible oxidation of the catalytic cysteine of USP19. Purified USP19CD WT or C506S was treated with the indicated concentrations of H₂O₂ for 10 min then analysed by either non-reducing or reducing SDS–PAGE electrophoresis. Asterisk indicates an oxidized species. The arrowhead indicated band likely represents disulphide-linked USP19 oligomer. (b) Ser510 in USP19 is required for the reversibility of the redox regulation. Equal amount of USP19CD WT and the S510G mutant treated with 0.5 mM H₂O₂ were assayed for DUB activity. Where indicated, DTT (1 mM) was added. (c,d) The catalytic cysteine of DUBs is preferentially oxidized by H₂O₂ in vivo. HEK293 cells transfected with plasmids expressing either USP19CD (c) or USP8CD (d) were treated with 1 mM H₂O₂ for 5 min followed by biotin labelling. The numbers indicate the relative levels of biotin-labelled DUBs. (e) H₂O₂ inhibits the labelling of endogenous DUBs by HA-tagged ubiquitin vinyl sulphone (HA-Ub-VS). Cells treated with either H₂O₂ or the DUB inhibitor PR-619 were incubated for 10 min at 37 °C in a labelling buffer containing 0.5% NP40 and HA-Ub-VS (2 μM). Where indicated, the buffer also contained 5 mM DTT. The whole-cell extracts were analysed by immunoblotting (IB). The brackets indicate DUBs that are sensitive to H₂O₂. A few Ub-VS reactive DUBs appear to be insensitive to H₂O₂ (indicated by asterisks), probably because their active sites are not organized unless treated with HA-Ub-VS. At this point, H₂O₂ has been removed. Whole cell extract (WCE).