Figure 2: Inhibition of DUBs by H2O2.
From: Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells
(a,b) Inhibition of USP19 by H2O2. USP19 purified under reducing conditions was treated with 0.5 mM H2O2 for 3 min and the DUB activity was assayed using either di-ubiquitin (a) or Ub-AFC (b) as the substrate. Where indicated, H2O2-treated USP19 (200 nM) was subsequently incubated with 2 mM DTT before the analysis. (c,d) The catalytic domain (CD) of USP19 is sensitive to inactivation by H2O2. (c) Di-ubiquitin was treated with USP19CD in the presence of the indicated concentrations of H2O2. (d), As in b, except that USP19CD (50 nM) was used. Note: The di-ubiquitin cleavage assay was performed in a smaller volume (20 μl) than the Ub-AFC assay (200 μl). Thus, more DTT was carried over by the enzyme. (e) The activity of USP8CD is reversibly inactivated by H2O2. (f) Oxidation significantly reduces the catalysis constant (Kcat) of USP19CD. USP19CD pretreated with 0, 200 or 500 μM H2O2 was incubated with the indicated concentrations of Ub-AFC. The DUB activities measured under each substrate concentration were plotted.
