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. 2013:4:1568.
doi: 10.1038/ncomms2532.

Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells

Jin-Gu Lee  1 , Kheewoong Baek, Nia Soetandyo, Yihong Ye
Affiliations
Free PMC article

Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells

Jin-Gu Lee et al. Nat Commun. 2013.
Free PMC article

Abstract

In eukaryotes, deubiquitinases (DUBs) remove ubiquitin conjugates from diverse substrates, altering their stabilities, localizations or activities. Here we show that many DUBs of the USP and UCH subfamilies can be reversibly inactivated upon oxidation by reactive oxygen species in vitro and in cells. Oxidation occurs preferentially on the catalytic cysteine, abrogating the isopeptide-cleaving activity without affecting these enzymes' affinity to ubiquitin. Sensitivity to oxidative inhibition is associated with DUB activation wherein the active site cysteine is converted to a deprotonated state prone to oxidation. We demonstrate that this redox regulation is essential for mono-ubiquitination of proliferating-cell nuclear antigen in response to oxidative DNA damage, which initiates a DNA damage-tolerance programme. These findings establish a novel mechanism of DUB regulation that may be integrated with other redox-dependent signalling circuits to govern cellular adaptation to oxidative stress, a process intimately linked to aging and cancer.

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Figures

Figure 1
Figure 1. Redox-dependent regulation of DUBs.
(a–c) Activation of DUBs in vitro. Shown are representative DUB activity curves. Purified DUBs were incubated with Ub-AFC and the fluorescence intensities were recorded as a function of time. Where indicated, the DUBs were pretreated with 1 mM DTT for 10 min. (d) DTT activates USP19. Purified USP19 was treated with or without 1 mM DTT for 10 min before incubated with Lys48-linked di-ubiquitin. The DUB activity was measured by SDS–PAGE analysis.
Figure 2
Figure 2. Inhibition of DUBs by H2O2.
(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.
Figure 3
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).
Figure 4
Figure 4. Sensitivity to oxidative inhibition is associated with DUB activation.
(a) Purified DUBs used in the assays. A known amount of BSA was used to estimate the concentration of the purified DUBs. (b) USP19 is highly sensitive to inhibition by H2O2. Purified USP19 or USP19CD was treated with the indicated concentrations of H2O2 at pH 7.4. The samples were diluted tenfold before the DUB activities were determined by the Ub-AFC assay. (c,d) The sensitivity of USP7 and UCH-L1 to inhibition by H2O2 is enhanced by high pH. USP7 or UCH-L1 was treated with H2O2 at the indicated pH and the DUB activity was determined by the Ub-AFC assay. (e) Ubiquitin activates USP7. USP7 was incubated at pH 7.4 in the absence or presence of ubiquitin (50 μM). The samples were diluted tenfold before Ub-AFC was added to determine the USP7 activities. (f) USP7 activation by ubiquitin sensitizes it to inhibition by H2O2. USP7 was pretreated in a buffer without or with ubiquitin for 5 min. Where indicated, H2O2 was also included. The USP7 activities were determined by the Ub-AFC assay at pH 7.4. The error bars indicate the mean of two independent experiments.
Figure 5
Figure 5. Oxidative stress attenuates DUB activities in cells.
(a) Treatment of HEK293 cells with H2O2 resulted in the loss of DUB activity. HEK293 cells either untreated or treated with 1 mM H2O2 for 30 min were lysed. The DUB activities in the indicated amount of whole-cell extracts were measured by the Ub-AFC assay. (b) DUB inhibition by oxidation is reversible. Where indicated, the extract of H2O2-treated cells was incubated with 1 mM DTT for 10 min before DUB activity was measured. (c) Inhibition of DUBs by H2O2 in vitro. The whole-cell extracts prepared from HEK293 cells were either untreated or treated with 200 μM H2O2 for 30 min. Where indicated, H2O2-treated extracts were further incubated with DTT before the DUB assay. (d–g) DUB activity was reversibly inhibited by endogenous ROS. (d) Whole-cell extracts were prepared from RAW264.7 cells treated with phorbol myristate acetate (PMA) plus Zymosan (Zym) (1 h). Untreated and H2O2-treated cells are controls. The DUB activity was measured as in a. (e), As in (d). Where indicated, 1 mM DTT was added to the extract of PMA/Zym-treated cells. (f) ROS generation was stimulated in HepG2 cells by insulin signalling. HepG2 cells treated with insulin (500 nM, 5 min) were stained with 10 μM DCFDA. Fluorescence intensity was measured by flow cytometry. Untreated cells were used to determine the background fluorescence. The error bars indicate the mean of two independent experiments. (g) Insulin signaling inhibits DUB activities in a redox-dependent manner. HepG2 cells treated with insulin or insulin together with NAC were lysed in a buffer containing Ub-AFC. The graph summarizes the results of five independent experiments (Error bars, standard deviation, n=5; P-values were calculated using paired Student’s t-test). (h) Dose-dependent inhibition of DUB activities in HepG2 cells by H2O2.
Figure 6
Figure 6. DUB inhibition in oxidative-stressed cells facilitates PCNA ubiquitination.
(a) The kinetics of DUB inhibition by H2O2. H2O2-treated HEK293 cells (1 mM) were injected into a deubiquitinating assay buffer containing 0.5% NP40 and Ub-AFC. The fluorescence intensity was recorded immediately after the addition of the cells. (b) PCNA ubiquitination is induced by H2O2 treatment. HEK293 cells were treated with 1 mM H2O2 for the indicated time periods. Cells were lysed directly in the Laemmli buffer before immunoblotting (IB) analyses. (c) A DUB inhibitor induces PCNA ubiquitination. HEK293 cells were treated with 20 μM PR-619 for the indicated time periods. A fraction of the cells were lysed in the Laemmli buffer for IB (top panel). The DUB activities in the remaining cells were also determined (bottom panel). (d) DNA damage and DUB inhibition synergistically induce PCNA monoubiquitination. The number indicates the intensity of the Ub-PCNA bands. (e) Quantification of ubiquitinated PCNA as per cent of total PCNA in untreated cells or cells treated with the indicated chemicals for 1 h (MMS 1 mM; PR-619, 20 μM; error bars represent means of two independent experiments). (f) H2O2 wash-out causes deubiquitination of PCNA. Cells treated with H2O2 (0.5 mM, 15 min) were incubated in a fresh medium in the absence or presence of the DUB inhibitor PR-619. Whole-cell extracts were analysed by IB. (g) Deubiquitination of PCNA after H2O2 removal requires USP1. As in f, except that cells treated with the indicated knockdown constructs were used. (h,i) H2O2-treated nuclear extract (NE) fails to cleave ubiquitin from PCNA in vitro. (h) In vitro ubiquitinated PCNA was incubated with NE, H2O2-treated NE or NE with the DUB inhibitor ubiquitin aldehyde (Ub-Al). (i) In vitro ubiquitinated PCNA was incubated with NE or H2O2-treated NE for the indicated time points.
Figure 7
Figure 7. Oxidative stress inhibits USP1 in complex with UAF1.
(a) The purified USP1–UAF1 complex. (b) The activity of the USP1–UAF1 complex is inhibited by H2O2. Where indicated, the USP1–UAF1 complex was treated with H2O2 (0.5 mM, 5 min). The DUB activities were determined by the Ub-AFC assay. (c) USP1 inhibition by H2O2 is reversed by DTT. The activity of the H2O2-treated USP1–UAF1 complex was measured. Where indicated, DTT was added to the reaction. (d) A model depicting the redox regulation of DUBs.
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