1. Deubiquitinases as a Signaling Target of Oxidative Stress
Xiomaris M. Cotto-Rios, Miklós Békés, Jessica Chapman, Beatrix Ueberheide, Tony T. Huang 
Cell Reports 
Volume 2, Issue 6, Pages (December 2012)
DOI: /j.celrep
Copyright © 2012 The Authors Terms and Conditions

2. Cell Reports 2012 2, 1475-1484DOI: (10.1016/j.celrep.2012.11.011)
Copyright © 2012 The Authors Terms and Conditions

3. Figure 1
Oxidation of USP1 Is Dependent on Its Catalytic Site Competency
(A) PCNA monoubiquitination is transiently induced by oxidative stress, whereas checkpoint activation is prolonged. T98G and U2OS cells were treated with H2O2 (final concentration 1 mM) for the indicated amount of time. Samples were collected, lysed, and analyzed by western blot with the indicated antibodies.
(B) Oxidative stress-induced PCNA monoubiquitination is dependent on an intact Lys 164 site but independent of USP1 degradation. U2OS cells stably expressing HA-PCNA wild-type (WT) or HA-PCNA K164R mutant were left untreated or treated with 50 J/M2 UVC for 3 hr or treated with 1 mM H2O2 for 30 min. Samples were collected, lysed, and analyzed by western blot with the indicated antibodies.
(C) Oxidative stress-induced PCNA monoubiquitination mainly occurs during S phase. T98G cells were synchronized in G0/G1 by serum deprivation for 72 hr. Cells were then replenished with fresh media and collected at the indicated time points. Cells were either left untreated or treated with a final concentration of 0.5 mM H2O2. Synchronized cells were treated at 0 hr after release from serum deprivation for G0, after 4 hr of serum deprivation release for G1 phase, or after 24 hr of release for early S phase. Samples were collected, lysed, and analyzed by western blot with the indicated antibodies. Asterisk indicates a nonspecific, cross-reactive band when probing with anti-phospho Chk1 antibody (p317).
(D) Depletion of Rad18 results in increased Chk1 activation. U2OS cells were transfected for 48 hr with a control (Ctrl) siRNA or Rad18 siRNA. Cells were then treated with H2O2 (final concentration 0.5 mM) for the indicated amount of time. Samples were collected, lysed, and analyzed by western blot with the indicated antibodies. Asterisk indicates a nonspecific, cross-reactive band.
(E) The antioxidant activity of Tempol reduces the levels of PCNA monoubiquitination after oxidative stress. U2OS cells in the presence or absence of Tempol (0.5 mM) were treated with H2O2 (final concentration 0.3 mM) for the indicated amount of time. Samples were collected, lysed, and analyzed by western blot with the indicated antibodies.
(F) Depletion of USP1 further elevates PCNA monoubiquitination in the presence of oxidative stress. U2OS cells were transfected for 48 hr with a control (Ctrl) siRNA or USP1 siRNA. Cells were then treated with H2O2 (final concentration 0.3 mM) for 30 min. Samples were collected, lysed, and analyzed by western blot with the indicated antibodies.
(G) Oxidation of USP1 catalytic Cys to the sulfenic acid intermediate can be captured by DCP-Bio1 probe. U2OS cells were transfected for 48 hr with Usp1-Myc WT or (DDAA)-Myc mutant along with Xpress-UAF1. Cells were then treated with H2O2 (final concentration 0.3 mM) for 30 min. Extracts were made and divided for two separate reactions: labeling with DCP-Bio1 probe or with the UbVME DUB activity probe. Input represents 30% of extracts used for the DCP-Bio1 reaction.
(H) Oxidative stress does not disrupt the binding between Usp1 and UAF1 in cells. U2OS cells were transfected with Xpress-UAF1 only or cotransfected with Usp1-Myc WT and Xpress-UAF1 and treated with H2O2 (final concentration 1 mM) for 30 min. Extracts were subjected to immunoprecipitation with anti-Myc antibody and probed with indicated antibodies.
See also Figure S1.
Cell Reports 2012 2, DOI: ( /j.celrep )
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4. Figure 2 Reversible Inhibition of DUBs by Oxidative Stress
(A) Temporal analysis of ROS levels inside cells after H2O2 treatment. T98G cells were treated with a final concentration of 1 mM H2O2 for the indicated amount of time (top and bottom left). Cells were trypsinized, collected, and incubated with CM-H2DCFDA for 30 min at 37°C prior to FACS analysis. Additionally, T98G cells were left untreated or treated with 0.3 mM H2O2 for 30 min, 50 J/M2 UVC for 30 min, 2 mM HU for 18 hr, or MMC for 18 hr (bottom right) and trypsinized, collected, and incubated with CM-H2DCFDA for FACS analysis. Error bars represent SD of the experiment done in triplicate.
(B) USP1 and USP7 are reversibly inactivated by oxidative stress and correlate with the ubiquitination status of their respective substrates. U2OS cells were left untreated or treated with a final concentration of 1 mM H2O2 for the indicated timecourse. Extracts were made in parallel to assess DUB activity by the UbVME probe and by whole-cell lysis to assess the ubiquitination status of PCNA and p53 proteins.
(C) Ectopically expressed DUBs are similarly affected by oxidative stress as their endogenous counterparts. 293T cells were either transfected or not for 48 hr with wild-type Flag-USP7 or Flag-USP28 expression constructs. Then cells were left untreated or treated with 1 mM H2O2 for 30 min or with the indicated timecourse. Samples were then collected and processed for DUB activity assay as in (B) and probed with the indicated antibodies for western blot. Additional bands that appear are probably different isoforms of USP28 (USP28 antibody).
(D) Tempol can reverse the DUB catalytic inhibition by oxidative stress. U2OS cells were left untreated or treated with 0.3 mM H2O2 for 30 min in the absence or presence of Tempol. Cells were then trypsinized, collected, and incubated with CM-H2DCFDA as in (A). In parallel analysis, samples were collected and processed for DUB activity assay as in (B).
(E) ROS-inactivated USP7 in U2OS cells can be reversed after in vitro DTT treatment. U2OS cells were treated with 1 mM H2O2 for 30 min. Extracts were then preincubated on ice with 20 mM DTT (or not) prior to the labeling with UbVME as in (B).
(F) Recombinant USP7 can be reversibly inhibited by ROS in vitro. His-tagged recombinant full-length USP7 is subjected to a ubiquitin cleavage assay of K48-linked tetraubiquitin chains in the presence or absence of H2O2 (1 mM) preincubation. DUB reaction was done in the presence or absence of 20 mM DTT.
See also Figure S1.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

5. Figure 3
The Metalloprotease AMSH Is Not Susceptible to ROS-Mediated Catalytic Inhibition
(A) Detecting the oxidation of the active site Cys of USP7 by mass spectrometry. Left: extracted ion chromatograms for the peptide harboring the catalytic Cys NQGAT(C)YMNSLLQTLFFTNQLR in the H2O2-treated (top) and untreated (left) sample. The m/z values for the doubly (monoisotopic m/z = ) and the triply (monoisotopic m/z = ) charged peptide carrying a sulfonic acid modification on the Cys residue and an oxidation on Met are extracted with a mass window of 0.1 Da. In the H2O2-treated sample, the doubly and triply charged precursor of the sulfonic acid-modified peptide with an oxidation on the Met residue could be detected (see also right panel). The mass error is within 2 ppm of the theoretical value. In the untreated sample, there are no ions detected for the doubly or triply charged peptide carrying a sulfonic acid modification and a methionine oxidation. In addition, an m/z value for the peptide carrying just the sulfonic acid modification could not be detected (data not shown). Right: MS/MS spectrum of the triply charged ion of peptide NQGAT(C)YMNSLLQTLFFTNQLR carrying a sulfonic acid modification on the Cys residue and an oxidation on Met. Observed peptide bond cleavage is indicated in the sequence. The corresponding theoretical N-terminal (b-type ion) and C-terminal (y-type ions) ion series for the observed fragment ions is shown above and below the sequence, respectively. Note that the mass error on the precursor ion and all fragment ions is better than 2 ppm. Neutral loss of water from y- and b-type ions is not indicated in the spectrum.
(B) UCH-L1, but not SENP1, can be inhibited by ROS in vitro. Recombinant UCH-L1 and USP2CD (catalytic domain) were labeled by UbVME, whereas recombinant SENP1CD was labeled by SUMO1-VS. Samples were preincubated either with or without H2O2 (1 mM). Reactions were analyzed by Coomassie-stained SDS-PAGE.
(C) AMSH cleavage of K63-linked tetraubiquitin chains is unaffected by ROS. Recombinant USP2CD, AMSH, or AMSH plus STAM1 are subjected to ubiquitin cleavage assay of K63-linked tetraubiquitin chains in the presence or absence of H2O2 (1 mM) preincubation. The DUB reaction was done in the presence or absence of 20 mM DTT. Samples were then analyzed by western blot with anti-ubiquitin antibody.
(D) Model depicting how reversible oxidation of USP1 could modulate PCNA monoubiquitination in the presence of oxidative DNA damage. Left: in the absence of DNA damage, USP1 associated with its catalytic cofactor, UAF1, is in the catalytically active state and can readily deubiquitinate or suppress monoubiquitinated PCNA to prevent aberrant recruitment of error-prone TLS polymerases. Right: in response to oxidative stress or oxidative DNA damage, USP1 becomes transiently inactivated by direct oxidation of its catalytic Cys to the sulfenic acid intermediate. This renders USP1 unable to deubiquitinate PCNA, allowing for monoubiquitinated PCNA to engage with TLS polymerases that are responsible for DNA damage tolerance and/or gap repair. In the absence of oxidative DNA damage-induced checkpoint response, the cells become more reliant on the DNA damage tolerance pathway to ensure that the genome is fully replicated in a timely manner during S phase.
Cell Reports 2012 2, DOI: ( /j.celrep )
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6. Figure S1
Confirmation of Monoubiquitinated PCNA upon Oxidative Stress, Related to Figures 1 and 2
Analysis of PCNA monoubiquitination-defective cells and their sensitivities to oxidative stress. Analysis of PCNA mono ubiquitination-defective cells by western blot. Measurement of recombinant USP7 DUB reactivity to UbVME probe.
(A) U2OS cells were transiently transfected with or without HA-ubiquitin and treated with or without H2O2 (1mM) for 30 min. Cell extracts were lyzed as described in Experimental Procedures and immunoprecipitated with anti-HA antibody. Western blot analysis was performed and probed with anti-PCNA antibody. Asterisks denote nonspecific band.
(B) PCNA K164R-expressing cells are marginally more sensitive to oxidative stress than its wild-type counterpart. U2OS cells were transfected with PCNA siRNA and then subsequently transfected one day later with siRNA-resistant HA-PCNA WT or HA-PCNA K164R expression constructs. Cells were then treated with increasing dose of H2O2 and then stained with the DNA dye Syto-60 to quantify levels of cell viability. Experiment was done in triplicate with error bars depicting standard error. Samples were also collected and analyzed by western blot (probed with anti-PCNA antibody) to show the efficiency of PCNA replacement with the siRNA resistant constructs. Samples analyzed by western blot were treated with H2O2 (0.2 mM).
(C) Recombinant USP7 can be inhibited by a wide range of H2O2 dose. Recombinant full-length His-tagged USP7 is subjected to the DUB activity UbVME probe in the presence or absence of H2O2 at different doses then analyzed by western blot with anti-His antibody.
Cell Reports 2012 2, DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions