OTUB1 stabilizes mismatch repair protein MSH2 by blocking ubiquitination
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100466
ISSN1083-351X
AutoresQiong Wu, Yaping Huang, Liya Gu, Zhijie Chang, Guo‐Min Li,
Tópico(s)DNA Repair Mechanisms
ResumoDNA mismatch repair (MMR) maintains genome stability primarily by correcting replication errors. MMR deficiency can lead to cancer development and bolsters cancer cell resistance to chemotherapy. However, recent studies have shown that checkpoint blockade therapy is effective in MMR-deficient cancers, thus the ability to identify cancer etiology would greatly benefit cancer treatment. MutS homolog 2 (MSH2) is an obligate subunit of mismatch recognition proteins MutSα (MSH2-MSH6) and MutSβ (MSH2-MSH3). Precise regulation of MSH2 is critical, as either over- or underexpression of MSH2 results in an increased mutation frequency. The mechanism by which cells maintain MSH2 proteostasis is unknown. Using functional ubiquitination and deubiquitination assays, we show that the ovarian tumor (OTU) family deubiquitinase ubiquitin aldehyde binding 1 (OTUB1) inhibits MSH2 ubiquitination by blocking the E2 ligase ubiquitin transfer activity. Depleting OTUB1 in cells promotes the ubiquitination and subsequent degradation of MSH2, leading to greater mutation frequency and cellular resistance to genotoxic agents, including the common chemotherapy agents N-methyl-N'-nitro-N-nitrosoguanidine and cisplatin. Taken together, our data identify OTUB1 as an important regulator of MSH2 stability and provide evidence that OTUB1 is a potential biomarker for cancer etiology and therapy. DNA mismatch repair (MMR) maintains genome stability primarily by correcting replication errors. MMR deficiency can lead to cancer development and bolsters cancer cell resistance to chemotherapy. However, recent studies have shown that checkpoint blockade therapy is effective in MMR-deficient cancers, thus the ability to identify cancer etiology would greatly benefit cancer treatment. MutS homolog 2 (MSH2) is an obligate subunit of mismatch recognition proteins MutSα (MSH2-MSH6) and MutSβ (MSH2-MSH3). Precise regulation of MSH2 is critical, as either over- or underexpression of MSH2 results in an increased mutation frequency. The mechanism by which cells maintain MSH2 proteostasis is unknown. Using functional ubiquitination and deubiquitination assays, we show that the ovarian tumor (OTU) family deubiquitinase ubiquitin aldehyde binding 1 (OTUB1) inhibits MSH2 ubiquitination by blocking the E2 ligase ubiquitin transfer activity. Depleting OTUB1 in cells promotes the ubiquitination and subsequent degradation of MSH2, leading to greater mutation frequency and cellular resistance to genotoxic agents, including the common chemotherapy agents N-methyl-N'-nitro-N-nitrosoguanidine and cisplatin. Taken together, our data identify OTUB1 as an important regulator of MSH2 stability and provide evidence that OTUB1 is a potential biomarker for cancer etiology and therapy. DNA mismatch repair (MMR) is a highly conserved DNA repair pathway across species that plays a vital role in maintaining replication fidelity. The typical MMR reaction in eukaryotic cells involves mismatch recognition, mismatch removal, and repair DNA synthesis. Mismatch recognition is carried out by MutSα (MSH2-MSH6) or MutSβ (MSH2-MSH3), which triggers a series of downstream reactions including interacting with proliferating cell nuclear antigen and recruiting MutLα (MLH1-PMS2) and exonuclease 1 (Exo1) to a nearby strand break on the nascent DNA strand. Mismatch removal is conducted by Exo1, which excises the nascent strand from the nick up to and beyond the mismatch to generate a single-strand gap. Finally, DNA polymerase δ fills this gap, and the repair is concluded by nick ligation catalyzed by ligase I (1Li G.M. Mechanisms and functions of DNA mismatch repair.Cell Res. 2008; 18: 85-98Crossref PubMed Scopus (762) Google Scholar). In addition to correcting replication errors, the MMR system also maintains genome stability by processing nonmismatch DNA lesions induced by genotoxic agents (1Li G.M. Mechanisms and functions of DNA mismatch repair.Cell Res. 2008; 18: 85-98Crossref PubMed Scopus (762) Google Scholar, 2Li G.M. The role of mismatch repair in DNA damage-induced apoptosis.Oncol. Res. 1999; 11: 393-400PubMed Google Scholar). In this process, MutSα recognizes these DNA adducts to provoke strand-specific MMR (3Duckett D.R. Drummond J.T. Murchie A.I. Reardon J.T. Sancar A. Lilley D.M. Modrich P. Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6443-6447Crossref PubMed Scopus (368) Google Scholar, 4Li G.-M. Wang H. Romano L.J. Human MutSa specifically binds to DNA containing aminofluorene and acetylaminofluorene adducts.J. Biol. 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Cell. 2014; 55: 31-46Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) lead to a hypermutable phenotype and the development of various types of human cancer. Conversely, overexpression of key MMR factors, such as MSH2 and MLH1, results in an increased mutation frequency (17Shcherbakova P.V. Kunkel T.A. Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations.Mol. Cell. Biol. 1999; 19: 3177-3183Crossref PubMed Scopus (152) Google Scholar) or apoptotic cell death (18Zhang H. Richards B. Wilson T. Lloyd M. Cranston A. Thorburn A. Fishel R. Meuth M. Apoptosis induced by overexpression of hMSH2 or hMLH1.Cancer Res. 1999; 59: 3021-3027PubMed Google Scholar). Therefore, the precise regulation of the homeostasis of cellular MMR proteins is critical for maintaining genome stability. The ubiquitination-proteosome system (UPS) regulates the protein proteostasis of a wide range of substrates under normal or stress conditions in human cells in a posttranslational manner. Previous studies have shown that the stability of MSH2 is regulated by HDAC6-mediated ubiquitination (16Zhang M. Xiang S. Joo H.Y. Wang L. Williams K.A. Liu W. Hu C. Tong D. Haakenson J. Wang C. Zhang S. Pavlovicz R.E. Jones A. Schmidt K.H. Tang J. et al.HDAC6 deacetylates and ubiquitinates MSH2 to maintain proper levels of MutSalpha.Mol. Cell. 2014; 55: 31-46Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and USP10-mediated deubiquitination (19Zhang M. Hu C. Tong D. Xiang S. Williams K. Bai W. Li G.M. Bepler G. Zhang X. Ubiquitin-specific peptidase 10 (USP10) deubiquitinates and stabilizes MutS homolog 2 (MSH2) to regulate cellular sensitivity to DNA damage.J. Biol. Chem. 2016; 291: 10783-10791Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). It has been noted that the rate of UPS-mediated MutSα degradation varies across different cell lines (20Hernandez-Pigeon H. Laurent G. Humbert O. Salles B. Lautier D. Degadration of mismatch repair hMutSalpha heterodimer by the ubiquitin-proteasome pathway.FEBS Lett. 2004; 562: 40-44Crossref PubMed Scopus (21) Google Scholar), and multiple motifs that can be ubiquitinated are present in MSH2, which implies that other ubiquitinases/deubiquitinases might also regulate MSH2 proteostasis. A recent mass spectrometry study of MMR protein interactomes revealed a potential interaction between MSH2 and OTUB1 (21Chen Z. Tran M. Tang M. Wang W. Gong Z. Chen J. Proteomic analysis reveals a novel mutator S (MutS) partner involved in mismatch repair pathway.Mol. Cell. Proteomics. 2016; 15: 1299-1308Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), a deubiquitilating enzyme that belongs to the ovarian tumor (OTU) superfamily of predicted cysteine proteases (22Balakirev M.Y. Tcherniuk S.O. Jaquinod M. Chroboczek J. Otubains: A new family of cysteine proteases in the ubiquitin pathway.EMBO Rep. 2003; 4: 517-522Crossref PubMed Scopus (202) Google Scholar, 23Saldana M. VanderVorst K. Berg A.L. Lee H. Carraway K.L. Otubain 1: A non-canonical deubiquitinase with an emerging role in cancer.Endocr. Relat. Cancer. 2019; 26: R1-R14Crossref PubMed Scopus (27) Google Scholar). This suggests that MSH2 could be a substrate of OTUB1. OTUB1 is a highly specific ubiquitin iso-peptidase that functions in two different ways. In the canonical way, OTUB1 acts as a cysteine protease to directly deubiquitinate its substrates (22Balakirev M.Y. Tcherniuk S.O. Jaquinod M. Chroboczek J. Otubains: A new family of cysteine proteases in the ubiquitin pathway.EMBO Rep. 2003; 4: 517-522Crossref PubMed Scopus (202) Google Scholar). OTUB1 also works in a noncanonical way by interacting with E2 enzymes to inhibit the ubiquitin transfer to substrates that are involved in diverse biological pathways (24Juang Y.C. Landry M.C. Sanches M. Vittal V. Leung C.C. Ceccarelli D.F. Mateo A.R. Pruneda J.N. Mao D.Y. Szilard R.K. Orlicky S. Munro M. Brzovic P.S. Klevit R.E. Sicheri F. et al.OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function.Mol. Cell. 2012; 45: 384-397Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 25Nakada S. Tai I. Panier S. Al-Hakim A. Iemura S. Juang Y.C. O'Donnell L. Kumakubo A. Munro M. Sicheri F. Gingras A.C. Natsume T. Suda T. Durocher D. Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1.Nature. 2010; 466: 941-946Crossref PubMed Scopus (251) Google Scholar). Here, we demonstrate that OTUB1 interacts with MSH2 to prevent MSH2 from ubiquitination through the noncanonical way. Depleting OTUB1 impairs the cellular proteostasis of MutSα and leads to MMR deficiency–associated resistance to genotoxic agents. Therefore, this study has established OTUB1 as a novel regulator of MMR and DNA damage response. To determine whether OTUB1 interacts with MSH2, we expressed Flag-tagged MSH2 (Flag-MSH2) and HA-tagged OTUB1 (HA-OTUB1) in HEK293T cells and performed reciprocal pulldown experiments using Flag agarose and HA magnetic beads. We found that Flag-MSH2 and HA-OTUB1 pulled each other down (Fig. 1A), which suggests a specific interaction between MSH2 and OTUB1. We obtained similar results in a co-immunoprecipitation experiment to determine endogenous interactions between these two proteins by using an MSH2-specific or an OTUB1 antibody (Fig. 1B). Although the interaction between MSH2 and OTUB1 is relatively weak, it is much more specific than the control reactions in both the pulldown and the co-immunoprecipitation assays. OTUB1 mainly contains three domains (Fig. 1C): the Ub-binding domain (1–47aa), the linker domain (48–85aa), and the OTU domain (85–271aa) (26Messick T.E. Russell N.S. Iwata A.J. Sarachan K.L. Shiekhattar R. Shanks J.R. Reyes-Turcu F.E. Wilkinson K.D. Marmorstein R. Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein.J. Biol. Chem. 2008; 283: 11038-11049Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). To determine which domain interacts with MSH2, we constructed GST-tagged full-length OTUB1 (FL) and truncated or residue-altered OTUB1s—OTUB1(1–47), OTUB1(48–271), OTUB1(48–159), OTUB1(159–271), OTUB1-C91A, and OTUB1-D88A (Fig. 1C)—and detected the interactions between these OTUB1 polypeptides and MSH2. GST pull-down assays showed that the FL OTUB1 strongly interacted with MSH2 (Fig. 1D). We also observed strong interactions between MSH2 and OTUB1(48–271) and OTUB1(48–159), but not between MSH2 and OTUB1(1–47) or OTUB1(159–271) (Fig. 1D). The deubiquitylation function of OTUB1 relies on the catalytic center containing amino acids C91, D88, and H265 (26Messick T.E. Russell N.S. Iwata A.J. Sarachan K.L. Shiekhattar R. Shanks J.R. Reyes-Turcu F.E. Wilkinson K.D. Marmorstein R. Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein.J. Biol. Chem. 2008; 283: 11038-11049Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), which could form a catalytic triad. To determine the role of this activity center in MSH2 deubiquitination, we measured MSH2's interactions with the catalytic-dead mutants OTUB1-C91A and OTUB1-D88A. We found that both the C91A and D88A mutants could interact with MSH2 (Fig. 1D). Taken together, these results indicate that OTUB1 physically interacts with MSH2 via its middle C1 domain, including its catalytic center. To determine which MSH2 domain interacts with OTUB1, we first generated GST-tagged full-length MSH2 and three GST-tagged MSH2 mutants: MSH2 (1–378), MSH2 (200–700), and MSH2 (624–934) (Fig. 1E), as described previously (19Zhang M. Hu C. Tong D. Xiang S. Williams K. Bai W. Li G.M. Bepler G. Zhang X. Ubiquitin-specific peptidase 10 (USP10) deubiquitinates and stabilizes MutS homolog 2 (MSH2) to regulate cellular sensitivity to DNA damage.J. Biol. Chem. 2016; 291: 10783-10791Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). We used these GST-tagged proteins to pull down HA-OTUB1 in HeLa cell lysates. As expected, FL-MSH2 could pull down HA-OTUB1 (Fig. 1F). Among the three mutants, MSH2 (200–700) and MSH2 (624–934) could precipitate OTUB1, which suggests that the ubiquitylated residues in MSH2 that are regulated by OTUB1 are located in the central and C-terminal domains. To narrow down the OTUB1 interaction domain(s), we further divided MSH2 fragments S2 and S3 into several smaller peptides (Fig. 1E, S4-S10) and performed the same pull-down assay. The results showed that all peptides except S10, which failed to pull down OTUB1, more or less interacted with OTUB1 (Fig. 1F). Therefore, we conclude that OTUB1 interacts with MSH2 mostly through the central domain. To determine whether the OTUB1–MSH2 interaction regulates the stability of MSH2, we measured Flag-MSH2 levels in HEK293T cells with or without the overexpression of HA-OTUB1. We found that the Flag-MSH2 level was much higher in cells expressing HA-OTUB1 than in those without HA-OTUB1 expression (Fig. 2, A and B), which suggests that OTUB1 does indeed stabilize MSH2. To further explore this, we measured the half-life of MSH2 by treating cells with protein synthesis inhibitor cycloheximide (CHX). Consistently, the half-life of Flag-MSH2 was significantly higher in cells overexpressing OTUB1 (Fig. 2, C and D). Interestingly, the catalytic-dead mutant OTUB1 (C91A) also extended the half-life of Flag-MSH2; however, we did not observe MSH2 stabilization in cells expressing the OTUB1 (D88A) mutant (Fig. 2, C and D). Because the catalytic Cys91 and Asp88 are essential for the OTUB1 deubiquitination activity (27Zhao L. Wang X. Yu Y. Deng L. Chen L. Peng X. Jiao C. Gao G. Tan X. Pan W. Ge X. Wang P. OTUB1 protein suppresses mTOR complex 1 (mTORC1) activity by deubiquitinating the mTORC1 inhibitor DEPTOR.J. Biol. Chem. 2018; 293: 4883-4892Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and OTUB1's inhibition of E2 ligases (24Juang Y.C. Landry M.C. Sanches M. Vittal V. Leung C.C. Ceccarelli D.F. Mateo A.R. Pruneda J.N. Mao D.Y. Szilard R.K. Orlicky S. Munro M. Brzovic P.S. Klevit R.E. Sicheri F. et al.OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function.Mol. Cell. 2012; 45: 384-397Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 25Nakada S. Tai I. Panier S. Al-Hakim A. Iemura S. Juang Y.C. O'Donnell L. Kumakubo A. Munro M. Sicheri F. Gingras A.C. Natsume T. Suda T. Durocher D. Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1.Nature. 2010; 466: 941-946Crossref PubMed Scopus (251) Google Scholar), respectively, these results suggest that OTUB1 stabilizes MSH2 via noncanonical inhibition of E2 ligase activity. To verify this, we performed similar half-life analysis using a known OTUB1 noncanonical substrate, DEPTOR (27Zhao L. Wang X. Yu Y. Deng L. Chen L. Peng X. Jiao C. Gao G. Tan X. Pan W. Ge X. Wang P. OTUB1 protein suppresses mTOR complex 1 (mTORC1) activity by deubiquitinating the mTORC1 inhibitor DEPTOR.J. Biol. Chem. 2018; 293: 4883-4892Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The stability of Flag-DEPTOR was essentially the same as that of Flag-MSH2, i.e., OTUB1 (C91A), but not OTUB1(D88A), could stabilize DEPTOR (Fig. 2E). To further test whether OTUB1 is essential for maintaining MSH2 stability, we stably knocked down (KD) OTUB1 by two different small hairpin RNAs (shRNAs) in both HeLa and SW620 cells. We treated these cells with cycloheximide to inhibit the endogenous protein synthesis over a specific course of time and quantified the relative expression levels of MSH2 by western blot. The results showed that MSH2, as well as its partner MSH6, degraded much faster in OTUB1-KD cells than in control cells (Fig. 2, F and G). To rule out the off-target effects of shRNAs, we restored the expression of OTUB1 in OTUB1-KD cells and found that OTUB1 restoration indeed efficiently increased the half-life of both MSH2 and MSH6 in these cells (Fig. 2H). The observed stability correlation between MSH6 and MSH2 is consistent with the fact that MSH6 is unstable in the absence of MSH2 (28Chang D.K. Ricciardiello L. Goel A. Chang C.L. Boland C.R. Steady-state regulation of the human DNA mismatch repair system.J. Biol. Chem. 2000; 275: 29178Abstract Full Text Full Text PDF PubMed Google Scholar, 29de Wind N. Dekker M. Claij N. Jansen L. van Klink Y. Radman M. Riggins G. van der Valk M. van't Wout K. te Riele H. HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.Nat. Genet. 1999; 23: 359-362Crossref PubMed Scopus (175) Google Scholar, 30Marra G. Iaccarino I. Lettieri T. Roscilli G. Delmastro P. Jiricny J. Mismatch repair deficiency associated with overexpression of the MSH3 gene.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8568-8573Crossref PubMed Scopus (172) Google Scholar). However, whether MSH6's stability is regulated independently by OTUB1 remains to be investigated. Nevertheless, the data presented here indicate that OTUB1 stabilizes MSH2 probably by inhibiting E2 activity. To test our hypothesis that OTUB1 stabilizes MSH2 by preventing its ubiquitination, we measured the ubiquitination levels of MSH2 in HeLa cells treated with MG132 and stably transfected with shRNA against OTUB1 or scrambled shRNA in the presence of HA-Ub. Under these conditions, ubiquitinated proteins are protected from degradation via the proteasome. Even though a similar amount of ubiquitinated proteins was present in all cells, significantly more ubiquitinated MSH2 was pulled down in OTUB1-KD cells (OTUB1-sh1 and OTUB1-sh2) than in cells with scrambled shRNA (Fig. 3A). Consistently, restoring OTUB1 to OTUB1-KD cells significantly reduced the ubiquitination level of MSH2 (Fig. 3B), as compared with cells transfected with an empty vector (V). Collectively, these results suggest that MSH2 is a substrate of OTUB1 and that OTUB1 inhibits MSH2 ubiquitination. To further determine which OTUB1 function is involved in protecting MSH2 from being ubiquitinated, we performed in vitro assays to examine the individual OTUB1 proteins (WT, C91A, and D88A) for their ability either to directly deubiquitinate MSH2 or to inhibit MSH2 ubiquitination by E2 enzymes. The deubiquitination-specific substrate FOXM1 (31Karunarathna U. Kongsema M. Zona S. Gong C. Cabrera E. Gomes A.R. Man E.P.S. Khongkow P. Tsang J.W.H. Khoo U.S. Medema R.H. Freire R. Lam E.W.F. OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance.Oncogene. 2016; 35: 1433-1444Crossref PubMed Scopus (71) Google Scholar) and the noncanonical substrate DEPTOR were used as positive controls in these assays. In the in vitro ubiquitination assay, individual Flag-tagged substrates (MSH2, FOXM1, and DEPTOR) were incubated with GST-OTUB1 (WT, C91A, or D88A) and HA-Ub in a ubiquitination system containing rabbit reticulocyte lysate (RRL), as described previously (19Zhang M. Hu C. Tong D. Xiang S. Williams K. Bai W. Li G.M. Bepler G. Zhang X. Ubiquitin-specific peptidase 10 (USP10) deubiquitinates and stabilizes MutS homolog 2 (MSH2) to regulate cellular sensitivity to DNA damage.J. Biol. Chem. 2016; 291: 10783-10791Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and ubiquitinated substrates were visualized by western blotting using a Ub antibody. As shown in Figure 3C (in vitro Ub), substrates MSH2 (lanes 1–4) and DEPTOR (lanes 9–12) exhibited much less ubiquitination in reactions containing WT OTUB1 (lanes 2 and 10) and OTUB1-C91A (lanes 3 and 11) than those containing OTUB1-D88A (lanes 4 and 12), but we observed little difference in ubiquitination among various OTUB1 proteins when the deubiquitination-specific substrate FOXM1 was used in the same assay (lanes 5–8, in vitro Ub). Conversely, the in vitro deubiquitination assays revealed that both OTUB1(WT) and OTUB1(D88A), but not OTUB1(C91A), could efficiently deubiquitinate FOXM1 (Fig. 3C, in vitro Deub, lanes 5–8), as expected. However, there was essentially no difference in ubiquitination removal among all reactions when MSH2 (lanes 1–4) and DEPTOR (lanes 9–12) were used in the deubiquitination assays; in other words, OTUB1 fails to remove established ubiquitination on MSH2 and noncanonical substrate DEOTOR. These results strongly suggest that OTUB1 blocks MSH2 ubiquitination by inhibiting the E2 ligase activity, rather than by directly deubiquitinating MSH2. To further verify the in vitro ubiquitination and deubiquitination results, we expressed HA-Ub, His-MSH2, and three forms of Flag-OTUB1s (WT, C91A, and D88A) in HEK293T cells and performed His-tag pull-down assays, and we measured the ubiquitination levels of His-MSH2. We found less ubiquitinated MSH2 in HEK293T cells expressing both WT OTUB1 and OTUB1-C91A, but not OTUB1-D88A (Fig. 3D). These results agree with those depicted in Figure 3C. We therefore conclude that OTUB1 prevents MSH2 ubiquitination by suppressing the E2 ligase activity. MSH2 is an obligate subunit of both mismatch recognition proteins, MutSα and MutSβ, and depleting MSH2 leads to a mutator phenotype characterized by an elevated mutation frequency (1Li G.M. Mechanisms and functions of DNA mismatch repair.Cell Res. 2008; 18: 85-98Crossref PubMed Scopus (762) Google Scholar, 32Modrich P. Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology.Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1295) Google Scholar). Since OTUB1 regulates MSH2 stability, we postulated that depleting OTUB1 would cause a mutator phenotype. To test this hypothesis, we performed HPRT assays to determine the spontaneous mutation frequency in WT and OTUB1-KD cells, as described previously (14Li F. Mao G. Tong D. Huang J. Gu L. Yang W. Li G.M. The histone mark H3K36me3 regulates human DNA mismatch repair through its interaction with MutSalpha.Cell. 2013; 153: 590-600Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). OTUB1 knockdown resulted in a 4- to 20-fold increase in mutation frequency in HeLa and SW620 cells, and restoring OTUB1 in OTUB1-KD cells rescued the mutator phenotype in both HeLa OTUB1-KD and SW620 OTUB1-KD cells (Table 1). Therefore, OTUB1's regulation of MSH2 is critical to MMR and genome maintenance.Table 1Depletion of OTUB1 induces a mutator phenotypeCell lineMutation frequency (×107)Fold of increase in MFp valueHeLaScramble1.67 ± 0.021OTUB1-sh16.83 ± 0.074.1<0.01OTUB1-sh1+Vector16.5 ± 0.111.6 0.05SW620Scramble0.17 ± 0.081OTUB1-sh24.6 ± 0.325.98<0.01OTUB1-sh2+Vector3.63 ± 0.1320.47 0.05 Open table in a new tab MMR proteins, particularly MutSα, also play important roles in DNA damage signaling, which leads to apoptotic cell death (2Li G.M. The role of mismatch repair in DNA damage-induced apoptosis.Oncol. Res. 1999; 11: 393-400PubMed Google Scholar). Thus, defects in MSH2 render cells tolerant to many genotoxic agents, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and cisplatin (1Li G.M. Mechanisms and functions of DNA mismatch repair.Cell Res. 2008; 18: 85-98Crossref PubMed Scopus (762) Google Scholar, 2Li G.M. The role of mismatch repair in DNA damage-induced apoptosis.Oncol. Res. 1999; 11: 393-400PubMed Google Scholar). To test whether OTUB1 deficiency compromises MMR's role in DNA damage signaling and promotes drug tolerance, we treated control and OTUB1-KD SW620 cells with MNNG and cisplatin and measured their survival after treatment. We found that OTUB1-KD cell lines, whether OTUB1-shRNA1 or OTUB1-shRNA2, were more tolerant of cisplatin or MNNG treatment than control SW620 cells (Fig. 4A). To rule out potential off-target effects in the knockdown cells, we performed the survival assay in OTUB1-restored SW620-OTUB1-KD cells. The results showed that restoring OTUB1 expression to SW620-OTUB1-KD also restored drug sensitivity (Fig. 4B), which confirms that OTUB1 is involved in DNA damage–induced response, likely by stabilizing cellular MSH2. To determine whether SW620 cells undergo apoptotic cell death in response to treatment, we measured the cleavage of poly(ADP-ribose) polymerase 1 (PARP1), a hallmark of apoptosis (33Kaufmann S.H. Desnoyers S. Ottaviano Y. Davidson N.E. Poirier G.G. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: An early marker of chemotherapy-induced apoptosis.Cancer Res. 1993; 53: 3976-3985PubMed Google Scholar, 34Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase.Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2236) Google Scholar). The levels of cleaved PARP1 increased in a time-dependent manner in both control and OTUB1-KD SW620 cells treated with cisplatin (Fig. 4C). We quantified the PARP1 cleavage as previously described (35Castri P. Lee Y.J. Ponzio T. Maric D. Spatz M. Bembry J. Hallenbeck J. Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kappaB-dependent signaling.Biochim. Biophys. Acta. 2014; 1843: 640-651Crossref PubMed Scopus (30) Google Scholar), and the results showed that the cleaved PARP1 level in OTUB1-KD cells was lower than in control cells (Fig. 4D). We performed the same analysis in OTUB1-rescued OTUB1-KD cells and found that restoring the express
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