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SET7 methylates the deubiquitinase OTUB1 at Lys 122 to impair its binding to E2 enzyme UBC13 and relieve its suppressive role on ferroptosis

2023; Elsevier BV; Volume: 299; Issue: 4 Linguagem: Inglês

10.1016/j.jbc.2023.103054

ISSN

1083-351X

Autores

Hongyan Deng, Shuke Jia, Jinhua Tang, Fangjing Rong, Chenxi Xu, Xiaoyun Chen, Zixuan Wang, Chunchun Zhu, Xueyi Sun, Qian Liao, Wen Liu, Wenhua Li, Wuhan Xiao, Xing Liu,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

The deubiquitinating enzyme OTUB1 possesses canonical deubiquitinase (DUB) activity and noncanonical, catalytic-independent activity, which has been identified as an essential regulator of diverse physiological processes. Posttranslational modifications of OTUB1 affect both its DUB activity and its noncanonical activity of binding to the E2 ubiquitin-conjugation enzyme UBC13, but further investigation is needed to characterize the full inventory of modifications to OTUB1. Here, we demonstrate that SET7, a lysine monomethylase, directly interacts with OTUB1 to catalyze OTUB1 methylation at lysine 122. This modification does not affect DUB activity of OTUB1 but impairs its noncanonical activity, binding to UBC13. Moreover, we found using cell viability analysis and intracellular reactive oxygen species assay that SET7-mediated methylation of OTUB1 relieves its suppressive role on ferroptosis. Notably, the methylation-mimic mutant of OTUB1 not only loses the ability to bind to UBC13 but also relieves its suppressive role on Tert-Butyl hydroperoxide–induced cell death and Cystine starvation/Erastin–induced cellular reactive oxygen species. Collectively, our data identify a novel modification of OTUB1 that is critical for inhibiting its noncanonical activity. The deubiquitinating enzyme OTUB1 possesses canonical deubiquitinase (DUB) activity and noncanonical, catalytic-independent activity, which has been identified as an essential regulator of diverse physiological processes. Posttranslational modifications of OTUB1 affect both its DUB activity and its noncanonical activity of binding to the E2 ubiquitin-conjugation enzyme UBC13, but further investigation is needed to characterize the full inventory of modifications to OTUB1. Here, we demonstrate that SET7, a lysine monomethylase, directly interacts with OTUB1 to catalyze OTUB1 methylation at lysine 122. This modification does not affect DUB activity of OTUB1 but impairs its noncanonical activity, binding to UBC13. Moreover, we found using cell viability analysis and intracellular reactive oxygen species assay that SET7-mediated methylation of OTUB1 relieves its suppressive role on ferroptosis. Notably, the methylation-mimic mutant of OTUB1 not only loses the ability to bind to UBC13 but also relieves its suppressive role on Tert-Butyl hydroperoxide–induced cell death and Cystine starvation/Erastin–induced cellular reactive oxygen species. Collectively, our data identify a novel modification of OTUB1 that is critical for inhibiting its noncanonical activity. Ovarian tumor domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) is a member of OUT family of deubiquitylating enzymes (DUBs) (1Liao Y. Yang M. Wang K. Wang Y. Zhong B. Jiang N. Deubiquitinating enzyme OTUB1 in immunity and cancer: good player or bad actor?.Cancer Lett. 2022; 526: 248-258Crossref PubMed Scopus (15) Google Scholar). As the funding member of the OUT family, OTUB1 has been identified as an essential regulator of diverse physiological processes through deubiquitylating K48-linked ubiquitin chains (1Liao Y. Yang M. Wang K. Wang Y. Zhong B. Jiang N. Deubiquitinating enzyme OTUB1 in immunity and cancer: good player or bad actor?.Cancer Lett. 2022; 526: 248-258Crossref PubMed Scopus (15) Google Scholar, 2Wang T. Yin L. Cooper E.M. Lai M.Y. Dickey S. Pickart C.M. et al.Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1.J. Mol. Biol. 2009; 386: 1011-1023Crossref PubMed Scopus (113) Google Scholar, 3Mevissen T.E. Hospenthal M.K. Geurink P.P. Elliott P.R. Akutsu M. Arnaudo N. et al.OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis.Cell. 2013; 154: 169-184Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 4Wiener R. Zhang X. Wang T. Wolberger C. The mechanism of OTUB1-mediated inhibition of ubiquitination.Nature. 2012; 483: 618-622Crossref PubMed Scopus (186) Google Scholar, 5Juang Y.C. Landry M.C. Sanches M. Vittal V. Leung C.C. Ceccarelli D.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 (146) Google Scholar, 6Sato Y. Yamagata A. Goto-Ito S. Kubota K. Miyamoto R. Nakada S. et al.Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13.J. Biol. Chem. 2012; 287: 25860-25868Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To date, OTUB1 has been shown to catalyze deubiquitination of multiple targets including p100, ERα, UBE2E1, Snail, DEPTOR, YB-1, SMAD2/3, c-IAP1, p53, AKT, SOCS1, UBC13, PD-L1, Cyclin E1, MSH2, SLC7A11, TRAF3, TRAF6, and Nur77, resulting in positively or negatively modulating these targets' function (4Wiener R. Zhang X. Wang T. Wolberger C. The mechanism of OTUB1-mediated inhibition of ubiquitination.Nature. 2012; 483: 618-622Crossref PubMed Scopus (186) Google Scholar, 7Li Y. Yang J.Y. Xie X. Jie Z. Zhang L. Shi J. et al.Preventing abnormal NF-kappaB activation and autoimmunity by Otub1-mediated p100 stabilization.Cell Res. 2019; 29: 474-485Crossref PubMed Scopus (27) Google Scholar, 8Pasupala N. Morrow M.E. Que L.T. Malynn B.A. Ma A. Wolberger C. OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination.J. Biol. Chem. 2018; 293: 18285-18295Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 9Zhou H. Liu Y. Zhu R. Ding F. Cao X. Lin D. et al.OTUB1 promotes esophageal squamous cell carcinoma metastasis through modulating Snail stability.Oncogene. 2018; 37: 3356-3368Crossref PubMed Scopus (64) Google Scholar, 10Zhao L. Wang X. Yu Y. Deng L. Chen L. Peng X. et al.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 (42) Google Scholar, 11Dong W. Wang H. Shahzad K. Bock F. Al-Dabet M.M. Ranjan S. et al.Activated protein C Ameliorates renal ischemia-reperfusion injury by restricting Y-box binding protein-1 ubiquitination.J. Am. Soc. Nephrol. 2015; 26: 2789-2799Crossref PubMed Scopus (62) Google Scholar, 12Herhaus L. Al-Salihi M. Macartney T. Weidlich S. Sapkota G.P. OTUB1 enhances TGFbeta signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3.Nat. Commun. 2013; 4: 2519Crossref PubMed Scopus (96) Google Scholar, 13Goncharov T. Niessen K. de Almagro M.C. Izrael-Tomasevic A. Fedorova A.V. Varfolomeev E. et al.OTUB1 modulates c-IAP1 stability to regulate signalling pathways.EMBO J. 2013; 32: 1103-1114Crossref PubMed Scopus (89) Google Scholar, 14Sun X.X. Dai M.S. Deubiquitinating enzyme regulation of the p53 pathway: a lesson from Otub1.World J. Biol. Chem. 2014; 5: 75-84PubMed Google Scholar, 15Zhou X. Yu J. Cheng X. Zhao B. Manyam G.C. Zhang L. et al.The deubiquitinase Otub1 controls the activation of CD8(+) T cells and NK cells by regulating IL-15-mediated priming.Nat. Immunol. 2019; 20: 879-889Crossref PubMed Scopus (54) Google Scholar, 16Wang X. Mulas F. Yi W. Brunn A. Nishanth G. Just S. et al.OTUB1 inhibits CNS autoimmunity by preventing IFN-gamma-induced hyperactivation of astrocytes.EMBO J. 2019; 38e100947Crossref Scopus (25) Google Scholar, 17Nakada S. Tai I. Panier S. Al-Hakim A. Iemura S. Juang Y.C. et al.Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1.Nature. 2010; 466: 941-946Crossref PubMed Scopus (282) Google Scholar, 18Mulas F. Wang X. Song S. Nishanth G. Yi W. Brunn A. et al.The deubiquitinase OTUB1 augments NF-kappaB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13.Cell Mol. Immunol. 2021; 18: 1512-1527Crossref PubMed Scopus (31) Google Scholar, 19Zhu D. Xu R. Huang X. Tang Z. Tian Y. Zhang J. et al.Deubiquitinating enzyme OTUB1 promotes cancer cell immunosuppression via preventing ER-associated degradation of immune checkpoint protein PD-L1.Cell Death Differ. 2021; 28: 1773-1789Crossref PubMed Scopus (65) Google Scholar, 20Liao Y. Wu N. Wang K. Wang M. Wang Y. Gao J. et al.OTUB1 promotes progression and proliferation of prostate cancer via deubiquitinating and stabling Cyclin E1.Front. Cell Dev. Biol. 2020; 8617758Google Scholar, 21Wu Q. Huang Y. Gu L. Chang Z. Li G.M. OTUB1 stabilizes mismatch repair protein MSH2 by blocking ubiquitination.J. Biol. Chem. 2021; 296100466Abstract Full Text Full Text PDF Scopus (12) Google Scholar, 22Liu T. Jiang L. Tavana O. Gu W. The deubiquitylase OTUB1 mediates ferroptosis via stabilization of SLC7A11.Cancer Res. 2019; 79: 1913-1924Crossref PubMed Scopus (228) Google Scholar, 23Xie M. Yin Y. Chen L. Yin A. Liu Y. Liu Y. et al.Scavenger receptor A impairs interferon response to HBV infection by limiting TRAF3 ubiquitination through recruiting OTUB1.FEBS J. 2020; 287: 310-324Crossref PubMed Scopus (19) Google Scholar, 24Pei H.Z. Huang B. Chang H.W. Baek S.H. Ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 inhibits inflammation by regulating Nur77 stability.Cell Signal. 2019; 59: 85-95Crossref PubMed Scopus (7) Google Scholar, 25Li S. Zheng H. Mao A.P. Zhong B. Li Y. Liu Y. et al.Regulation of virus-triggered signaling by OTUB1- and OTUB2-mediated deubiquitination of TRAF3 and TRAF6.J. Biol. Chem. 2010; 285: 4291-4297Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 26Stanisic V. Malovannaya A. Qin J. Lonard D.M. O'Malley B.W. OTU Domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) deubiquitinates estrogen receptor (ER) alpha and affects ERalpha transcriptional activity.J. Biol. Chem. 2009; 284: 16135-16145Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In addition to canonical DUB activity, the noncanonical, catalytic-independent activity of OTUB1 has been discovered (1Liao Y. Yang M. Wang K. Wang Y. Zhong B. Jiang N. Deubiquitinating enzyme OTUB1 in immunity and cancer: good player or bad actor?.Cancer Lett. 2022; 526: 248-258Crossref PubMed Scopus (15) Google Scholar, 27Saldana 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 (51) Google Scholar). OTUB1 inhibits the ubiquitination of target proteins by binding to and inhibiting the E2 ubiquitin-conjugation enzymes instead of directly removing K48-linked polyubiquitin chains on target proteins (4Wiener R. Zhang X. Wang T. Wolberger C. The mechanism of OTUB1-mediated inhibition of ubiquitination.Nature. 2012; 483: 618-622Crossref PubMed Scopus (186) Google Scholar, 5Juang Y.C. Landry M.C. Sanches M. Vittal V. Leung C.C. Ceccarelli D.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 (146) Google Scholar, 6Sato Y. Yamagata A. Goto-Ito S. Kubota K. Miyamoto R. Nakada S. et al.Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13.J. Biol. Chem. 2012; 287: 25860-25868Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 17Nakada S. Tai I. Panier S. Al-Hakim A. Iemura S. Juang Y.C. et al.Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1.Nature. 2010; 466: 941-946Crossref PubMed Scopus (282) Google Scholar). OTUB1 also inhibits DNA damage repair and promotes transforming growth factor-β signaling pathway independently of its DUB activity (12Herhaus L. Al-Salihi M. Macartney T. Weidlich S. Sapkota G.P. OTUB1 enhances TGFbeta signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3.Nat. Commun. 2013; 4: 2519Crossref PubMed Scopus (96) Google Scholar, 17Nakada S. Tai I. Panier S. Al-Hakim A. Iemura S. Juang Y.C. et al.Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1.Nature. 2010; 466: 941-946Crossref PubMed Scopus (282) Google Scholar). Recently, it is reported that the DUB activity is not required for OTUB1 to enhance SLC7A11 stabilization and inhibit ferroptosis (22Liu T. Jiang L. Tavana O. Gu W. The deubiquitylase OTUB1 mediates ferroptosis via stabilization of SLC7A11.Cancer Res. 2019; 79: 1913-1924Crossref PubMed Scopus (228) Google Scholar). The posttranslational modifications (PTMs) have been widely recognized for affecting target protein activity and function (28Bradley D. The evolution of post-translational modifications.Curr. Opin. Genet. Dev. 2022; 76101956Crossref PubMed Scopus (14) Google Scholar). Given that OTUB1 possesses deubiquitinase activity and noncanonical function activity, PTMs in affecting OTUB1 function at these two aspects have been identified (27Saldana 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 (51) Google Scholar). Factor inhibiting HIF regulates OTUB1 deubiquitinase activity by catalyzing OTUB1 hydroxylation in an oxygen-dependent manner (29Scholz C.C. Rodriguez J. Pickel C. Burr S. Fabrizio J.A. Nolan K.A. et al.FIH regulates cellular metabolism through hydroxylation of the deubiquitinase OTUB1.PLoS Biol. 2016; 14e1002347Crossref PubMed Scopus (69) Google Scholar, 30Pickel C. Gunter J. Ruiz-Serrano A. Spielmann P. Fabrizio J.A. Wolski W. et al.Oxygen-dependent bond formation with FIH regulates the activity of the client protein OTUB1.Redox Biol. 2019; 26101265Crossref PubMed Scopus (14) Google Scholar). The ubiquitin-like modifier FAT10 stimulates OTUB1 deubiquitinase activity in both covalent modification and a noncovalent manner (31Bialas J. Boehm A.N. Catone N. Aichem A. Groettrup M. The ubiquitin-like modifier FAT10 stimulates the activity of deubiquitylating enzyme OTUB1.J. Biol. Chem. 2019; 294: 4315-4330Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Meanwhile, PTMs in affecting the nonenzymatic activity OTUB1 have also been reported. UBCH5-mediated monoubiquitination of OTUB1 is required for its noncanonical regulation of p53 (32Li Y. Sun X.X. Elferich J. Shinde U. David L.L. Dai M.S. Monoubiquitination is critical for ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 (Otub1) to suppress UbcH5 enzyme and stabilize p53 protein.J. Biol. Chem. 2014; 289: 5097-5108Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Casein kinase 2 triggers OTUB1 nuclear localization by phosphorylating OTUB1 at Ser16 (33Herhaus L. Perez-Oliva A.B. Cozza G. Gourlay R. Weidlich S. Campbell D.G. et al.Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization.Sci. Signal. 2015; 8: ra35Crossref PubMed Scopus (44) Google Scholar). S-Nitrosylation of OTUB1 alters its stability and UBC13 binding (34Kumari R. Kumar R. Dey A.K. Saha S. Maiti T.K. S-nitrosylation of OTUB1 alters its stability and Ubc13 binding.ACS Chem. Neurosci. 2022; 13: 1517-1525Crossref PubMed Scopus (4) Google Scholar). Further investigating PTMs of OTUB1 will shed new lights on the molecular regulation of canonical and noncanonical activity of OTUB1. SET7 (also known as SETD7, SET9, and SET7/9) is a monomethylase, which can methylate histone and nonhistone proteins, resulting in either negatively or positively modulating their target proteins' function (35Nishioka K. Chuikov S. Sarma K. Erdjument-Bromage H. Allis C.D. Tempst P. et al.Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation.Genes Dev. 2002; 16: 479-489Crossref PubMed Scopus (474) Google Scholar, 36Wang H. Cao R. Xia L. Erdjument-Bromage H. Borchers C. Tempst P. et al.Purification and functional characterization of a histone H3-lysine 4-specific methyltransferase.Mol. Cell. 2001; 8: 1207-1217Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 37Chuikov S. Kurash J.K. Wilson J.R. Xiao B. Justin N. Ivanov G.S. et al.Regulation of p53 activity through lysine methylation.Nature. 2004; 432: 353-360Crossref PubMed Scopus (664) Google Scholar, 38Kouskouti A. Scheer E. Staub A. Tora L. Talianidis I. Gene-specific modulation of TAF10 function by SET9-mediated methylation.Mol. Cell. 2004; 14: 175-182Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 39Kontaki H. Talianidis I. Lysine methylation regulates E2F1-induced cell death.Mol. Cell. 2010; 39: 152-160Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 40Kurash J.K. Lei H. Shen Q. Marston W.L. Granda B.W. Fan H. et al.Methylation of p53 by Set7/9 mediates p53 acetylation and activity in vivo.Mol. Cell. 2008; 29: 392-400Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 41Subramanian K. Jia D. Kapoor-Vazirani P. Powell D.R. Collins R.E. Sharma D. et al.Regulation of estrogen receptor alpha by the SET7 lysine methyltransferase.Mol. Cell. 2008; 30: 336-347Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 42Ea C.K. Baltimore D. Regulation of NF-kappaB activity through lysine monomethylation of p65.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 18972-18977Crossref PubMed Scopus (191) Google Scholar, 43Yang X.D. Huang B. Li M. Lamb A. Kelleher N.L. Chen L.F. Negative regulation of NF-kappaB action by Set9-mediated lysine methylation of the RelA subunit.EMBO J. 2009; 28: 1055-1066Crossref PubMed Scopus (177) Google Scholar, 44Liu X. Chen Z. Xu C. Leng X. Cao H. Ouyang G. et al.Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.Nucl. Acids Res. 2015; 43: 5081-5098Crossref PubMed Scopus (79) Google Scholar, 45Kim Y. Nam H.J. Lee J. Park D.Y. Kim C. Yu Y.S. et al.Methylation-dependent regulation of HIF-1alpha stability restricts retinal and tumour angiogenesis.Nat. Commun. 2016; 710347Google Scholar). In this study, we found that that OTUB1 contains a SET7 targeting motif [(K/R) (S/T) K]. Further assays show that SET7 mediated the monomethylation of OTUB1 on lysine 122, leading to the impairment of its binding to UBC13 and the relief of its suppressive role on ferroptosis. This study reveals an essential role of SET7 in affecting the noncanonical activity of OTUB1. In addition to histone H3, SET7 was also found to monomethylate nonhistone proteins by targeting a conserved core motif [(K/R) (S/T) K] in these proteins (37Chuikov S. Kurash J.K. Wilson J.R. Xiao B. Justin N. Ivanov G.S. et al.Regulation of p53 activity through lysine methylation.Nature. 2004; 432: 353-360Crossref PubMed Scopus (664) Google Scholar, 44Liu X. Chen Z. Xu C. Leng X. Cao H. Ouyang G. et al.Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.Nucl. Acids Res. 2015; 43: 5081-5098Crossref PubMed Scopus (79) Google Scholar, 46Tang J. Deng H. Wang Z. Zha H. Liao Q. Zhu C. et al.EGLN1 prolyl hydroxylation of hypoxia-induced transcription factor HIF1alpha is repressed by SET7-catalyzed lysine methylation.J. Biol. Chem. 2022; 298101961Abstract Full Text Full Text PDF Scopus (3) Google Scholar). After searching the amino acid sequences of OTUB1 from different organisms, the ' KSK ' residues were identified in OTUB1 (Fig. 1A), in which lysine 122 might be methylated by SET7. To validate this hypothesis, we developed an antibody (anti-OTUB1-K122me) to specifically recognize lysine 122 monomethylation of human OTUB1. The specificity of this antibody was validated by dot blot assay (Fig. 1B). Monomethylation of OTUB1 was readily detected by the antibody in WT H1299 cells (OTUB1+/+) but not in OTUB1-deficient H1299 cells (OTUB1−/−) (Fig. 1C). However, reconstitution of Flag-tagged WT OTUB1 in OTUB1−/− H1299 cells restored the methylated OTUB1 to be detected (Fig. 1D). By contrast, reconstitution of methylation-site mutant of OTUB1 (OTUB1-K122A) could not restore the methylated OTUB1 (Fig. 1D). Subsequently, we examined whether OTUB1 methylation at K122 was mediated by SET7. When Flag-tagged WT OTUB1 (Flag-OTUB1) was cotransfected with empty vector, Myc-SET7 or its enzymatically deficient mutant (Myc-SET7-H297A) (37Chuikov S. Kurash J.K. Wilson J.R. Xiao B. Justin N. Ivanov G.S. et al.Regulation of p53 activity through lysine methylation.Nature. 2004; 432: 353-360Crossref PubMed Scopus (664) Google Scholar) in OTUB1−/− H1299 cells, monomethylation of OTUB1 in the cells transfected with WT SET7 was higher than in the cells transfected with empty vector control, while monomethylation of OTUB1 in the cells transfected with Myc-SET7-H297A was similar to the cells transfected with empty vector control, suggesting that OTUB1 might be methylated by endogenous SET7 in these cells or OUTB1 might be methylated at K122 by another unknown methyltransferase (Fig. 1E). However, the methylated OTUB1 was not detected when Flag-tagged OTUB1-K122A (Flag-OTUB1-K122A) was cotransfected with SET7 or its enzymatically deficient mutant (SET7-H297A) in OTUB1−/− H1299 cells (Fig. 1E). To further determine whether endogenous OTUB1 was methylated by SET7, we examined methylation of OTUB1 in SET7+/+ and SET7−/− H1299 cells after coimmunoprecipitation with anti-OTUB1 antibody. Monomethylation of OTUB1 in SET7+/+ H1299 cells was higher than that in SET7−/− H1299 cells (Fig. 1F). To further validate these results, we performed mass spectrometry assay. As shown in Figure 1G, the monomethylated K122 was identified in OTUB1 when SET7 was coexpressed (Fig. 1G). Next, we examined whether OTUB1 interacts with SET7 by coimmunoprecipitation assay. Ectopically expressed OTUB1 interacted with ectopically expressed SET7 and vice versa (Fig. 2, A and B). In H1299 cells, endogenous OTUB1 was coimmunoprecipitated with endogenous SET7 (Fig. 2C), while endogenous coimmunoprecipitation between OTUB1 and SET7 was not detected in OTUB1-deficient H1299 cells (Fig. 2D). Escherichia coli–expressed GST-tagged OTUB1 or GST-OTUB1-K122R interacted with E. coli–expressed His-tagged SET7 in vitro (Fig. 2E). These data indicated that OTUB1 directly associated with SET7. In addition, it appeared that the N-terminal domain (E2/UBD) of OTUB1 did not bind to SET7, while the C-terminal domain of OTUB1 could bind to SET7 (Fig. 2, F and G). Taken together, these data suggest that SET7 interacts with OTUB1 to catalyze monomethylation of OTUB1 at K122. Given that OTUB1 is a typical deubiquitinase, we initially sought to know whether SET7-mediated methylation of OTUB1 at K122 could affect deubiquitinase activity of OTUB1. We compared the deubiquitinase activity between WT OTUB1 and its methylation-mimic mutant (OTUB1-K122F) by enzymatic activity assay. The WT OTUB1 had the same capability as its methylation-mimic mutant (OTUB1-K122F) to catalyze deubiquitination of K48-linked di-ubiquitin (Fig. 3, A–C). Then, we examined the effect of OTUB1 on ubiquitination of TRAF3, a well-defined target deubiquitinated by OTUB1 (47Mao A.P. Li S. Zhong B. Li Y. Yan J. Li Q. et al.Virus-triggered ubiquitination of TRAF3/6 by cIAP1/2 is essential for induction of interferon-beta (IFN-beta) and cellular antiviral response.J. Biol. Chem. 2010; 285: 9470-9476Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). As shown in Figure 3D, the deubiquitination activity of WT OTUB1 on TRAF3 was similar to that of the two mutants, OTUB1-K122A and OTUB1-K122F. In addition, coexpression of SET7 had no obvious effect on the deubiquitination of TRAF3 by OTUB1 (Fig. 3E). These data suggest that SET7-mediated methylation of OTUB1 does not affect deubiquitinase activity of OTUB1. Subsequently, we sought to know whether SET7-mediated methylation of OTUB1 at K122 could affect OTUB1 protein stability. Overexpression of either SET7 or its enzymatically deficient mutant (SET7-H297A) with increasing amounts together with HA-tagged OTUB1 in HEK293T cells had no obvious effect on OTUB1 protein stability (Fig. 4A). Endogenous OTUB1 protein level was not changed when Myc-SET7 or its enzymatically deficient mutant (SET7-H297A) were transfected into HEK293T cells with increasing amounts (Fig. 4B). Moreover, endogenous OTUB1 protein level was almost the same between SET7+/+ and SET7−/− H1299 cells (Fig. 4C). Moreover, overexpression of WT OTUB1 and its methylation-mimic mutant (OTUB1-K122F) also did not alter endogenous SET7 protein level in HEK293T cells (Fig. 4D). In OTUB1−/− H1299 cells, reconstitution of WT OTUB1 or its methylation-mimic mutant (OTUB1-K122F) had no effect on endogenous SET7 protein levels (Fig. 4E). Consistently, by cycloheximide pulse chase assay, SET7 deficiency had no effect on endogenous OTUB1 protein levels (Fig. 4F), meanwhile, OTUB1 deficiency also did not affect endogenous SET7 protein levels (Fig. 4G). These data suggest that SET7 and OTUB1 do not affect each other at protein level although they directly associate in cells. As reported previously, phosphorylation of OTUB1 at Serine 16 by Casein kinase 2 causes nuclear accumulation of OTUB1 (33Herhaus L. Perez-Oliva A.B. Cozza G. Gourlay R. Weidlich S. Campbell D.G. et al.Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization.Sci. Signal. 2015; 8: ra35Crossref PubMed Scopus (44) Google Scholar), we sought to know whether SET7-mediated methylation of OTUB1 at K122 could alter subcellular localization of OTUB1. By immunofluorescent staining, we detected that WT HA-OTUB1 mainly located in cytosol when overexpressed in HEK293T cells (Fig. 5A). In agreement with the previous report (33Herhaus L. Perez-Oliva A.B. Cozza G. Gourlay R. Weidlich S. Campbell D.G. et al.Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization.Sci. Signal. 2015; 8: ra35Crossref PubMed Scopus (44) Google Scholar), the phosphomimic mutant of OTUB1, HA-OTUB1-S16E, localized almost exclusively to nuclei when overexpressed in HEK293T cells, whereas the nonphosphorylatable mutant of OTUB1, HA-OTUB1-S16A located entirely in cytosol when overexpressed in HEK293T cells (Fig. 5A). However, the methylation-deficient mutants of OTUB1, HA-OTUB1-K122A or HA-OTUB1-K122R, mainly located in cytosol, exactly the same as the methylation-mimic mutant of OTUB1, HA-OTUB1-K122F (Fig. 5A). Moreover, OTUB1 was detected primarily in the cytoplasmic fraction, while overexpression of SET7 did not cause its nuclear translocation (Fig. 5B). In fact, the subcellular localization of OTUB1 was almost the same between SET7+/+ and SET7−/− H1299 cells (Fig. 5C). These data suggest that SET7-mediated methylation of OTUB1 does not affect the subcellular localization of OTUB1. It is well-defined that OTUB1 also possesses a noncanonical function independently of its deubiquitinase activity, which inhibits the ubiquitination of target proteins by binding to and inhibiting the E2 ubiquitin-conjugation enzymes (4Wiener R. Zhang X. Wang T. Wolberger C. The mechanism of OTUB1-mediated inhibition of ubiquitination.Nature. 2012; 483: 618-622Crossref PubMed Scopus (186) Google Scholar, 5Juang Y.C. Landry M.C. Sanches M. Vittal V. Leung C.C. Ceccarelli D.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 (146) Google Scholar, 6Sato Y. Yamagata A. Goto-Ito S. Kubota K. Miyamoto R. Nakada S. et al.Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13.J. Biol. Chem. 2012; 287: 25860-25868Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Thus, we sought to know whether SET7-mediated methylation of OTUB1 can affect OTUB1 binding to the E2 enzyme, UBC13. Ectopically expressed SET7 inhibited the interaction between reconstituted OTUB1 with endogenous UBC13 in OTUB1−/− H1299 cells (Fig. 6A). Furthermore, in OTUB1−/− H1299 cells, reconstitution of WT OTUB1 bound to more endogenous UBC13 than reconstitution of the methylation-mimic mutant of OTUB1 (K122F) (Fig. 6B). In addition, coimmunoprecipitation assay showed that endogenous Ubc13 bound to more endogenous Otub1 in Set7-deficient mouse embryo fibroblast (MEF) cells (Set7−/−) than in Set7-intact MEF cells (Set7+/+) (Fig. 6C). These data suggest that SET7-mediated methylation of OTUB1 attenuates OTUB1 binding to UBC13. Recently, OTUB1 was identified as a negative regulator of ferroptosis by directly interacting with and stabilizing SLC7A11, which was independent of its DUB activity (22Liu T. Jiang L. Tavana O. Gu W. The deubiquitylase OTUB1 mediates ferroptosis via stabilization of SLC7A11.Cancer Res. 2019; 79: 1913-1924Crossref PubMed Scopus (228) Google Scholar). Since OTUB1 possesses a noncanonical function independently of its DUB activity by binding to and inhibiting the E2 ubiquitin-conjugation enzymes, such as UBC13 (4Wiener R. Zhang X. Wang T. Wolberger C. The mechanism of OTUB1-mediated inhibition of ubiquitination.Nature. 2012; 483: 618-622Crossref PubMed Scopus (186) Google Scholar, 5Juang Y.C. Landry M.C. Sanches M. Vittal V. Leung C.C. Ceccarelli D.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 (146) Google Scholar, 6Sato Y. Yamagata A. Goto-Ito S. Kubota K. Miyamoto R. Nakada S. et al.Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13.J. Biol. Chem. 2012; 287: 25860-25868Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), SET7-mediated methylation of OTUB1 attenuates OTUB1 binding to UBC13. Therefore, we sought to determine whether SET7-mediated methylation of OTUB1 has an i

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