The mechanism of OTUB1 inhibition of ubiquitination

Department of Biophysics and Biophysical Chemistry and Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Nature (Impact Factor: 41.46). 02/2012; 483(7391):618-22. DOI: 10.1038/nature10911
Source: PubMed


Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13∼Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13∼Ub and inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13∼Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13∼Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13∼Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13∼Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes in a non-catalytic manner.

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    • "So far, however, just several studies showed the limited understanding of the DDR roles for DUBs that mediate the processing and removal of ubiquitin . Although these DUBs have been previously suggested DDR connections (Clerici et al., 2014; Huang et al., 2006; Jacq et al., 2013; Nakada et al., 2010; Nicassio et al., 2007; Nijman et al., 2005; Nishi et al., 2014; Wiener et al., 2012), all of them were not directly involved in DNA end resection. The genetic screen in search of DUBs that are involved in DDR showed that USP4 perhaps is involved in DDR, but the detailed function and mechanism is not clear (Nishi et al., 2014). "
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    ABSTRACT: DNA end resection is a highly regulated and critical step in DNA double-stranded break (DSB) repair. In higher eukaryotes, DSB resection is initiated by the collaborative action of CtIP and the MRE11-RAD50-NBS1 (MRN) complex. Here, we find that the deubiquitylating enzyme USP4 directly participates in DSB resection and homologous recombination (HR). USP4 confers resistance to DNA damage-inducing agents. Mechanistically, USP4 interacts with CtIP and MRN via a specific, conserved region and the catalytic domain of USP4, respectively, and regulates CtIP recruitment to sites of DNA damage. We also find that USP4 autodeubiquitylation is essential for its HR functions. Collectively, our findings identify USP4 as a key regulator of DNA DSB end resection.
    Full-text · Article · Sep 2015 · Cell Reports
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    • "The over expression of CK2α has been shown to increase the degradation of Ikaros protein (i.e., a tumor suppressor in ALL) via the ubiquitin pathway [3,32,33]. Interestingly in the t(12, 21)ALLs, not only CK2α is under expressed but OTUB1, a deubiquitinating enzyme, is found slightly over expressed (see Table 2, rank 12) [34]. OTUB1 hydrolase can specifically remove ‘Lys-48”-linked conjugated ubiquitin from proteins and plays an important regulatory role at the level of protein turnover by preventing degradation. "
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    Full-text · Article · Aug 2014 · Clinical Proteomics
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    • "Moreover, OTUB1 was found to antagonize SMAD2/3's ubiquitination independent of its catalytic activity as it interacts with E2 enzymes and inhibits efficient ubiquitin transfer from E2 to E3. This mechanism is reminiscent to the mechanism described in an earlier study on OTUB1-medi- ated inhibition of ubiquitination (Wiener et al., 2012). "
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    Full-text · Article · Apr 2014 · Protein & Cell
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