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  • Another pathway that requires proper DUB recruitment


    Another pathway that requires proper DUB recruitment is the DNA damage response (DDR). After ultraviolet (UV)-induced DNA damage, monoubiquitylated proliferating cell nuclear antigen (PCNA) mediates signaling that leads to repair. The DUB complex USP1/USP1-associated factor 1 (UAF1) deubiquitylates PCNA after the complex is recruited to the substrate by recruitment factor human ELG1 [14]. BRCA1/BRCA2-containing complex, subunit 36 (BRCC36) is another DUB in the DDR, where it deubiquitylates several proteins as a catalytic subunit of the BRCA1-A complex. In this complex, specialized ubiquitin and small ubiquitin-like modifier (SUMO)-binding domains recruit BRCC36 to sites of damage 15, 16, 17, 18, 19. Finally, also in the nucleus, transcription factor forkhead box K2 (FOXK2) targets the UCH class DUB BAP1 (BRCA1-associated protein 1) to 5416 to facilitate histone H2A deubiquitylation [20]. In NF-κB signaling, ubiquitin conjugation has multiple roles. Different components of the pathway recruit the DUBs USP10 and CYLD (cylindromatosis). The protein MCPIP-1 (monocyte chemotactic protein induced protein 1) recruits USP10, whereas CYLD is recruited by the E3 ligase HOIP (HOIL interacting protein) (Figure 1). Moreover, CYLD contains a B-box domain that promotes its cytoplasmic localization 21, 22. The previous examples illustrated how external proteins can recruit DUBs to relevant pathways or substrates. While DUBs in general can recognize the ubiquitin part of a substrate via their catalytic domains (CDs), sometimes extra specificity is achieved by specialized domains that are present in DUBs themselves. One of the best-studied examples is the DUB USP7. Here, the N-terminal TRAF domain of USP7 binds small peptide motifs in its targets EBNA-1 (Epstein–Barr nuclear antigen 1), p53 and MDM2 (Mouse double minute 2 homolog) to facilitate their deubiquitylation (Figure 1) 23, 24. USP15 uses its DUSP-Ubl domain to recruit and deubiquitylate the E3 ligase BRCA1-associated protein (BRAP) [25], while the H2A deubiquitinase USP3 requires its intact Zinc finger domain to bind H2A [26].
    Substrate-mediated regulation Besides recruitment, some DUBs require further activation. Surprisingly, the cognate Ubl itself can affect the activity of the DUB or ubiquitin-like protease by rearranging the catalytic triad. Early structural studies on the CD of USP7 revealed that its catalytic triad can exist in an inactive configuration [27]. Binding of a ubiquitin derivative ‘realigned’ the catalytic triad towards an active configuration and also changed the conformation of the ‘switching loop’, a surface loop close to the active site that is important for activation 27, 28. These effects suggest that USP7 CD is only active when ubiquitin is correctly bound. In UCH-L1 (Figure 1), ubiquitin binding at an ‘exosite’ (i.e., distant from the active site), also induces a cascade of conformational changes that rearranges the catalytic triad [29]. This type of allosteric activation also occurs in the sentrin-specific protease (SENP) class of SUMO proteases, where it has been elegantly quantified. When SENPs are incubated with the tail-less SUMO β-grasp domain, this increases the catalytic turnover against a model peptide substrate 30, 31, 32. However, this type of ubiquitin/Ubl-induced rearrangement is not generically present, because other inactive DUBs contain correctly aligned active sites, even in the absence of ubiquitin. When present, this regulation by a Ubl itself cannot give much specificity. Thus, it is not surprising that regulation of USP7 for example, has further layers of complexity, as discussed later. Substrates can give rise to more complex types of activation, generating high specificity. In OTULIN (OTU domain-containing deubiquitinase with linear linkage specificity), a member of the OTU class DUBs, the substrate actively assists in catalysis. OTULIN regulates NF-κB signaling by its exclusive ability to disassemble linear ubiquitin chains [33]. In these chains, ubiquitin moieties are linked via the amino terminus at Met1 instead of via one of its lysines. They are made by the HOIP E3 ligase in the linear chain assembly (LUBAC) complex. Interestingly, OTULIN uses this unique linkage to sense its substrate. Normally, OTULIN Asp336 functions as an autoinhibitory element that favors an unproductive catalytic triad conformation, but Glu16 of the proximal ubiquitin (the target ubiquitin) in linear chains reorganizes the catalytic triad towards an active state. Mutation of this substrate Glu16 reduces the kcat 240 times but hardly affects the binding, indicating that actively it promotes catalysis. Of all ubiquitin chain types, only a linear chain can bind such that the Glu16 in the proximal ubiquitin is correctly positioned to assist in catalysis, explaining how OTULIN activity is specifically restricted to cellular pathways that feature linear polyubiquitin signaling.