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  • A different example of substrate dependent activation is see


    A different example of substrate-dependent activation is seen in CSN5, a JAMM-type DUB found in the eight subunit COP9 signalosome (CSN) [34]. The CSN deconjugates NEDD8 (neural precursor cell expressed developmentally downregulated 8) from Cullin Ring ubiquitin E3 ligases (CRLs). Through this activity, CSN decreases the ubiquitin E3 ligase activity of CRLs. A recent structural analysis of CSN rationalized how interaction between DUB and its neddylated CRL substrate leads to activation [35]. The catalytic subunit CSN5 forms a subcomplex with CSN6 and CNS4. In absence of substrate, a loop in CSN5, Ins1, occludes the active site, leading to autoinhibition. In the presence of neddylated CRLs, the CSN4 subunit undergoes a large conformational change to bind the substrate, at the expense of its interaction with the so-called ‘Ins-2’ loop of CSN6. These substrate-induced conformational changes alleviate the autoinhibition and prime CSN for deconjugation. Point mutations in the Ins1 and Ins2 loops can activate CSN even in the absence of neddylated CRLs, confirming that they hedgehog signaling act as autoinhibitory elements [35]. The ability of CSN4 to sense neddylated CRLs ensures that activation only occurs in the presence of the substrate.
    DUB regulation by intramolecular and external factors The next layer of regulation is the direct regulation of DUB catalytic activity. In this mode of regulation, the CD of DUBs can be viewed as core units whose catalytic activity is modulated by interaction with other protein modules, either external, or within the DUB itself [2]. A common form of regulation of this type is that given by a large molecular machine. There are several examples of DUBs that only attain optimal activity and localization within the structural integrity of such multisubunit molecular machines, such as the COP9 signalosome. Other notable examples of this type include USP14 and RPN11 in the proteasome 11, 36, 37, BRCC36 within the BRCA1-A and BRISC complexes 15, 16, 38 and Ubp8 in the yeast SAGA DUB module 39, 40, 41. In these cases, the DUBs display low activity in isolation, but are robustly activated within the complex. For Ubp8, complex formation likely stabilizes the active site and the ubiquitin-binding surface 40, 42. In general, the exact molecular mechanism of activation in these large complexes remains poorly understood, but may depend on a combination of regulatory influences. Apart from these examples where DUBs are activated as part of large macromolecular assemblies, many well-studied examples exist where simple domains, within the DUB or from outside, specifically modulate the activity of CDs. USP5 contains several ubiquitin binding domains (UBDs) that assist in the disassembly of polyubiquitin chains of a variety of linkages [43]. The crystal structure of full-length USP5 revealed a previously unpredicted domain, named nUBP, that packs tightly against the CD and allosterically activates pelagic zone 1000-fold (Figure 1) [44]. Moreover, addition of free ubiquitin to USP5 can further stimulate USP5 activity through binding to the ZnF-UBP domain via an as yet unknown mechanism [43]. A second example where additional domains are important is USP4, a DUB with roles in TGF-β (transforming growth factor beta) signaling and splicing. The isolated USP4 CD was found to have an unusually high affinity for ubiquitin (low nanomolar range), suggesting that it could be constitutively product inhibited in cells, where the ubiquitin concentration is in the range of 4–20μM [45]. However, the N-terminal DUSP-Ubl domain present in full-length USP4 can allosterically promote product release, thereby increasing kcat of USP4. This effect involves the ‘switching loop’, a loop close to the catalytic triad. This ‘switching loop’ also has a role in the hedgehog signaling activity of USP7 or its yeast homolog Ubp15. The C-terminal HUBL domain of USP7 can dynamically fold back onto the CD to allow contact of a C-terminal peptide at the end of the HUBL domain with the ‘switching loop’. This intramolecular interaction increases both kcat and KM of the CD [28]. Unlike USP7, the N-terminal TRAF domain of Ubp15 also affects intrinsic activity [46]. Apparently, the ‘switching loop’ has a conserved regulatory function in multiple USPs, although the details of the activation differ.