External factors such as protein
External factors, such as protein partners, can also activate DUBs by either reinforcing the stimulatory effects of intramolecular factors or by other means. The intramolecular activation of the USP7 CD by its HUBL domain is allosterically potentiated by external factor GMP synthase (GMPS), which consolidates the active state leading to an additional increase in kcat 28, 47.
One of the best-studied examples of DUB activation by external factors is USP1. This DUB controls DNA repair signaling by deubiquitylating FANCD2 (Fanconi anemia group D2) and PCNA 48, 49 after DNA cross-linking and UV damage, respectively. USP1 is regulated by autocleavage  and its kcat is strongly stimulated by the WD40 repeat protein UAF-1 . UAF-1 achieves this by increasing the basicity of the histidine general bromodomain inhibitor in the USP1 catalytic triad . Two closely related DUBs, USP12 and USP46, are similarly activated by UAF-1, but unlike USP1, these can be hyperactivated by another WD40 repeat protein, named WDR20 52, 53, 54.
DUB activation also occurs in endocytosis and autophagy pathways. The JAMM class enzyme AMSH is recruited to endosomes by the adaptor protein STAM . Besides recruitment, STAM can also directly activate AMSH hydrolysis of polyubiquitin chains on endosome-targeted proteins. The exact mechanism of this activation is unclear, but both kcat and KM effects were suggested 13, 55. In autophagy, the activity of USP10 and USP13 is modulated by Beclin-1, which is a subunit of the essential Vps34 complexes that have a role in phagosome nucleation. These complexes can be rapidly degraded by ubiquitylation of Beclin-1. However, Beclin-1 prevents this by binding USP10 and USP13, and stimulating these DUBs to remove ubiquitin from itself, both in cells and in vitro.
A particularly interesting example of DUB regulation by external proteins involves stimulation of OTUB1 activity by ubiquitin E2 enzymes. OTUB1 can noncatalytically interfere with polyubiquitin synthesis by specifically inhibiting E2 enzyme Ubc13 linked to ubiquitin at its active site (charged E2) 57, 58. Conversely, a subset of charged and uncharged E2s, including UbcH5B, stimulate Lys48-linked polyubiquitin hydrolysis by OTUB1 by increasing substrate affinity . Crystal structures indicate that UbcH5B achieves this increased affinity by stabilizing a ubiquitin-binding site on OTUB1. Whether charged E2s stimulate OTUB1 activity, or whether OTUB1 inhibits polyubiquitin synthesis of the E2 depends on the relative concentrations of charged E2 and free ubiquitin. Therefore, the authors suggested an elegant model wherein OTUB1-E2 complexes dynamically regulate the level of polyubiquitin chains in cells by either activating chain hydrolysis or inhibiting E2-mediated chain synthesis.
The activities of the UCH family DUBs UCH-L5 and BAP1 are regulated by related deubiquitinase adaptor (DEUBAD) domains . BAP1 is a tumor suppressor that is activated ASX (additional sex combs) to deubiquitylate H2A in Polycomb gene repression . In the proteasome, the DEUBAD domain of RPN13 activates UCH-L5 by increasing the affinity for substrates 62, 63, 64, 65, 66. This occurs through a combination of mild effects, including allosteric stabilization of the so-called ‘active site crossover loop’ and restriction of the inhibitory mobility of the C-terminal ULD domain of UCH-L5 65, 66.
Negative regulation of DUBs While all DUBs discussed so far are activated by intramolecular domains or external proteins, in a limited number of rare cases DUBs are directly inhibited by other proteins. The first example of this type is UCH-L5 inhibition by INO80G. INO80G is a metazoan-specific subunit of INO80 chromatin-remodeling complexes and is associated with UCH-L5 during DNA repair 67, 68. This interaction strongly inhibits UCH-L5 . Similar to the UCH-L5 activator RPN13 (discussed earlier), INO80G contains a DEUBAD domain  and recent structural analyses revealed that it inhibits UCH-L5 by occupying the ubiquitin-docking site on the enzyme through an unique hairpin structure that is absent in the DEUBAD domain of activator RPN13 65, 66. While the DEUBAD domain of RPN13 activates UCH-L5 by increasing its affinity for substrates, in INO80G it does the opposite and dramatically decreases the affinity for substrates. Thus, the regulation of UCH-L5 is achieved at the level of substrate affinity, leading to a change in KM rather than kcat, as observed in USP1 regulation for instance . The dual mode of regulation of UCH-L5 suggests that strict spatial and temporal control should exist to ensure the right activity at the right place. For example, the inhibition of UCH-L5 by INO80G must be alleviated during DNA repair because the catalytic activity of UCH-L5 is required in this pathway .