© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 1997-2006 doi:10.1242/jcs.201855 COMMENTARY Mechanisms of regulation and diversification of deubiquitylating enzyme function Pawel Leznicki* and Yogesh Kulathu* ABSTRACT been linked to the pathogenesis of a number of human diseases, Deubiquitylating (or deubiquitinating) enzymes (DUBs) are such as cancer, and neurodegenerative, inflammatory and proteases that reverse protein ubiquitylation and therefore metabolic disorders (Heideker and Wertz, 2015). modulate the outcome of this post-translational modification. Approximately 100 DUBs are encoded in the human genome DUBs regulate a variety of intracellular processes, including (Nijman et al., 2005b). Depending on the organization of the protein turnover, signalling pathways and the DNA damage catalytic domain, DUBs can be classified into distinct families response. They have also been linked to a number of human (Table 1), the vast majority of which are cysteine proteases. These diseases, such as cancer, and inflammatory and neurodegenerative include ubiquitin-specific proteases (USPs), ubiquitin C-terminal – disorders. Although we are beginning to better appreciate the role of hydrolases (UCHs), ovarian tumour proteases (OTUs), Machado DUBs in basic cell biology and their importance for human health, Joseph disease proteases (MJDs) and the recently discovered MIU- there are still many unknowns. Central among these is the containing novel DUB family (MINDY) proteases (Abdul Rehman conundrum of how the small number of ∼100 DUBs encoded in et al., 2016). Additionally, there is also a metalloproteinase family the human genome is capable of regulating the thousands of of DUBs, the JAB1/MPN/Mov34 (JAMM) domain proteases ubiquitin modification sites detected in human cells. This (Nijman et al., 2005b). Strikingly, although DUBs have been Commentary addresses the biological mechanisms employed to implicated in many cellular processes, in most cases their precise modulate and expand the functions of DUBs, and sets directions for function is still either poorly characterized or completely unknown. future research aimed at elucidating the details of these fascinating Importantly, there are at least 600 E3 ubiquitin ligases, and at ∼ processes. steady-state conditions, 20,000 ubiquitylation sites on thousands This article is part of a Minifocus on Ubiquitin Regulation and of intracellular proteins can be detected (Clague et al., 2015; Kim Function. For further reading, please see related articles: et al., 2011; Udeshi et al., 2013). How a relatively small number of ‘Exploitation of the host cell ubiquitin machinery by microbial DUBs manages to regulate such a vast number of modifications is effector proteins’ by Yi-Han Lin and Matthias P. Machner (J. Cell one of the key unsolved questions in the field. In this Commentary, Sci. 130, 1985–1996). ‘Cell scientist to watch – Mads Gyrd-Hansen’ we will discuss the different layers of regulation of DUB activity that (J. Cell Sci. 130, 1981–1983). ensure that DUBs are activated, regulated and targeted to their appropriate substrates to achieve precise spatio-temporal control of KEY WORDS: Deubiquitinase, Deubiquitylase, Signal transduction, ubiquitin signals within a specific physiological pathway. We will Ubiquitylation discuss these concepts with an emphasis on how functional diversification of DUBs can be achieved. Introduction Post-translational attachment of ubiquitin, or ubiquitylation, Regulation of DUB abundance controls most intracellular processes, including protein turnover, Conceptually, the simplest mechanism for modulating the activity, intracellular signalling, endocytosis and the DNA damage and hence biological function, of a given protein is by regulating response (Clague and Urbe, 2017; Heideker and Wertz, 2015; its intracellular concentration. This also applies to DUBs, whose Kee and Huang, 2015). Ubiquitin is most often conjugated to abundance can be controlled both via transcription or translation lysine residues of target proteins in a reaction catalysed by an and via degradation. For example, levels of several DUBs are enzymatic cascade that involves the E1 activating, E2 conjugating regulated in a cell cycle-dependent manner and are directly related and E3 ligating enzymes. Importantly, ubiquitin can also be ligated to their function. USP37 is one such DUB, and its transcription to any of the seven internal lysine residues or the N-terminal is controlled by the E2F transcription factors, resulting in high methionine of another ubiquitin, forming chains whose linkage USP37 levels during the G1/S transition (Huang et al., 2011). type defines the outcome of protein ubiquitylation. Ubiquitylation This in turn allows for the stabilization of cyclin A and cell can be reversed by deubiquitylating (or deubiquitinating) enzymes cycle progression into S phase. Interestingly, USP37 is degraded in (DUBs) that cleave ubiquitin off the substrate protein or within mitosis by the anaphase-promoting complex (APC) containing ubiquitins in a polyubiquitin chain (Fig. 1). The importance of the CDH1 (also known as FZR1) (APCCDH1) (Huang et al., 2011). components of the ubiquitin conjugation and deconjugation Similarly, levels of USP33 oscillate during the cell cycle, which has systems is underscored by the fact that their deregulation has implications for USP33-mediated stabilization of CP110, a centriolar protein that controls cell cycle-dependent centrosome MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, duplication (Li et al., 2013). Transcription of A20 (also known as University of Dundee, Dow Street, Dundee DD1 5EH, UK. TNFAIP3) and cylindromatosis (CYLD), modulators of nuclear factor (NF)-κB signalling, is also tightly regulated and induced in *Authors for correspondence ([email protected]; [email protected]) response to NF-κB activation in a feedback loop to inhibit the Y.K., 0000-0002-3274-1642 pathway (Pahl, 1999; Sun, 2010). Moreover, several DUBs are Journal of Cell Science 1997 COMMENTARY Journal of Cell Science (2017) 130, 1997-2006 doi:10.1242/jcs.201855 (>600) (40) E3 ATP E2 Substrate Ub (2) E1 E1 Ub E2Ub E2 Ub E3 AMP+PPi Activation Conjugation E1 E2 Substrate Ligation Substrate Ub Ub Substrate Substrate Substrate Ub Ub Ub Ub Ub Ub Ub Ub Deubiquitylation Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub DUB DUB Multi- Monoubiquitylation monoubiquitylation Polyubiquitylation (~100) Ubiquitylation sites: ~ 20,000 Fig. 1. Overview of protein ubiquitylation. Ubiquitin (Ub) is activated and transferred to a substrate protein in a cascade reaction catalysed by E1 activating, E2 conjugating and E3 ligating enzymes. This can lead to the formation of distinct types of modifications, all of which can potentially be reversed by deubiquitylating enzymes (DUBs). Numbers in brackets indicate the number of members for each family of enzymes. ubiquitylated and some, such as USP4 (Zhang et al., 2012), can these DUBs not only enhances their catalytic activity but also auto-deubiquitylate and hence counteract their own ubiquitin- places them within the context of the proteasome, where they can mediated degradation. Therefore, transcriptional and post- influence different stages of protein degradation. Hence, USP14 translational mechanisms regulate the abundance of DUBs and and UCH37 can potentially modulate the ubiquitylation status of thus their functions. This regulation can also be cell type-specific, proteins prior to their commitment for degradation, whereas Rpn11 enabling fine tuning of ubiquitin signalling. acts at later stages (Worden et al., 2014). Interestingly, UCH37 is also part of another complex, the chromatin-remodelling INO80 Interacting partners of DUBs complex, in which its activity is inhibited (Yao et al., 2008). Elegant Interacting partners are key regulators of the biological functions of structural studies have revealed that equivalent DEUBiquitylase DUBs and can affect their catalytic activity and facilitate substrate ADaptor (DEUBAD) domains present in both Rpn13 (also known recognition. Indeed, in some cases, a DUB is essentially inactive as ADRM1 in mammals) and INO80G (also known as NFRKB) unless it is incorporated into a protein complex. This is particularly induce distinct structural rearrangements in UCH37 that result in its evident in the case of large macromolecular assemblies that contain activation at the proteasome and inhibition at the INO80 complex, DUBs as functional subunits. For example, all three proteasome- respectively (Sahtoe et al., 2015; VanderLinden et al., 2015). associated DUBs, the metalloproteinase Rpn11 (also known as Akin to the recruitment of UCH37 into two distinct complexes, PSMD14) and the two cysteine proteases USP14 and UCH37 (also the proteasome and the INO80 complex, BRCC36 (a member of the known as UCHL5), are significantly activated upon incorporation JAMM DUB family) is incorporated into two alternative into the 19S regulatory particle (Borodovsky et al., 2001; Qiu et al., complexes, the nuclear Abraxas complex (ARISC) and cytosolic 2006; Worden et al., 2014; Yao et al., 2006). Recruitment of BRCC36 isopeptidase complex (BRISC). Both the ARISC and BRISC complexes share the core subunits, BRCC45 and MERIT40 Table 1. DUBs are divided into several families (also known as BRE and BABAM1 , respectively), but in addition Family Members Active they also contain complex-specific components, namely, Abro1 (also known as KIAA0157 and FAM175B) for the BRISC complex USP 57 51 OTU