Introduction Protein ubiquitination is a posttranslational m

Introduction Protein ubiquitination is a posttranslational modification that plays a major role in almost all cellular processes in eukaryotes (Hochstrasser, 2009; Komander & Rape, 2012). It involves the covalent attachment of ubiquitin (Ub) via its C-terminal glycine carboxylate to a primary amine of a target protein, generally to a lysine side chain resulting in an isopeptide bond. Ub itself can also be ubiquitinated and as such give rise to polyUb chains. This conjugation occurs at the side chain of one of the seven internal lysine residues (Lys-6, 11, 27, 29, 33, 48, 63), resulting in an isopeptide bond, or at the N-terminus (Met-1), resulting in a linear Ub chain, and it has been shown that all eight linkages coexist in atp enzyme (Xu et al., 2009). Counteraction of the build-up of (poly-)ubiquitinated proteins is achieved by a group of deubiquitinating proteases (DUBs) that remove or trim the ubiquitin modification, liberating the substrate protein, recycling Ub and ending the Ub-induced signal. Nearly a hundred genes encoding DUBs have been identified in the human genome, which can be classified in seven distinct families. The subfamilies of ubiquitin-specific proteases (USP), ubiquitin C-terminal hydrolases (UCH), Ovarian TUmor domain proteases (OTU), Machado-Joseph disease proteases (MJD), Motif interacting with ubiquitin-containing novel DUB family (MINDY), and Zinc finger with UFM1-specific peptidase domain protein (ZUFSP) are cysteine proteases, whereas JAB1/MPN/MOV34 proteases (JAMMs) are zinc-dependent metalloproteases (Abdul Rehman et al., 2016; Komander, Clague, & Urbe, 2009; Kwasna et al., 2018; Nijman et al., 2005; Reyes-Turcu, Ventii, & Wilkinson, 2009). As distinct Ub linkages result in distinct biological signals (Komander & Rape, 2012; Yau & Rape, 2016), the determination of the linkage specificities of DUBs gives fundamental insights into the biological pathways they are involved in. It has been shown that some DUBs, mainly USPs, are able to process all isopeptide linked chains (Faesen et al., 2011) whereas others, especially OTUs, display a preference for one or a few Ub chain types (Mevissen et al., 2013). Another level of complexity is based on the existence of Ub-like proteins (Ubls) (Kerscher, Felberbaum, & Hochstrasser, 2006). These posttranslational modifiers share structural homology to Ub as well as a highly similar system for conjugation and deconjugation. The most studied examples are the small ubiquitin-like modifiers (SUMO), the neural precursor cell-expressed developmentally downregulated 8 (Nedd8) and interferon-stimulated gene of 15kDa (ISG15). SUMOylation plays a key role in genome stability, and many of its protein targets are involved in DNA-damage responses (e.g., PCNA and BRCA1) (Flotho & Melchior, 2013). Nedd8 plays an important role in cell cycle control and its main targets are Cullin proteins, which are Ub ligase subunits (Soucy, Smith, & Rolfe, 2009). ISG15 is strongly induced by Type-I interferons as part of the innate immune response to viral and bacterial infections (Zhang & Zhang, 2011). Similar to the Ub system, specific proteases deconjugate Ubls from their targets. These include SENPs acting on SUMO (Hickey, Wilson, & Hochstrasser, 2012), USP18 acting on ISG15 (Malakhov, Malakhova, Kim, Ritchie, & Zhang, 2002) and DENs acting on Nedd8 (Gan-Erdene et al., 2003) and we refer to this group of proteases as Ubl-specific proteases.