The ubiquitin (Ub) proteasome system and Ub signalling networks are crucial to cell biology and disease development. Deubiquitylases (DUBs) control cell signalling by removing mono-Ub and polyubiquitin chains from substrates. DUBs take part in almost all processes that regulate cellular life and are frequently dysregulated in disease. We have catalogued 99 currently known DUBs in the human genome and sequence conservation analyses of catalytic residues suggest that 11 lack enzyme activity and are classed as pseudo-DUBs. These pseudoenzymes play important biological roles by allosterically activating catalytically competent DUBs as well as other active enzymes. Additionally, pseudoenzymes act as assembly scaffolds of macromolecular complexes. We discuss how pseudo-DUBs have lost their catalytic activity, their diverse mechanisms of action and their potential as therapeutic targets. Many known pseudo-DUBs play crucial roles in cell biology and it is likely that unstudied and overlooked pseudo-DUB genes will have equally important functions.
Ubiquitylation of proteins serves as a post-translational signal to regulate virtually all cellular processes through the precise spatial and temporal control of protein stability, activity and/or localisation . Enzymes involved in the ubiquitin (Ub) system are frequently dysregulated in cancer, neurodegeneration, autoimmunity and other human diseases [2–5]. Ubiquitylation is a versatile post-translational modification aptly suited for a cellular communication system, with the essential ‘writer, reader and eraser’ core signalling modules (Figure 1).
Overview of Ub signalling.
Ub processing enzymes (E1, E2 and E3) write the Ub signalling code by adding Ub to substrates (writer module). A single Ub can be conjugated to lysine residues on the surface of substrate proteins (this is known as mono-ubiquitylation), or conjugated further to lysine residues on the surface of Ub itself, thus leading to polyubiquitin (poly-Ub) chains. Seven lysine residues (K6, K11, K27, K29, K33, K48 and K63) and the free amino terminus (M1) on Ub can be targeted for poly-ubiquitylation, and thus eight different Ub chain types can be generated in total, all having different topologies and unique signalling properties . Ub chains can be ‘read out’ by ubiquitin-binding domains (UBDs), which allow signal decoding and transmission (reader module). Ubiquitylation is a reversible process, and Ub is removed by deubiquitylating (DUB) enzymes (eraser module). DUB actions allow the cell to produce monomeric Ub, recycle Ub from chains and reverse signalling events resulting from ubiquitylation [6,7]. To date, 99 DUBs have been identified in the human genome, comprising six major structural families, namely (1) the ubiquitin-specific proteases (USPs), (2) the ubiquitin C-terminal hydrolases (UCHs), (3) the Machado–Joseph domain proteases (Josephins), (4) the ovarian tumour proteases (OTUs), (5) the Jab1/MPN/Mov34 metalloproteases (JAMM/MPNs) and (6) the newly discovered MIU-containing novel DUB family proteases (MINDYs) (Figure 2) [8,9].
Catalogue of known human DUBs and pseudo-DUBs.
The JAMM/MPN family is unique in using zinc during catalysis, whereas all other DUBs are cysteine proteases. Cysteine-dependent DUBs contain a common catalytic triad with a cysteine, a histidine and an acidic residue, although their structural folds differ markedly [6,10]. Nevertheless, four of the DUB families (USPs, UCHs, Josephins and OTUs) can be assigned to the papain-like structural fold, according to Pfam and SCOP databases. Representative structures for each DUB family have been reported and our understanding of how the families function as proteases has greatly improved in recent years . However, fundamental questions remain as to how DUB function is regulated (i.e. switched on and off) and how substrate specificity is controlled. For example, while some DUBs display exquisite selectivity for specific Ub chain types in vitro and in vivo, others appear highly promiscuous, at least in vitro. Similarly, the catalytic and non-catalytic functions of some DUBs are highly regulated, while others appear constitutively active or permanently repressed (e.g. pseudo-DUBs). This variability of function is underpinned by diverse regulatory molecular mechanisms, even within the same structural family. As such, DUBs represent a fascinating family of enzymes that require a close inspection of mechanism of action as well as biological function.
Overview of pseudoenzymes and pseudo-DUBs
The past 10–15 years have seen a surge in studies showing how pseudoenzymes (enzymes predicted to lack catalytic activity) employ innovative and unexpected mechanisms to regulate biological functions. Pseudoenzymes perform these important roles by allosterically activating catalytically competent enzymes, acting as crucial scaffolds and as molecular switches. The best studied pseudoenzymes are pseudokinases, which have been covered in numerous reviews [11–15]. Around 50 proteins containing the kinase fold have been classed as pseudokinases, representing ∼10% of the human kinome . While some predicted pseudokinases have shown vestigial kinase activity in vitro, the majority are classed as true pseudokinases with no catalytic activity, and perform important biological roles as pseudoenzymes. It is now clear that almost every enzyme family contains some inactive ‘rogue’ members. The precise fraction may vary depending on the enzyme class, but in the relatively small number of families studied, ∼5–15% of proteins are inactive pseudoenzymes [17–20]. This is true for pseudokinases, pseudoproteases, pseudo-E2 Ub ligases and pseudo-DUBs [21–23].
The human genome contains 99 known DUBs (Figure 2) and this number is likely to increase as new members and families are discovered . Of the 99 known DUB proteins, 11 are predicted to be catalytically inactive pseudoenzymes and classed as pseudo-DUBs. Interestingly, the number of pseudo-DUBs differs widely for each family, and two families, namely UCH and Josephins, have no pseudo-DUBs. The JAMM/MPN family contains the largest number (seven pseudo-DUBs), followed by the USP family with two inactive pseudo-DUBs, while the OTU and MINDY families each contain one pseudo-DUB (Figure 2). Despite the lack of catalytic residues, pseudo-DUBs perform regulatory roles in many biological functions, such as gene expression and regulation [24–26], DNA damage repair [27–29], protein turnover [30,31], cytokine signalling and immune responses .
The JAMM/MPN family of DUBs contains 14 members in humans. They share a common metalloprotease domain architecture, denoted the MPN domain, harbouring a zinc chelating active site [33–35]. Seven out of the 14 family members are predicted to be catalytically active (denoted MPN+), while the others, which bear substitutions to essential catalytic residues, are predicted to be catalytically dead (denoted MPN−). MPN family proteases typically function within larger multi-subunit complexes, which imparts great potential for multi-layer regulation.
The MPN family DUBs regulate diverse aspects of cellular biology, most notably DNA repair through BRCC36 (MPN+) in the BRCA1-A complex (BRCC36–ABRAXAS1–BRCC45–MERIT40–RAP80 complex) [29,36–38], interferon receptor signalling through BRCC36 (MPN+) in the BRISC–SHMT2 complex (BRCC36–ABRAXAS2–BRCC45–MERIT40 complex) [32,39] (Figure 3), and protein degradation through Rpn11/PSMD14 (MPN+) in the proteasome lid complex . The CSN5–CSN6 MPN+–MPN− pair is part of the Cop9 signallosome (CSN) complex that deneddylates cullin proteins modified with the ubiquitin-like (UBL) small protein NEDD8. Thus, CSN5 is a deneddylase rather than a DUB.
BRCC36 functions as part of two distinct protein complexes.
Interestingly, many of the MPN− pseudo-DUBs directly interact with active MPN+ domains to form stable heterodimers (Figure 4). In a remarkable analogy to kinase-pseudokinase heterodimers where a pseudokinase allosterically activates a kinase [12,41,42], some pseudo-DUBs also allosterically activate DUBs (Figures 4A and 5A).
Heterodimerisation of DUBs with inactive pseudo-DUBs.
Mechanism of MPN domain heterodimerisation.
BRCC36–ABRAXAS1 and BRCC36–ABRAXAS2 (BRCC36–KIAA0157)
The active DUB BRCC36 (MPN+) is found in two macromolecular complexes in the cell (Figure 3), depending on which partnering MPN− pseudo-DUB it interacts with (ABRAXAS1 or ABRAXAS2). The BRCC36–ABRAXAS1 complex translocates to the nucleus via the ABRAXAS1 nuclear localisation signal (NLS) and is part of a larger DUB complex called the BRCA1-A complex. This complex localises to sites of DNA damage (e.g. a double-strand break) through RAP80 anchoring to K63-linked poly-Ub chains (Figure 3). Therefore, the BRCA1-A complex plays at least two roles that ultimately lead to DNA damage repair: (1) binding and cleaving K63-linked poly-Ub chains and (2) recruitment of BRCA1 to sites of DNA damage.
In the cytoplasm, BRCC36 interacts with ABRAXAS2 (aslo known as KIAA0157) and is part of a larger complex called BRISC–SHMT2 (Figure 3). This cytoplasmic DUB complex interacts with and deubiquitylates interferon receptors 1 and 2 (IFNAR1/2) and prevents the receptors from being prematurely endocytosed and entering the lysosomal degradation pathway. Thus, the BRISC–SHMT2 complex regulates interferon-dependent immune response by stabilising the interferon receptors and ensuring their availability at the membrane .
BRISC deficiency in mice due to genetic deletion of the BRISC specific pseudo-DUB ABRAXAS2 resulted in resistance to bacterial lipopolysaccharide . Interestingly, the mice did not display any adverse phenotypes or deficiency in the DNA damage response, raising the possibility that inhibitors of pseudo-DUB function in the BRISC may have clinical utility against disease processes stemming from elevated inflammatory cytokine signals.
Biochemical studies demonstrated that BRCC36 is inactive in isolation and that heterodimerisation with the pseudo-DUB ABRAXAS2 was required for DUB activity [43–45]. The crystal structure and biochemical studies of BRCC36–ABRAXAS2 (aslo known as BRCC36–KIAA0157) provided insights into the mechanism of BRCC36 activation . BRCC36 forms an obligate heterodimer with ABRAXAS2 and interactions are mediated via MPN domains and C-terminal helices from both proteins. The C-terminal helical bundle participates in higher order interactions to form a dimer of heterodimers (super dimer), which constitutes the minimally active assembly capable of cleaving Lys63-linked poly-Ub chain substrates (Figures 4A and 5A). A forced BRCC36–BRCC36 homodimer was also observed by X-ray crystallography; however, the BRCC36 active site residues in this crystal structure were not optimally positioned for catalysis (Figure 5A). Instead, BRCC36 requires direct interactions with ABRAXAS2 to attain an active conformation (Figure 5A) .
The JAMM motif is typical of MPN domain-containing proteases and is required for catalysis. It contains five conserved residues from four key structural motifs, namely:
(1) E-loop, which houses a glutamic acid residue (E30, Figure 4A). Its role is to co-ordinate a catalytic water molecule.
(2) H-strand, which houses the His-X-His motif with two histidine residues (H94 and H96, Figure 4A) involved in Zn2+ binding.
(3) S-loop, which houses a serine residue (S104, Figure 4A) involved in stabilising the transition state intermediate.
(4) D-helix, which houses an aspartic acid residue (D107, Figure 4A) involved in Zn2+ binding.
ABRAXAS1 and 2 both have several changes in the JAMM motif that preclude function as an active DUB, with changes in the E-loop, H-strand and D-helix (Supplementary Figure S1). The structure of BRCC36–ABRAXAS2 complex revealed that ABRAXAS2 is incapable of interacting with Zn2+ and is therefore a pseudo-DUB . Structural comparison of active BRCC36 with the pseudo-DUB ABRAXAS2 revealed that while there are non-conservative changes in the active site residues, the overall MPN fold and the position of the JAMM motif residues are the same for both active and inactive MPN domains (Figure 4A). As a result, the MPN fold maintains its structural integrity, despite the loss of Zn2+ and several changes to conserved catalytic residues. Interestingly, the positive charge lost by the absence of the Zn2+ ion is compensated by a guanidinium group from Arg103 (ABRAXAS2) which replaces the Zn2+-interacting His96 (BRCC36) residue (Figure 4A).
The biochemical and structural data thus far support the notion that ABRAXAS2 and the related pseudo-DUB ABRAXAS1 serve as allosteric activators of BRCC36 and have a non-catalytic role as molecular scaffolds for the correct assembly of BRCA1-A and BRISC–SHMT2 complexes.
Rpn11–Rpn8 (PSMD14–PSMD7 in humans)
Yeast Rpn11 and Rpn8 (PSMD14 and PSMD7, respectively, in humans) are part of the 19S regulatory particle of the proteasome and play a key role in Ub processing and recycling. The MPN+ domain protein Rpn11 removes poly-Ub chains from ubiquitylated substrates before they translocate to the 20S proteasome. Similar to BRCC36, Rpn11 partners with the pseudo-DUB Rpn8 (Figure 4B), and is part of a larger macromolecular complex. Rpn8 supports the role of Rpn11 through direct interactions and is required for full Rpn11 catalytic activity [31,46].
The yeast pseudo-DUB Rpn8 contains three notable changes in catalytic residues: E-loop (Glu to Arg), H-strand (His-X-His motif changed to His-X-Gly) and S-loop (Ser to Arg). Changes in the H-X-H motif render Rpn8 incapable of binding the catalytic Zn2+ (Figure 4B). Interestingly, the overall fold and integrity of the MPN domain remains unaffected by compensating interactions in the form of a salt bridge between Asp103 (D-loop) and Arg32 (E-loop), and Arg100 (S-loop) hydrogen bonding with a backbone carbonyl group from the E-loop.
Collectively, these active site residue changes abrogate Rpn8 catalytic activity. It is still unclear if Rpn8 allosterically activates Rpn11 or if its main role is to stabilise the Rpn11 fold within the multiprotein complex.
The COP9 signalosome (CSN complex) has eight subunits amounting to ∼450 kDa . CSN5, the active protease subunit, removes Nedd8 from cullins and thus regulates the activity of cullin RING E3 ligases [48,49]. CSN5 and CSN6 genes are overexpressed in many cancers, and the Cop9 signalosome is implicated in Ub-mediated degradation of several cancer-related proteins: p27, p53, Mdm2, Smad7, Runx3, Id1, Skp2 and HIF1 . Despite the fact that CSN5 is not specific for cleaving Ub, CSN5 (MPN+) and CSN6 (MPN−) heterodimers are similar in topology to Rpn11–Rpn8 complexes (Figures 4B,C and 5B) and represent a recurring theme in MPN domain heterodimerisation.
The pseudo-DUB CSN6 contains four notable changes in the E-loop (Glu to Gln), H-strand (His-X-His motif changed to Thr-X-Gly) and S-loop (Ser to Asn). Changes in the H-X-H motif render CSN6 incapable of binding the catalytic Zn2+ (Figure 4C). Interestingly, the position of the D-helix remains unchanged by compensating interactions between Asp135 and Lys153 from a neighbouring β-strand. This ensures minimal disruption to the CSN6 MPN fold in the absence of the Zn2+ ion.
Collectively, these active site residue changes abrogate CSN6 catalytic activity. It is still unclear if CSN6 allosterically activates CSN5 or if its main role is to stabilise the CSN5 fold within the multiprotein complex.
Heterodimerisation of CSN5–CSN6, Rpn11–Rpn8 and eIF3f–eIF3h (discussed below) is topologically similar, but differs from the heterodimer formed by BRCC36–ABRAXAS2 (Figure 4, left panels and Figure 5). This may be because BRCC36–ABRAXAS1/2 require further assembly into a dimer of heterodimers (super dimer), which represents the active form of the enzyme (Figures 4A and 5A) . This diverse mode of activation could be exploited to develop selective BRCC36 inhibitors by targeting the dimerisation and higher order super-dimerisation sites, instead of the common MPN+ active site.
Pseudo-DUBs as scaffolding modules of large complexes
Pseudo-DUBs can also act as molecular scaffolds of many macromolecular complexes, which are central to cellular biology. Examples include the spliceosome, eukaryotic initiation factor 3 (eIF3) complex and the RNA deadenylating complex Pan2–Pan3 (Figure 6).
Pseudo-DUBs as molecular scaffolds.
Prp8 and USP39
Splicing of pre-mRNA is an essential process performed by the spliceosome complex and a crucial step in gene expression and protein translation. Correct processing of introns from pre-mRNAs requires a high degree of accuracy and the spliceosome machinery is highly regulated. The pseudoenzyme Prp8 is central to the assembly of this large multiprotein–nucleic acid complex (Figure 6A). Prp8 is a large protein (human Prp8 has 2335 amino acids) and contains four different inactive pseudoenzyme domains: (1) a restriction endonuclease (RE) homology domain (pseudo-RE), (2) a reverse transcriptase (RT) homology domain (pseudo-RT), (3) an RNaseH homology domain (pseudo-RNAseH) and (4) an MPN− domain (pseudo-DUB). Each of these domains has a role in binding to either a protein or nucleic acid component of the spliceosome.
The Prp8 MPN− domain contains two active site residue changes, one in the E-loop (Glu changed to Gln) and the other in the H-strand (His-X-His motif changed to His-X-Gln). It was shown that the single His to Gln change in the H-X-H motif of yeast Prp8p is sufficient to completely abolish Zn2+ binding [51,52] and potentially catalysis; however, the Prp8 MPN− domain retains the ability to interact with Ub . It is as yet unclear if the Prp8 pseudo-DUB domain has other roles in the spliceosome beyond acting as a macromolecular scaffold.
Interestingly, the spliceosome complex also contains the pseudo-DUB USP39 (Sad1 in yeast) , although the role of this pseudoenzyme in the splicing reaction is not clear. Mutations in the active site of key catalytic residues (Cys to Asp and His to Ser) render USP39 inactive (Supplementary Figure S2). Similarly, yeast Sad1 is devoid of catalytic activity, although Sad1 is still capable of interacting with Ub . As with the Prp8 MPN− domain, the exact biological role of Sad1 Ub interaction is not clear, although they could serve as readers of Ub signalling by interacting with Ub and/or Ub chains.
eIF3f and eIF3h are MPN− domain pseudo-DUBs that are part of the eIF3 complex. Binding of initiation factors is required to initiate protein synthesis and recruit transfer and messenger RNAs to the 40S subunit before assembly of active ribosomes. eIF3 is involved in both ribosome biogenesis and protein synthesis . The full eIF3 complex consists of 13 subunits and amounts to ∼800 kDa . Interestingly, eIF3 displays a significant degree of similarity with the CSN complex and both contain an MPN–MPN heterodimeric core. In the eIF3 complex, eIF3f and eIF3h are pseudoenzymes with their main role thought to be of a scaffolding nature (Figure 6B). Both eIF3f and eIF3h are involved in direct binding to other members of the eIF3 complex and although they are devoid of catalytic activity, their scaffolding roles are essential [57,58].
The pseudo-DUB eIF3f contains five notable residue changes in the catalytic motifs: E-loop (Glu to Arg), H-strand (His-X-His motif changed to Ala-X-Gly), S-loop (Ser to Thr) and D-Helix (Asp to Ser). Changes in the H-X-H motif and D-helix render eIF3f incapable of binding the catalytic Zn2+ and therefore inactive.
The pseudo-DUB eIF3h also contains five notable residue changes in the catalytic motifs: E-loop (Glu to Val), H-strand (His-X-His motif changed to Gln-X-Thr), S-loop (Ser changed to Thr) and D-Helix (Asp to Leu). Similar to eIF3f, changes in the H-X-H motif and D-helix render eIF3h incapable of binding the catalytic Zn2+ and inactive.
Taken together, active site amino acid substitutions in eIF3f and eIF3h, which collectively abrogate catalytic function, suggest that their primary function is to serve as molecular scaffolds for assembly of the eIF3 complex.
The yeast Pan2–Pan3 deadenylating complex takes part in shortening of poly(A) tails of mRNA . The Pan2 orthologue in humans is the pseudo-DUB USP52 and many of the functions are evolutionarily conserved. USP52 was recently described as an important regulator of the HIF1A-mediated hypoxic response, by regulating Hif1A mRNA stability .
USP52/Pan2 lack the catalytic cysteine (Cys mutated to Ala) and histidine (His mutated to Ser) residues required for DUB activity and are classed as pseudo-DUBs. However, Pan2 contains a catalytically active RNAse domain fused to the pseudo-DUB domain (Figure 6C). Interestingly, Pan3 contains a pseudokinase domain that dimerises, resulting in a Pan2–Pan3 complex with 1 : 2 stoichiometry (Figure 6C). Dimerisation of the Pan3 pseudokinase and the interactions of the Pan2 pseudo-DUB domain are both required for RNAse activity and biological function [60,61]. Furthermore, it is possible that USP52 binds Ub and acts as a UBD and a reader of Ub signalling.
Predicted pseudo-DUBs with unknown functions
FAM105A is an OTU domain containing protein that is closest to OTULIN (aslo known as FAM105B or Gumby) (Figure 2). The latter is an active DUB, which specifically cleaves Met1-linked linear Ub chains involved in cellular signalling [62,63]. FAM105A lacks the catalytic cysteine residue, which has been mutated to an Asp (Supplementary Figure S3). FAM105A is conserved as an inactive pseudo-DUB in vertebrate species, including mammals, birds, frogs and a turtle species (Pelodiscus sinensis), where the active site Cys is replaced by Asp or Ser (data not shown). Therefore, FAM105A is a predicted pseudo-DUB, the function of which remains to be identified. Interestingly, the Drosophila genome contains two OTU domain pseudo-DUBs, although these are related to OTUD4 and are involved in regulation of apoptosis . Similar pseudo-OTUD4 proteins are found in the tunicate Ciona and in the brachiopod Lingula.
The MINDY DUB family was only recently identified and contains four members, MINDY1–4 . MINDY DUBs are active against K48-linked poly-Ub chains, displaying exquisite specificity . Interestingly, a previously unannotated member (MINDY4B) is predicted to have the same fold, but lacks all three catalytic residues (Supplementary Figure S4). MINDY4B appears conserved as an inactive pseudo-DUB across vertebrates as well as in some invertebrates, for example in the tunicate Ciona intestinalis, in the aphid Acyrthosiphon pisum and in the crab Limulus polyphemus. The function of MINDY4B predicted pseudo-DUBs remains unknown.
Evolution of pseudo-DUBs
One striking observation is that 50% of JAMM/MPN DUBs are classed as pseudo-DUBs, leading to questions as to how these evolved. A possible explanation is that most JAMMs have a propensity to function as dimers with examples of JAMM/MPN functional dimers observed in many eukaryotes (e.g. yeast, ants, rodents and humans), as well as archaea . It is possible that after gene duplication events, accumulation of mutations in active site residues of one protomer is tolerated, since dimerisation ensures that at least 50% enzyme activity is always present. Over time, the supporting role as an allosteric activator or as a scaffolding protein may be more important than catalytic activity, thus leading to the evolution of pseudoenzymes. This would represent a gain in evolutionary terms, since repurposing an established fold may be more advantageous than creating a new one.
Pseudo-DUBs as drug targets
Given that many pseudo-DUBs take part in influencing major cellular functions, including activity of the proteasome (Rpn11–Rpn8), protein synthesis (Prp8 and USP52), Cop9 signallosome (CSN5–CSN6), DNA damage repair (ABRAXAS1) and interferon signalling (ABRAXAS2), they present an opportunity for therapeutic intervention in disease conditions. Developing inhibitors of active DUBs represents challenges, since compounds that target JAMM domain DUBs have so far only targeted the active site Zn2+ moiety [66,67], rendering them relatively non-specific. Similarly, many compounds that target other DUB families react with the active site cysteine [4,68]. Therefore, developing small molecule inhibitors against pseudo-DUBs by exploiting their allosteric mode of action rather than their active site may be a more selective way of inhibiting active DUB complexes such as Rpn11–Rpn8, CSN5–CSN6 and BRCC36–ABRAXAS1/2.
The DUB families, like most other enzyme families, contain a fraction of pseudoenzyme members that lack catalytic activity. These pseudo-DUBs have important biological functions involved in cell signalling, protein turnover and Ub recycling. Despite their lack of catalytic activity, they have evolved different ways to influence cell signalling and cell biology. One key observation is the various mechanisms of action that pseudo-DUBs employ, in many cases still influencing Ub signalling systems by partnering with active DUBs. Pseudo-DUBs are often found as part of large macromolecular complexes where they act as allosteric activators of other enzymes, but also as molecular scaffolds required for complex assembly.
While the roles of most pseudo-DUBs are known, many questions still remain. For instance, what is the functional relevance of Prp8 and USP39/Sad1 interaction with Ub? Do other pseudo-DUBs retain Ub-binding ability and Ub chain selectivity, thereby acting as readers of Ub signalling? Pseudo-DUBs that interact with mono-Ub or poly-Ub chains may still have the same effect as active DUBs through a ‘substrate trapping’ mechanism. For instance, recent studies show that alanine substitutions of active site residues can convert DUBs into avid UBDs . By interacting with Ub or poly-Ub, pseudo-DUBs can sterically hinder binding of other proteins that act as readers of Ub signals, thus reversing the signal in the same way that an active DUB can by removing Ub.
Finally, some pseudo-DUBs like FAM105A and MINDY4B remain unstudied with no assigned functional roles, despite their evolutionary conservation. As new DUBs continue to be discovered, the pseudo-DUB field is also likely to see additional new members. These have the potential to reveal new mechanisms of action of how pseudoenzymes work as well as important functional and biological insights.
MIU-containing novel DUB family proteases
ovarian tumour proteases
ubiquitin C-terminal hydrolases
Our work on pseudoenzymes is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [200523/Z/16/Z] to E.Z., and a grant from the Polish National Science Centre [2014/15/B/NZ1/03359] to K.P.
We thank Roger Greenberg, Richard Bayliss and Derek Ceccarelli for critically reading of this manuscript and useful discussions.
The Authors declare that there are no competing interests associated with the manuscript.