Ubiquitylation is a major posttranslational modification that controls most complex aspects of cell physiology. It is reversed through the action of a large family of deubiquitylating enzymes (DUBs) that are emerging as attractive therapeutic targets for a number of disease conditions. Here, we provide a comprehensive analysis of the complement of human DUBs, indicating structural motifs, typical cellular copy numbers, and tissue expression profiles. We discuss the means by which specificity is achieved and how DUB activity may be regulated. Generically DUB catalytic activity may be used to 1) maintain free ubiquitin levels, 2) rescue proteins from ubiquitin-mediated degradation, and 3) control the dynamics of ubiquitin-mediated signaling events. Functional roles of individual DUBs from each of five subfamilies in specific cellular processes are highlighted with an emphasis on those linked to pathological conditions where the association is supported by whole organism models. We then specifically consider the role of DUBs associated with protein degradative machineries and the influence of specific DUBs upon expression of receptors and channels at the plasma membrane.
The ubiquitin-proteasome system (UPS) is firmly established as an all-pervasive regulator of cellular function in eukaryotes (73). The tagging of proteins with the 76-amino acid polypeptide ubiquitin not only provides a signal for protein degradation, but also a reversible posttranslational modification used to modulate enzymatic activity and protein interactions. The deubiquitylases (DUBs) are a family of ∼90 enzymes that control the cellular flux of ubiquitin by its removal from substrate proteins (13, 120, 187, 209, 210). In this review, we shall detail the five subfamilies of mammalian DUBs. We provide an updated analysis of their sequence and structural features. We discuss their cellular and organismal expression patterns and highlight associations with disease. Finally, we turn to their control of the cellular response to its environment, through influences upon the trafficking and activity of receptors and channels.
The seven internal lysine residues intrinsic to ubiquitin (Lys 6, 11, 27, 29, 33, 48 and 63) all provide sites for the generation of an isopeptide bond with the COOH terminus of another ubiquitin (Figure 1A). Linear ubiquitin chains, linked by peptide bonds between the NH2 and COOH termini, can be generated de novo as well as being directly translated from two of the four pro-ubiquitin genes (269). Types of polyubiquitin chains display varying topologies, illustrated by the dispositions of the proximal (free COOH terminus) ubiquitin compared with the distal ubiquitin in the overlaid crystal structures for five varieties of di-ubiquitin molecules so far determined (Figure 1B) (75, 133). Structures of linear ubiquitin and Lys63-linked di-ubiquitin suggest open chain configurations that have been likened to “beads on a string.” Others are more compacted, providing restricted access to some surfaces. This is illustrated in Figure 1C for a hydrophobic patch centered around Ile44, which is often critical for protein interactions. In solution, a number of conformations likely exist in dynamic equilibrium for a given chain type, which can be selected for and stabilized by protein-protein interactions (26, 263, 288).
Polyubiquitin chains may be homogeneous assemblies of a single linkage type or heterogeneous linkages, which can include more than one linkage type to a common proximal ubiquitin (branched chains). In principle, DUBs can hydrolyze ubiquitin chains from the ends (exo-activity) or from within the polymer (endo-activity). Throughout this review the standard protease nomenclature of Schechter and Berger is adopted; interaction sites associated with the substrate are denoted as P1, P2 etc NH2 terminal to the scissile bond and P1′, P2′ etc towards the COOH terminus, with the corresponding sites on the enzyme denoted S1, S2 and S1′, S2′ etc (226). The ubiquitin conjugated to substrate protein defines the proximal ubiquitin of a chain. Thus, when the proximal ubiquitin is being cleaved, the P1′, P2′ sites correspond to DUB binding sites on the ubiquitylated protein itself.
The complexity of ubiquitin chain types bears analogy to protein glycosylation, but its significance is still under active consideration (122, 133). Proteomic studies reveal that each of the isopeptide linkage types is represented to a significant extent in both yeast and mammalian cells (54, 117, 196, 282, 298). The attachment of ubiquitin chains with any isopeptide linkage excepting Lys63 appears to target substrates to the proteasome in vivo (54, 172). Lys63 linkages may be particularly important in targeting endosomal proteins to the lysosome (38, 142), whilst linear ubiquitin chains play a critical role in the NFκB signaling pathway (269). One prevailing notion is that these different linkage types may provide some specificity for interaction with proteins containing different ubiquitin binding domains, particularly when such domains are organized in tandem (182, 204).
II. CATALYTIC MECHANISM
The DUBs can be subdivided into five families based on the architecture of their catalytic domains: ubiquitin specific proteases (USPs), ubiquitin COOH-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Josephins, and the JAB1/MPN/MOV34 family (JAMMs). The first four families are cysteine proteases, which rely on a catalytic triad of conserved amino acids, in common with classical cysteine proteases such as papain (245). A nearby histidine lowers the pKa of the catalytic cysteine residue facilitating a nucleophilic attack, whilst a third residue (Asp or Asn) is normally required for alignment and polarization of this His residue. One feature of this mechanism is the formation of an acyl intermediate by the covalent linkage between the cysteine and the carboxyl group, which will be generated upon scission (Figure 2A). Whilst the cysteine protease DUBs have divergent catalytic domain structure, once bound to the ubiquitin COOH terminus, the catalytic residues superpose with little deviation (119). In distinction, JAMMs are Zn2+ metalloproteases, in which invariant His, Asp, and Ser residues coordinate the catalytic zinc. The reaction mechanism is predicted to be similar to other metalloproteases such as thermolysin (Figure 2B). The catalytic zinc ion is coordinated by two His, an Asp, and a water molecule. A neighboring Glu accepts a proton from the water molecule leaving a hydroxyl ion, which attacks the substrate bond at the carbonyl carbon. The transient tetrahedral intermediate, which is formed, collapses resulting in scission, with the hydroxyl group from the water replacing the leaving group at the COOH terminus of the distal ubiquitin (Figure 2B).
Aside from the catalytic domain, DUBs often contain other domains and shorter structural motifs, which may regulate activity and govern interactions. This is most prominent for the USP family. We provide an updated set of annotations across the DUB superfamily arranged according to sequence homology of their catalytic domains (Figure 3). This includes several newly recognized structures, such as PH domains in USPs 26, 29, and 37, specific insertions within the catalytic domain (289) and intrinsically disordered regions that are statistically more likely to provide sites for posttranslational modifications and protein-protein interactions. Table 1 provides an overview of the counterparts to human DUBs that are found in other species routinely used in experimental biology, revealing a core set of 16 DUB family members common to humans, Danio rerio (zebrafish), Drosophila melanogaster, Caenorhabditis elegans, and at least one or other commonly used strain of yeast (additional accession information is provided in Supplementary Table 1. The online version of this article contains supplementary material.). We provide a comprehensive analysis of the evolutionary relationships between family members in Supplementary Figure 1. A systematic study of the 41 Drosophila DUBs has revealed key roles in development, adult motility, and longevity (260), while a morpholino-based functional screen has identified 57 DUBs that play key roles in early development of zebrafish (259).
Quantitative proteomic analysis reveals that protein copy numbers of individual DUBs span several orders of magnitude in model cell lines such as Swiss 3T3 cells (79, 231) (Figure 4A). Highly expressed DUBs (>300,000 copies/cell) include components of the proteasome and COP9 signalosome, UCHL1 and UCHL3, which suppress the formation of ubiquitin adducts, and OTUB1, which is the most abundant active DUB represented in 3T3 cells. With the exception of UCHL1, these proteins tend to be highly expressed across all cell lines that have been scrutinized at the proteomic level (79, 231). While some expressed DUBs may be below the detection limit by current mass spectrometry, copy numbers in the low hundreds have been determined. Examples include the mitochondrial DUB, USP30, and the sumoylation target USP25 (173, 181, 231).
Three systematic studies have mapped the subcellular distributions of human DUBs in cultured cells and the complement of DUBs found in Saccharomyces pombe (128, 244, 261). Figure 4B illustrates prominent subcellular sites of accumulation for individual mammalian DUBs, derived from these studies and from more specific investigations. At the organismal level, based on transcriptional profiling, many DUBs are relatively overexpressed in the brain, hematopoietic system, and reproductive organs. Figure 5 provides a preliminary map of tissue “hot spots” for particular DUBs. Examples of transcriptional control of DUBs include vasopressin and aldosterone induction of USP10 and USP2-45, respectively (24, 68).
IV. CLEAVAGE SPECIFICITY
A. Chain Linkage Specificity
One aspect of the appendage of particular polyubiquitin chain types to substrate proteins is that it can then restrict their processing to chain-specific deubiquitylase activities (Table 2). Early studies indicated a stringent preference of the JAMM family member, AMSH, for Lys63-linked chains over Lys48 chains (170, 171), which has now been extended to exclude activity towards other chain types (27, 123). Several other JAMM family members such as AMSH-LP and BRCC36 have similar selectivity for Lys63-linkages (45, 70, 225). A corollary of this stringency is that these DUBs may be unable to efficiently remove the proximal ubiquitin moiety that is directly attached to substrate proteins. However, other JAMMs such as MYSM1 and POH1 are capable of making this cleavage (284, 297).
The availability of substrates bearing all eight simple ubiquitin chain linkage types has prompted more extensive surveys of DUB specificity in vitro (27, 67, 123, 154, 268) (Table 2). In general, while the USP family of proteins displays variations in kcat and Km over orders of magnitude, they exhibit little specificity for particular isopeptide chain linkages (67, 123). Notable exceptions are provided by USPL1 which is a SUMO-specific protease (230), USP18 which may be specific for the ubiquitin-like modifier, ISG15 (164) and CYLD which shows a marked preference for the open conformation presented by Lys63 and linear ubiquitin chains (27, 123). The ubiquitin binding site of USP family enzymes such as USP21 contact Lys6, which precludes endoactivity for Lys6-linked chains. USPs can only interact with the distal end of this particular ubiquitin chain type and process it sequentially. In contrast, the OTU family protein OTUD3 can cleave such chains at any position (95). Remarkably, the OTU family catalytic domains display a wide diversity in chain preferences, despite a high degree of structural conservation. For example, OTUB1 and OTUB2 show opposite proclivities for Lys63- and Lys48-linked chains, respectively, while Cezanne/OTU7B and TRABID are most active towards Lys11- and Lys29-linked chains, respectively (27, 60, 154, 268, 270). One study has suggested that the Josephin protein Ataxin-3 shows a preference for Lys63- over Lys48-linked chains, but that branched chains containing both linkages provide a preferred substrate, from which Lys63 linkages are preferentially processed (279).
B. Discrimination Between Ubiquitin and Ubiquitin-like Modifications
Several ubiquitin-like proteins including ISG15, Nedd8, and SUMO modify proteins using similar mechanisms to ubiquitin itself (94). Whilst these all present ubiquitin-like folds, DUBs are nevertheless able to discriminate between these molecules. In some cases (SUMO, Atg12, FAT10), this is determined by divergent amino acid sequences adjacent to the COOH-terminal Gly-Gly residues, a region which corresponds to 71LRLRGG76 in ubiquitin. However, a high degree of similarity to ubiquitin in this region for Nedd8 (contains Ala72 for Arg72) and identity in the case of ISG15 allow for cross-reactivity (5). USP21 shows activity towards ubiquitin and ISG15 but not Nedd8. The structure of USP21 in complex with a noncleavable form of linear di-ubiquitin aldehyde provides an explanation of this specificity that can be generalized to the entire USP family. The Arg72 residue of ubiquitin interacts with an invariant Glu residue (Glu304 in USP21) to enhance affinity but furthermore, modeling of Nedd8 into the ubiquitin S1 site also results in steric clashes and charge repulsion (287).
V. REGULATION OF DUB ACTIVITY
Like most cellular enzymes, the activity of DUBs can be controlled through multiple mechanisms. Several DUBs require assembly into large multimolecular complexes for full activation, exemplified by the proteasomal DUBs which are discussed in detail below. Another example is provided by the allosteric activation of USP22 by multiple components of the SAGA coactivator complex (118, 138, 223). Simpler instances of allosteric regulation are found with the increase in kcat following interaction of USP1, USP12, and USP46 with UAF1 (WDR48) (40, 41). However, the interaction between USP1 and UAF1 is itself regulated by CDK1-mediated serine phosphorylation of USP1 (267). The COOH-terminal region of USP7 contains 5 Ubiquitin-like (Ubl) domains organized into 2-1-2 Ubl units, the last pair of which (HUBL-45) activate USP7 by 100-fold (66, 71). The metabolic enzyme GMPS binds to the first three Ubl domains and hyperactivates USP7 by stabilization of the HUBL-45 interaction with the catalytic domain (66). Other examples of intramolecular domains influencing activity include an increase in Km of USP4 mediated by a Ubl domain inserted within the catalytic domain itself (160) and an increase in kcat of USP16 attributable to its ZnF-UBP domain (67). Interaction of DUBs with proteins bearing ubiquitin binding domains, exemplified by the interaction between the endosomal DUBs AMSH and USP8 with components of the ESCRT-0 complex, can enhance activity by providing more effective capture of substrate and reducing the apparent Km (171, 218).
Cross-talk between phosphorylation and ubiquitin modification is a significant aspect of intracellular signaling networks (101, 174). In a most direct case, the enzymatic activity of OTUD5 (DUBA) is entirely contingent on phosphorylation at a single serine residue, which interacts directly with the COOH-terminal tail of ubiquitin (97). Differential phosphorylation of USP8 at S680 and dephosphorylation of USP37 during M-phase of the cell cycle correspond with enhanced and reduced activity, respectively (100, 176, 190). USP8 also undergoes translocation to endosomes following acute EGFR stimulation (219), whilst USP4 translocates from nucleus to cytoplasm following phosphorylation by Akt (290). These examples illustrate a further mechanism of regulation, by dynamically changing the palette of associates and substrates to colocalizing proteins. Other posttranslational modifications are emerging as modifiers of activity, including ubiquitylation itself. Ubiquitylation of Ataxin-3 in the vicinity of the catalytic site enhances its activity (255), whilst sumoylation of USP25 inhibits activity (173). Interestingly, the catalytic cysteine of the cysteine protease DUBs is widely subject to reversible inactivation by modification with reactive oxygen species (ROS), similarly to protein tyrosine phosphatases (48, 62, 132, 144, 217).
VI. THE FIVE DUB FAMILIES: ROLES IN HEALTH AND DISEASE
In the following section we discuss the major characteristics of each subfamily of DUBs, highlighting examples of particular physiological significance or where linkage to disease is well established. Generically DUB catalytic activity may be used to 1) maintain free ubiquitin levels, 2) rescue proteins from any of the ubiquitin-mediated degradation pathways (proteasomal, endosomal, and autophagosomal), and 3) control the dynamics of ubiquitin-mediated signaling events (120). Figure 6 and Supplementary Table 2 provide a summary of DUBs linked to disease through mutational or expression analysis. We also highlight examples where mouse models have provided significant physiological insight and further collate this information in Table 3.
A. USP Family
The USPs are the largest family of DUBs and contain an assortment of accessory domains (Figure 3). Several of these are drawn from inserts within the catalytic domain (289), which have been variously shown to influence activity (160) or localization (121, 254). Multiple USPs carry a ZnF-UBP binding domain, of which a subset (USP3, USP5, USP13, USP16, USP44, USP45, USP49), have been shown to (or by homology are predicted to) specifically recognize the free COOH terminus Gly-Gly motif of ubiquitin (21, 183, 193, 207). This interaction underpins the central function of the abundant USP5 (isopeptidase T) in processing newly synthesized linear polyubiquitin chains. It provides selectivity for unanchored ubiquitin and is necessary for optimal catalytic activity (207), whilst in combination with other ubiquitin binding domains it contributes to a high avidity for tetra-ubiquitin (208). However, it is possible that the ZnF-UBP domain also presents a protein interaction module for the 72 other human proteins which possess COOH-terminal di-Gly motifs. One interesting member of this list is histone H4, which could serve to recruit both USP3 and USP16 to histone complexes, where they have been proposed to deubiquitylate histone H2A (87, 110, 183).
USP7/HAUSP has garnered a great deal of attention because of the prominence of some well-characterized substrates that are associated with tumor suppression (p53/MDM2, FOXO4, PTEN, INK4a) (50, 51, 151, 152, 163, 242, 262). USP7 −/− mice suffer embryonic lethality, in part due to p53 activation, as embryonic development is extended in USP7/p53 double-knockout embryos (124, 125).
Several USPs are associated with DNA repair pathways, most prominently USP1. Its deubiquitylating activity regulates Fanconi anemia, complementation group D2 (FANCD2) and proliferating cell nuclear antigen (PCNA), which are both components of the cross-link repair pathway (41, 98, 145, 186). Recently, USP1 has also been shown to control the stability of ID (inhibitors of DNA binding) proteins, which inhibit differentiation and maintain stem cell characteristics in osteosarcoma. USP1 overexpression impairs osteoblastic differentiation of mesenchymal precursors, while depletion of USP1 induced such differentiation in osteosarcoma cells (277). Accordingly, USP1 −/− mice show defects in skeletal development including ossification of the cranial and long bones, further to a genomic instability and Fanconi anemia phenotype previously reported (116, 277).
B. UCH Family
Structural studies indicate that UCHL1 and UCHL3 substrates are limited by a requirement for the leaving group to pass through a loop region, which sits directly over the active site (55, 108, 109, 175). Accordingly, UCHL1 and UCHL3 both show negligible in vitro activity against Lys48, Lys63, and linear ubiquitin chains (123). However, the ability to hydrolyze Lys48 and Lys63 linked chains can be conferred upon UCHL3 through expansion of this loop by the insertion of 5 or 10 glycine residues (201). Two classes of physiological substrate have been proposed, based on in vitro enzymatic analysis (140). 1) The proubiquitin genes in most organisms contain head to tail repeats of the ubiquitin sequence with an additional amino acid or short peptide capping the COOH terminus, which is highly variable between species. 2) All of the intermediates in the enzymatic activation of the ubiquitin COOH terminus are thioesters, which can form adventitious adducts by thiol or amine modification that may be recycled by UCHL1/3 action. This function is congruent with the high copy numbers observed for these enzymes in proteomics experiments and their lack of protein-protein interaction domains (Figures 3 and 4) (79, 231). A back of the envelope calculation suggests that without countervailing measures, all free ubiquitin would be converted to glutathione thiol ester or otherwise conjugated with intracellular polyamines within a matter of minutes (199).
The other two UCH family members, the proteasome associated UCHL5/UCH37 and the tumor suppressor BAP1, have more extended cross-over loops, permissive for cleavage of ubiquitin chains (136, 296). It is likely that these longer loops can accommodate di-ubiquitin substrates as a consequence of greater flexibility rather than providing an opening through which the leaving group must pass. For both BAP1 and UCHL5, the in vitro activity against ubiquitin chains observed for the isolated catalytic domain is held in check by inhibitory domains in the full-length proteins. Binding to the appropriate physiological partners relieves this inhibition, providing a control mechanism for the appropriate expression of activity (228, 286).
UCHL1 is one of the most abundant brain proteins, estimated at 1–2% total protein (58, 106, 275). Several lines of evidence link UCHL1 to neurodegenerative conditions. A homozygous missense mutation, identified in three siblings of a Turkish family, has reduced affinity and catalytic activity towards ubiquitin and is proposed to lead to childhood-onset multisystem neurodegenerative syndrome (19). A separate mutation showing reduced activity has been linked to increased risk of Parkinson's disease (PD), but mouse models suggest that this could represent gain, rather than loss, of function (148, 234). Conversely, a common polymorphism, S18Y, may reduce PD's susceptibility in certain populations (165). A proteomic analysis has revealed that UCHL1 is a major target of oxidative damage in Alzheimer's disease (AD) and idiopathic PD brains (35). Transduction of UCHL1, coupled to the HIV-transactivator protein, into mouse hippocampal brain slices alleviates defects induced by treatment with oligomeric Aß protein and in the mouse APP/PSI model of AD. Furthermore, intraperitoneal injections of the UCHL1 fusion protein improve the contextual memory of APP/PSI mice (80). Interestingly, the induced expression of a neuron-specific UCH enzyme has been associated with long-term facilitation in Aplysia (91). Aberrant expression of UCHL1 is observed in a variety of cancer types, including lung, colon, and pancreas (93, 253, 283) and has been functionally associated with the determination of cellular invasive properties and determination of chemosensitivity (28, 115).
Mice lacking UCHL1 due to an intragenic deletion exhibit defective spermatogenesis and gracile axonal dystrophy (Gad), which is thought to reflect defective axonal transport (221). Focal degeneration in the gracile fasciculus, observed in Gad mice, resembles the symptoms associated with chronic deprivation of the antioxidant vitamin E, but cannot be alleviated by vitamin E administration. However, it is interesting to note that upregulation of UCHL1 gene transcription is prominent in skeletal muscles of α-tocopherol (one form of vitamin E)-deficient mice (264). Gad mice show reduced levels of unconjugated ubiquitin in neurons, while expression of UCHL1 in cultured cells and mice enhances the free ubiquitin pool (191).
BAP1 may be the most commonly mutated DUB in cancer. Somatic inactivating mutations have been found at high incidence in uveal melanomas, clear cell renal carcinoma, and pleural malignant mesotheliomas (1, 82, 86). Germ-line mutations have been linked to a tumor predisposition syndrome for melanocytic tumors and mesothelioma (252, 274). BAP1 gene deletion in mice is embryonically lethal, but conditional knockout mice develop a myeloid disorder resembling chronic myelomonocytic leukemia (CMML) (57). BAP1 protein is largely confined to the nucleus, where it interacts with several transcriptional regulators including host cell factor-1 (HCF-1), the polycomb group proteins additional sex-combs like 1 and 2 (ASXL1 and ASXL2), and the DNA binding protein FOXK1, which are likely to form a modular complex (57, 162). Deubiquitylating activity of BAP1 maintains protein levels of the pleiotropic transcriptional regulator HCF-1 and its interacting partner O-linked N-acetylglucosamine transferase (OGT), which itself positively regulates HCF-1 activity by glycosylation (57). This complex plays a critical role in glucose sensing. Levels of the promoter of gluconeogenesis, peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, are increased in euglycemic conditions. It is proposed that N-acetylglucosylation of PGC-1α by HCF-1/OGT promotes its stability, through the recruitment of BAP1 deubiquitylating activity (220).
C. OTU Family
Linkage of OTU proteins to ubiquitin chain processing was first suggested by their binding to active site ubiquitin probes and their structural similarity to cysteine proteases (15, 22, 64). Despite structural conservation within the catalytic domain, the family shows diverse specificity for ubiquitin chain linkages (Table 2). The strongest vein of biological data for this family, which has been reviewed extensively elsewhere (34), links the OTU protein A20 (also known as TNFAIP3) to regulation of the proinflammatory NFκB pathway (88, 161, 236). The A20 gene can be induced by NFκB signaling and operates within a negative-feedback loop to restrict the duration or intensity of signaling. A20 function is particularly complex as it has been proposed to possess both intrinsic DUB and E3-ligase activities, which may coordinate both the assembly and disassembly of ubiquitin chains on substrate proteins. It can also antagonize interactions between various other E3 enzymes with the E2 enzymes UBC13 and UbcH5c (237). Furthermore, ubiquitin chain binding by intrinsic A20 ZnF domains influence ubiquitin dynamics on the NFκB pathway (23, 239). In vitro data indicate DUB specificity for Lys48- over Lys63-linked chains, but cellular models suggest a critical role in cleaving activating Lys63 chains en bloc from mediators of NFκB signaling, such as receptor interacting protein 1 (RIP1) (123, 156, 271).
Germ-line single-nucleotide polymorphisms of A20 in humans have been linked with susceptibility to a number of inflammatory conditions, including systemic lupus erythematosus, rheumatoid arthritis, and Crohn's disease (161). The widespread inflammation and perinatal mortality of A20-deficient mice has spurred the generation of lineage-specific, conditional knockout models that allow functional analysis in specific cell types. A20-deficient B cells show hypersensitivity to stimuli of the NFκB pathway and increased survival of germinal center B cells. This provides a framework for understanding the role of A20 in suppressing B-cell lymphomas, which is suggested by human genetic studies (36, 96, 161). Other studies have found critical functions for A20 in dendritic cells (83, 126), macrophages (167), and intestinal epithelial cells (265).
OTUB1 is one of the most highly expressed of all DUBs (Figure 4B; Refs. 79, 231). This may reflect a particular feature of this protein that is independent of its DUB activity. OTUB1 is a potent suppressor of Lys63-linked polyubiquitylation at DNA double-strand breaks, independent of its catalytic activity (111). This is accomplished by binding to and inhibiting transfer from the ubiquitin-charged E2 ubiquitin conjugating enzyme UBC13 (111). Furthermore, binding of OTUB1 to multiple E2 enzymes of the UBE2D and UBE2E class has been reported in a proteomic study (244). These include UbcH5, for which inhibition by OTUB1 is proposed to lead to p53 stabilization (246). Elegant structural and biochemical studies show that OTUB1 can also bind free ubiquitin. This binding induces conformational changes in the catalytic domain, which allow simultaneous binding to the E2-ubiquitin, such that both ubiquitins together mimic the configuration of a cleaved Lys48 di-ubiquitin. Thus OTUB1 is proposed to utilize a mechanism akin to product inhibition to inhibit the activity of associated E2 enzymes (111, 224, 273).
D. Josephin Family
Machado Joseph Disease (MJD) is the most common form of spinocerebellar ataxia worldwide. This progressive condition is characterized by polyQ expansion in the ataxin 3 (ATXN3) gene (168). The NH2-terminal Josephin domain possesses deubiquitylating activity (30, 227), while the COOH terminus of the most abundant isoform contains two UIM domains followed by the polyQ sequence and then a third UIM (89). The etiology of MJD is linked to the formation of cellular aggregates once a threshold of polyQ extension has been reached, in common with other polyQ diseases such as Huntington's. It is currently contentious if any specific attributes of MJD relate to associated depletion of enzymatic activity.
The biological function of ATXN3 remains poorly characterized. ATXN3 knockout in mice produces no obvious physiological changes, possibly because of redundancy with other DUBs. However, there is a general elevation in the amount of ubiquitylated protein (229). ATXN3 regulates transcription of multiple genes (65, 215), a property which may allow for a coordinated response to proteotoxic stresses, which have been shown to promote nuclear accummulation of ATXN3 (206). In C. elegans, ATXN3 has been linked to the control of longevity by the IGF-I signaling axis (131).
E. JAMM Family
The JAMM/MPN+ branch constitutes a subset of proteins from the broader MPN (Mpr1-Pad1-N-terminal) family which is conserved in bacteria and archaea. JAMMs contain a signature ‘H-x-H-P-x-S-x-D’ motif within the MPN domain that, through its invariant His and Asp residues, coordinates a zinc atom, which is required for activity (12, 47, 56, 169, 170, 225, 284). JAMMs are generally incorporated into large multimeric complexes such as the proteasome lid complex (POH1/Rpn11), COP9 signalosome (CSN5), and the endocytic ESCRT machinery (AMSH) (39, 46, 72). BRCC36 is associated with two complexes involved in DNA repair, BRISC and BRCA1-RAP180 (45, 235), whilst MYSM1 expresses its histone H2A deubiquitylating activity from within a complex containing the histone acetyltransferase (HAT) p300/CBP-associated factor (p/CAF) (297). Orchestration of histone modifications by MYSM1 has recently been shown to function as part of an epigenetic switch controlling B-cell development (107). Accordingly, mice deficient in MYSM1 show defects in lymphocyte and erythroid development (188).
Several members of this family show a strong preference for Lys63 polyubiquitin linkages including AMSH, AMSH-LP, BRCC36, and POH1 (45, 170, 171, 225). However, both MYSM1 and POH1 apparently cleave ubiquitin proximal to a protein substrate. In the case of MYSM1, this can be the monoubiquitin attached to histone H2A (297), while POH1 can cleave polyubiquitin chains en bloc, from unfolded proteasomal substrates (284). The structure of the catalytic domain of AMSH-LP in complex with Lys63 diubiquitin and later of the AMSH catalytic domain illustrated specific interactions of ubiquitin with the catalytic core, but also with two AMSH-specific insertions that interact with the proximal and distal ubiquitins, respectively (56, 225). However, these structures fail to clearly explain the Lys63 specificity of other JAMMs.
VII. DUBs AS INTEGRAL COMPONENTS OF PROTEIN MACHINERIES ASSOCIATED WITH DEGRADATION
In the following section, we discuss DUBs associated with two of the major protein degradation pathways, the proteasomal and lysosomal routes, for which some common principles related to ubiquitin homeostasis and protein rescue apply. DUBs associated with the ubiquitin-dependent degradative pathway of autophagy largely remain to be elucidated, although USP10 and USP13 have been implicated in the control of the stability of the autophagy gene product BECLIN1 (158).
A. Proteasomal DUBs
The 26S proteasome is a ∼2.5 MDa assembly of proteins, which is responsible for the degradation of most cytosolic proteins. It is comprised of two large subcomplexes corresponding to the 20S catalytic core (CP; core particle) and two copies of the 19S regulatory particle (RP; regulatory particle) that can be subdivided into base and lid components. The base contains a ring of 6 homologous ATPases that promote substrate unfolding and translocation into the 20S catalytic chamber through a narrow (∼13 Å) gated channel. The RP includes two ubiquitin receptors and three distinct DUB activities amongst its constituent proteins. One of these, POH1/PSMD14/Rpn11 (hereafter referred to as POH1), is constitutively incorporated in stoichiometric quantities and required for RP assembly. The other two, USP14 (Ubp6 in yeast) and UCH37/UCHL5 (not present in yeast), are reversibly associated (146).
Proteasomal DUBs have been suggested to be involved in the recycling of ubiquitin, even directly coupling this to protein degradation, or in “proof-reading” at the proteasome whereby certain proteins may be reprieved from degradation (72). Deubiquitylation is required for release of substrate from ubiquitin receptor proteins. If ubiquitin chain trimming outpaces substrate unfolding/translocation, it can result in dissociation from the proteasome and rescue of the protein. Conversely, retarded deubiquitylation leads to occlusion of substrate binding sites and clogs up the proteasome (291). Only siRNA knockdown of POH1 interferes with proteasome assembly, while depletion of USP14 or UCH37 alone both enhance protein degradation rates, but their combined depletion inhibits proteasomal activity (127). Yeast cells respond to ubiquitin depletion by upregulating the USP14 ortholog Ubp6, which restores ubiquitin levels (85). The ataxia (axj) mouse exhibits severe tremors at 2–3 wk of age, reflecting defective synaptic transmission, which results from an intronic mutation, leading to loss of full-length USP14 expression (14, 278). The observed phenotypes probably reflect depletion of the synaptic ubiquitin pool observed in axj mice, as they can be rescued by either neuron specific expression of Usp14 or ubiquitin itself (32, 33, 49).
Although crystal structures of proteasomal complexes are unavailable, sub-nanometer resolution structures derived from electron microscopy single-particle analyses provide information on the organization of constituent proteins (16, 53, 137, 141). POH1 is adjacent to the ubiquitin receptor Adrm1/Rpn13 and positioned directly above the AAA-ATPase N-ring (16). The activity of POH1 is enigmatic. Based on in vitro enzymatic and structural studies of AMSH, BRCC3, and POH1, it has been proposed that the JAMM family proteins may collectively possess a stringent specificity for Lys63 ubiquitin chain linkages (44, 45, 123, 171, 225, 235). However, elegant preceding work suggested that POH1 activity on proteasomal substrates 1) indirectly requires ATPase activity presumably for unfolding of the substrate, 2) is coupled to proteasomal degradation, and 3) completely removes ubiquitin by cleavage at the base of the ubiquitin chain (266, 284).
In fact, the catalytic activities of all three proteasomal DUBs are dependent on incorporation or association with the 19S particle. Proteasomal binding activates Ubp6/USP14 by several hundredfold (143, 147). Binding of the 19S component Adrm1 (Rpn13) to the COOH-terminal tail of UCH37 is proposed to remove an autoinhibitory barrier, leading to acceleration of ubiquitin-AMC hydrolysis (but see Ref. 29 for a note of caution on this). Full incorporation into the 19S complex is required for efficient processing of polyubiquitin chains by UCH37, which occurs from the distal end (203, 286).
Not all proteasomal DUB functions require catalytic activity. Binding of ubiquitin chains to USP14/Ubp6 or UCH37 opens the gate of the 20S channel, and in combination with an unfolded substrate domain stimulates proteasomal ATPase activity. Although this offers the possibility of coupling ubiquitin recycling with degradation, gate opening or ATPase stimulation does not require catalytic activity (197, 198). Expression of catalytically inactive USP14/Ubp6 has been shown to have either positive or negative effects on proteasomal degradative activity, and these observations remain to be fully reconciled (84, 143, 197). Deletion of 31 amino acids from the COOH terminus of yeast Rpn11 leads to cell cycle defects and altered mitochondrial morphology. These morphological changes can be suppressed by expression of the Rpn11 COOH-terminal fragment alone, without any indication that this interacts directly with the proteasome (213).
POH1 and UCH37 also present further moonlighting functions independent of proteasome assembly or degradation. UCH37 associates with the Ino180 chromatin remodeling complex, where it is held in an inactive state. Inhibition is relieved by transient interaction with the proteasome, leading to the suggestion of cooperation between these two complexes in either transcription or DNA repair, both processes to which each complex has been linked (285). POH1 DUB activity has also recently been proposed to contribute to the choreography of the double-strand break DNA repair response (31). Finally, UCH37 also associates with Mothers against Decapentaplegic proteins (SMAD) proteins, in particular SMAD7, and inhibits type I transforming growth factor (TGF)-β receptor degradation (272).
Owing to the success in the clinic of the proteasomal inhibitor Bortezomib in treating multiple myeloma, there is great interest in developing further modes of proteasomal inhibition. Small molecule inhibitors of POH1 represent one such possibility. The small molecule b-AP15 was first identified as a candidate proteasome inhibitor on the basis of a gene expression signature shared with other known proteasome inhibitors. Its mechanism of action proved to be through dual inhibition of both cysteine protease deubiquitylating activities associated with the proteasome, whilst total cellular DUB activity and the activity of several recombinant USP proteins is unaffected (52). One may presume that the mode of inhibition may be indirect, via drug-induced conformational changes within the 19S particle. Nevertheless, some promising effects of b-AP15 on the progression of tumors in mouse models were reported (52). A selective inhibitor of USP14, IU1, was identified using a small molecule screening approach. Application of the drug to cells leads to enhanced degradation rates for a variety of overexpressed proteins, by opposing ubiquitin chain trimming (143). These observations point to potential benefits in the treatment of certain neurological conditions, where proteasome activity may be limiting for the suppression of aggregate formation of misfolded proteins.
B. ESCRT DUBs
Trafficking to the lysosome provides the major degradative pathway for the majority of plasma membrane channels, pumps, and receptors (38, 200). In many cases this is achieved by the capture of ubiquitylated proteins, which have entered the sorting endosome, by the endosomal sorting complex required for transport (ESCRT) machinery (92, 102, 276). This machinery is comprised of four subcomplexes, ESCRT-0, I, II and III, which were originally proposed to act in sequential fashion. This view has become more nuanced with time, and a higher degree of integration between these components seems likely. Under the influence of this machinery, the sorting endosome matures into multivesicular bodies (MVBs) with the accrual of luminal vesicles, laden with cargo molecules that bud from the limiting membrane. MVBs then deliver cargo to lysosomes by direct fusion (76).
Two DUBs, AMSH and USP8/UBPY, form a network of interactions with various components of ESCRT-0 and ESCRT-III (39). Both contain MIT domains that promote endosomal association, through distinct but overlapping sets of interactions with the CHMP protein constituents of the ESCRT-III subcomplex (3, 171, 218, 241, 258). Furthermore, they share a binding site on the SH3 domain of the ESCRT-0 component STAM (113, 250). It is striking that both proteasomal and ESCRT complexes carry a JAMM family DUB (POH1 or AMSH, respectively) specific for Lys63-linked chains as well as a more promiscuous enzyme (USP14 or USP8). Accordingly, this parallel can be extended to consideration of function, in that ESCRT DUBs may couple ubiquitin recycling with commitment to degradation, i.e., inclusion into luminal vesicles of the MVB. Equally they may perform proofreading functions as detailed above for proteasomal DUBs (39). This function was first proposed for AMSH based on the observation that its depletion leads to enhanced rates of EGFR degradation (25, 39, 170, 195). By deubiquitylating receptor, AMSH inhibits inclusion into luminal vesicles and receptors recycle to the plasma membrane. The body of data around USP8 is more complex. Its depletion has more severe effects on the organization of the endocytic pathway that combine to inhibit EGFR downregulation (25, 177, 218, 219). One contributing factor to such defective EGFR trafficking is that USP8 controls the stability of ESCRT-0 components, Hrs and STAM in cells and in conditional knockout mice (185, 219). However, for Frizzled receptor (Fz) and Smoothened (Smo), key components of Wingless (Wnt) and Hedgehog signaling pathways, respectively, USP8 plays a negative regulatory role with regard to receptor degradation more akin to that originally proposed for AMSH (153, 180, 281).
VIII. INFLUENCE ON CELL PHYSIOLOGY THROUGH CONTROL OF RECEPTORS AND CHANNELS
The influence of DUBs on membrane trafficking has a profound effect on cell physiology through the regulation of receptor and channel densities at the plasma membrane. Here we highlight some examples in addition to effects on EGFR, Smo, and Fz receptors (described above), where the physiological consequences of this regulation are supported by whole organismal models. The further influence of DUBs on specific effector pathways, downstream of ligands such as Wnt and TGF-β, has recently been reviewed elsewhere (7, 37, 251).
Surface levels of glutamate neurotransmitter receptors can be regulated by both direct and indirect ubiquitylation (43, 130). Usp-46 was identified in a C. elegans RNAi screen for DUBs regulating the abundance of the glutamate receptor GLR-1 at synapses in the ventral chord. Follow-up studies indicated that usp-46 mutant worms show defects in glutamate-dependent behaviors and that the effect on GLR-1 is accomplished by direct deubiquitylation of the receptor at the level of the sorting endosome (129). USP46 has been further linked to GABAergic transmission in mouse models. Quantitative trait locus analysis of CS mice, which exhibit depressive behavior mapped to a 3-bp in-frame deletion of USP46, which reduces catalytic activity (256). Depressive behavioral effects have since been recapitulated in USP46 knockout mice, which can be alleviated by Nitazepam, which enhances GABA binding to receptors (103).
Four DUBs have been implicated in the regulation of TGF-β receptor stability, UCH37 and the highly related USP4, USP11, and USP15. The individual effects of these may be more or less pronounced depending on cellular context (2, 6, 61, 272, 290). USP15 has also been associated with many other cellular signaling events including the MAP kinase pathway (90), β-catenin stability (99), and the NFκB pathway (232). It is recruited to TGF-β receptors in complex with the E3-ligase SMURF2 and SMAD7, which acts as a scaffold. Its deubiquitylating activity promotes receptor expression through stabilization of the receptor (61), and it appears that USP15 can enhance the role of TGF-β signaling in glioblastoma multiforme (GBM). However, it has also been shown that USP15 empowers transcriptional activation of R-SMADs, the ultimate effectors of the TGF-β pathway, by removing inactivating monoubiquitin (104); so fully discriminating the physiological importance of each of these effects may prove challenging. One consideration is that USP15 also promotes bone morphogenic protein (BMP) signaling, which utilizes overlapping SMAD family members with the TGF-β pathway, as effectors of an entirely distinct receptor type (104). USP11 was also identified as the major SMAD7 interacting DUB by proteomics and is proposed to be recruited to TGF-β receptor, which it deubiquitylates, in a similar manner to USP15 (6). USP4 is closely related to USP11 and USP15 (Figure 3) and was identified as a top hit in a genome-wide gain-of-function screen for enhancers of TGF-β signaling (other hits included USP11, USP15, USP19 but were not substantively followed up). Depletion of USP4 inhibits TGF-β but not BMP signaling, and in the zebrafish embryo leads to early morphogenetic defects. It also impedes cell migration in vitro and metastasis in a zebrafish xenograft model (292). In common with USP15, USP4 can deubiquitylate the activated TGF-β receptor but in contrast binds directly to it, independent of SMAD7.
USP10 is localized to sorting endosomes in human airway epithelial cells where it is proposed to directly deubiquitylate the cystic fibrosis transmembrane conductance regulator (CFTR) (20). An alternative mode of action of vasopressin-induced USP10 has been posited for its control of the epithelial sodium channel ENaC in renal cells. In this case, the relevant substrate is suggested to be Sorting Nexin 3, a positive regulator of recycling, that is stabilized by USP10 expression (24), recalling the stabilization of ESCRT-0 sorting factors by USP8 (see above).
In some instances, ubiquitylation may provide a direct sorting signal for internalization of receptors from the plasma membrane in addition to sorting into MVBs described above (38). For example, it may be at this point that Cezanne exerts its negative regulation on EGFR downregulation (195). DUBs may affect this step by direct deubiquitylation of receptors or through influencing components of the vesicular entry routes. The ubiquitin-dependent interaction of cargo molecules with clathrin-coated vesicle (CCV) adaptor proteins, such as epsin, promotes endocytosis (257). The Drosophila DUB Fat Facets (Faf) (USP9X in humans) interacts directly with the epsin homolog Liquid facets (Laf). Deubiquitylation of Laf by Faf is proposed to facilitate Notch signaling by promoting the internalization of the Notch ligand Delta during fly development (192).
Interest in the DUB family of enzymes is burgeoning as the range of biological processes shown to be under their control expands. In the last few years, rapid strides have been made in cataloguing their varying specificities for different types of polyubiquitin chains, but in most cases this is not fully understood at the structural level. Screening strategies have identified DUB's associated with important regulatory pathways in cellular systems, which are now increasingly being supported by animal models that show corresponding developmental defects or disease states. One must bear in mind that a requirement for catalytic activity has not been shown in all cases, and some assays may reflect other aspects of a particular DUB's function. Nevertheless, several DUBs are emerging as attractive drug targets, for which first generation tool compounds have been developed (11, 42). The stage is now set for the development of clinically useful therapies that build upon this body of knowledge.
No conflicts of interest, financial or otherwise, are declared by the authors.
Address for reprint requests and other correspondence: M. J. Clague, Cellular and Molecular Physiology, Institute of Translational Medicine, Univ. of Liverpool, Liverpool, UK (e-mail:).
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