Biochemical Journal

Review article

Protein partners of deubiquitinating enzymes

Karen H. Ventii, Keith D. Wilkinson


Protein modification by ubiquitin and ubiquitin-like molecules is a critical regulatory process. Like most regulated protein modifications, ubiquitination is reversible. Deubiquitination, the reversal of ubiquitination, is quickly being recognized as an important regulatory strategy. Nearly one hundred human DUBs (deubiquitinating enzymes) in five different gene families oppose the action of several hundred ubiquitin ligases, suggesting that both ubiquitination and its reversal are highly regulated and specific processes. It has long been recognized that ubiquitin ligases are modular enzyme systems that often depend on scaffolds and adaptors to deliver substrates to the catalytically active macromolecular complex. Although many DUBs bind ubiquitin with reasonable affinities (in the nM to μM range), a larger number have little affinity but exhibit robust catalytic capability. Thus it is apparent that these DUBs must acquire their substrates by binding the target protein in a conjugate or by associating with other macromolecular complexes. We would then expect that a study of protein partners of DUBs would reveal a variety of substrates, scaffolds, adaptors and ubiquitin receptors. In the present review we suggest that, like ligases, much of the regulation and specificity of deubiquitination arises from the association of DUBs with these protein partners.

  • deubiquitinating enzyme (DUB)
  • deubiquitination
  • proteasome
  • ubiquitin
  • ubiquitin-specific protease


Over the last few decades, protein modification by ubiquitin and ubiquitin-like molecules has emerged as a critical regulatory process in virtually all aspects of cell biology. This importance is underscored by the fact that the 2004 Nobel Prize in Chemistry was awarded for the discovery of ubiquitin-mediated proteolysis [1].

Ubiquitination, which is the process of covalently conjugating ubiquitin to a target protein, is accomplished through a series of reactions (Figure 1). In the first step, an ubiquitin-activating enzyme (E1) forms a thiol ester bond with the ubiquitin molecule in an ATP-dependent manner. The ubiquitin moiety is then transferred to one of a few dozen ubiquitin-conjugating enzymes (E2), and then to a specific target protein via the action of one of several hundred ubiquitin ligases (E3), leading to the formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and the ϵ-amino group on a lysine residue of the target protein [24]. The conjugation of one ubiquitin molecule to a lysine residue on the target protein (monoubiquitination) has been implicated in different cellular processes, including membrane trafficking, histone function, transcription regulation, DNA repair and DNA replication [5]. Additional rounds of ubiquitination can modify the first ubiquitin to result in the formation of polyubiquitin chains linked through the C-terminus of one ubiquitin and a lysine residue of the preceding ubiquitin. Different chain topologies can be formed depending on which lysine residue on ubiquitin is used for conjugation. Different chain types have been implicated in signalling different outcomes, including protein degradation, directing the localization of the modified protein, or modifying its activity, macromolecular interactions or half-life [6,7] (Figure 2). Ubiquitin modifications can be reversed by the action of enzymes collectively known as deubiquitinating enzymes (DUBs).

Figure 1 The involvement of DUBs at different steps of the ubiquitination process

During the process of ubiquitination, ubiquitin (Ub) is first conjugated to an ubiquitin-activating enzyme (E1) via a thiol ester bond in an ATP-dependent manner. The ubiquitin moiety is then transferred to one of a few dozen ubiquitin-conjugating enzymes (E2) and then to a specific target protein via the action of one of several hundred ubiquitin ligases (E3). DUBs are involved at several stages of the ubiquitination process. (1) DUBs are involved in processing ubiquitin, which is translated as a pro-protein. (2) By removing the ubiquitin signal from target proteins DUBs can protect Lys48-linked polyubiquitin-conjugated proteins from degradation at the proteasome. (3) DUBs are also involved in disassembling ubiquitin chains to regenerate free ubiquitin for re-use by the conjugation system.

Figure 2 Different ubiquitin signals target proteins for different fates

The conjugation of one ubiquitin (Ub) to one or several lysine (K) residues on the target protein (monoubiquitination) has been implicated in different cellular processes, including membrane trafficking, histone function, transcription regulation, DNA repair and DNA replication. Additional rounds of ubiquitination can modify the first ubiquitin to result in the formation of polyubiquitin chains linked through the C-terminus of one ubiquitin and a lysine residue that can be used to form different chain topologies. Different chain types have been implicated in signalling different outcomes, including protein degradation, directing the localization of the modified protein or modifying its activity, macromolecular interactions, or half-life. Different DUBs would be expected to recognize these unique chain topologies. Lys48, K48; Lys63, K63; Lys6, K6; Lys29, K29.


Deubiquitination is performed by the action of DUBs, ubiquitin-specific proteases that can reverse target-protein ubiquitination by editing or disassembling polyubiquitin chains. DUBs play an important role in several aspects of the ubiquitin–proteasome system, and mutations in genes expressing DUBs have been implicated in a number of diseases, including hereditary cancer and neurodegeneration [811].

There are five families of DUBs. Four are cysteine proteases, and there is one family of metalloproteases. DUBs specifically cleave ubiquitin from ubiquitin-conjugated protein substrates, ubiquitin precursors, ubiquitin adducts and polyubiquitin [12]. The cysteine protease DUBs can be further organized into four subclasses based on their ubiquitin protease domains: USPs (ubiquitin-specific proteases), UCHs (ubiquitin C-terminal hydrolases), OTUs (otubain proteases) and MJDs (Machado–Joseph disease proteases). All DUBs that are metalloproteases have an ubiquitin-protease domain called a JAMM (JAB1/MPN/Mov34 metalloenzyme) domain. In addition, viruses [13] and bacteria [14] have both acquired or evolved deubiquitinating enzymes, probably to interfere with host-cell processes as part of their mechanisms of virulence.

Nijman et al. [10] used the ENSEMBLE human-genome database to retrieve all putative DUBs from the human genome by selecting genes whose transcripts encode one of the five ubiquitin-protease domains. This analysis indicated that the human genome encodes approx. 95 putative DUBs, including many that had not been previously reported. These were broken down into 58 USP, four UCH, five MJD, 14 OTU and 14 JAMM domain-containing genes, many of which were associated with multiple transcripts. The abundance of DUB family members presumably allows for diversity and specificity of DUB activity, although relatively little is known about the physiological substrates of individual members of the family.

Structural and biochemical studies on isolated DUBs have shown that many exist in conformations that require substrate binding [15,16] or association with binding partners [17] to achieve their active conformation. Also, although many DUBs bind ubiquitin with reasonable affinities (in the nM to μM range), a larger number have little affinity. A number of these poor ubiquitin-binders have a good catalytic capability, exhibiting kcat/Km values in excess of 105 M−1·s−1 (K. D. Wilkinson, unpublished work). Thus it is apparent that these DUBs must acquire their substrates by binding to the target protein in a conjugate or by associating with other macromolecular complexes [8]. We would then expect that a study of protein partners of DUBs would reveal a variety of substrates, scaffolds, adaptors and ubiquitin receptors.

The specific biological role of most DUBs remains unknown [18], and defining these roles may be facilitated by determining their protein partners. For instance, many DUBs have been shown to associate physically with ubiquitin ligases, and their role in the ubiquitin pathway has often been inferred from the roles of the associated ligase. Given that the modes of regulation of DUBs and their substrate specificity are not well understood, the present review collates the information currently available about DUBs and their interacting proteins. Some are biological substrates, wherease others modulate activity of the DUBs. The DUBs chosen for discussion are those for which interacting proteins (other than ubiquitin) have been identified, and we hope that this review will help shed light on the functions of DUBs based on their protein partners. Table 1 lists the DUBs mentioned in this paper and their respective binding partners.

View this table:
Table 1 DUBs and their protein partners

X indicates the role(s) of the interacting protein.




USP1 is a DUB that has been implicated in Fanconi-anaemia-related cancer [19]. USP1 is involved in the process of DNA repair through its regulation of ubiquitinated Fanconi anaemia protein FANCD2 (Fanconi anaemia complementation group D) levels [20,21].


USP1 was found to co-immunoprecipitate with FANCD2 in HEK-293 (human embryonic kidney) cell lysates, and the proteins are observed to co-localize on chromatin after DNA damage [20]. This study showed that knockdown of USP1 by small interfering RNA leads to hyper-accumulation of monoubiquitinated FANCD2. USP1 is proposed to deubiquitinate FANCD2 when cells exit S-phase or recommence cycling after DNA damage, and may play a critical role in the Fanconi anaemia pathway by recycling FANCD2 [20].

UAF1 (USP1-associated factor 1)

USP1 isolated from HeLa cells was found to contain stoichiometric amounts of UAF1, a WD40 repeat-containing protein [22]. USP1 deubiquitinating enzyme activity against purified monoubiquitinated FANCD2 protein could be reconstituted in vitro, demonstrating that UAF1 functions as an activator of USP1. UAF1 binding increases the catalytic turnover (kcat), but not the affinity of the USP1 enzyme for the substrate (Km). DNA damage rapidly shuts down transcription of the USP1 gene, and leads to a rapid decline in the amount of the USP1/UAF1 protein complex.


USP2a is a DUB with oncogenic properties that has been implicated in prostate cancer [23]. USP2a regulates the p53 pathway through its deubiquitinating activity [24], and has also been shown to deubiquitinate the anti-apoptotic protein FAS (fatty acid synthase) [23]. The finding that USP2a is over-expressed in human prostate tumours has made it a potential therapeutic target in prostate cancer [23]. USP2a has also been suggested to play a part in normal spermatogenesis [25], another process in which p53 is thought to be involved [26,27].

Mdm2 (murine double minute 2)

Mdm2 is an E3 ubiquitin ligase oncoprotein and a key regulator of the p53 tumour-suppressor protein. It acts to ubiquitinate p53 and target the tumour suppressor for proteasome-mediated degradation [28]. A yeast two-hybrid screen using Mdm2 as bait identified USP2a as a protein partner [24]. In this study, full-length USP2a was found to interact with and deubiquitinate Mdm2, thereby causing the accumulation of Mdm2 in a dose-dependent manner. Consequently, USP2a-medited deubiquitination and stabilization of Mdm2 promoted Mdm2-mediated p53 ubiquitination and degradation. p53 is one of the most important tumour-suppressor proteins and plays an essential role in regulating the cell cycle and apoptosis by sensing the integrity of the genome [29]. The fact that USP2a regulates p53 implies a role for the protein in human cancers. The action of USP2a within the cell is antagonistic to that of another DUB called USP7/HAUSP (herpesvirus-associated USP) (Figure 3). USP7 interacts with and deubiquitinates p53, thereby stabilizing and protecting it from Mdm2-mediated degradation [30]. A second USP2 isoform (USP2b) results from alternative splicing, and this isoform may also interact with Mdm2 [31].

Figure 3 The regulation of p53 by the ubiquitin system illustrates the complex interplay between DUBs and E3 ligases

By regulating p53 levels through their deubiquitinating activities, USP7 and USP2a may contribute to cancer pathogenesis. Therapeutic strategies that target these p53-specific DUBs are becoming important as cancer treatments. Ub, ubiquitin.


FAS, a protein expressed from an androgen-regulated gene [23], is often over-expressed in biologically aggressive human tumours [32] and promotes tumour growth by increasing the resistance to apoptosis [33]. FAS was found to interact with histidine-tagged USP2a via affinity chromatography and MS of an LNCaP cell (human prostate cancer cell) lysate, and this interaction was confirmed by co-immunoprecipitation [32]. The findings from this study suggest that the USP2a enzyme acts by inhibiting FAS protein degradation by the proteasome, thereby resulting in the protection of prostate cancer cells from apoptosis. The finding that USP2a is involved in human prostate cancer, coupled with information derived from the crystal structure of the USP2a catalytic domain [34], has made it a potential target for therapeutic drugs [23].


USP4 is a DUB with a suggested role as an oncoprotein because USP4 protein levels have been found to be elevated in small-cell lung carcinomas and adenocarcinomas of the lung, and overexpression of a mouse cDNA encoding Unp (USP4) leads to oncogenic transformation of NIH 3T3 cells [35,36].


USP4 was found to interact with the protein Ro52 (also known as the tripartite motif-containing protein TRIM21) in a yeast two-hybrid assay, and the interaction was confirmed by a mammalian two-hybrid interaction in COS-1 cells [12]. Ro52 is an E3 ubiquitin ligase that can ubiquitinate itself (self-ubiquitination) as well as ubiquitinate USP4 in vitro and in vivo. In contrast, USP4 can deconjugate ubiquitin from itself (self-deubiquitination) as well as deubiquitinate self-ubiquitinated Ro52 [37]. In other words, the two proteins trans-regulate each other by ubiquitination and deubiquitination. The two proteins were shown to co-localize to cytoplasmic rod-like structures in HEK-293 cells, suggesting that Ro52 interacts with USP4 in mammalian cells to form a heteromeric protein complex that mediates both ubiquitination and deubiquitination of substrates [38].

Adenosine A2A receptor

Adenosine A2A receptors are G-protein-coupled receptors that have been shown to interact with USP4 through their C-terminal tail [39]. Intracellular A2A receptors are thought to be ubiquitinated, presumably because of misfolding and subsequent intervention by the endoplasmic-reticulum quality-control mechanism, thereby leading to degradation of the receptors by proteasomes [39]. The importance of the USP4–adenosine A2A receptor interaction lies in the ability of USP4 to stimulate receptor deubiquitination, thereby increasing cell-surface expression of functional receptors [40].


USP6/Tre2 is expressed from the Tre2 oncogene, which is derived from the chimeric fusion of two genes: USP32 (NY-REN-60), encoding a USP, and TBC1D3, encoding a RabGAP (where GAP is GTPase-activating protein) [41]. USP6 is structurally related to the Ypt/RabGAPs and is involved in various human cancers, including Ewing's sarcoma [42].

Myl2 (light regulatory chain of myosin II) and LOC91256

In order to identify proteins that interact with the GAP domain of USP6, a yeast two-hybrid screen was done with two cDNA libraries from human tissues (skeletal muscle and placenta). Two components of the cytoskeleton were identified: Myl2 and LOC91256 [43]. Myl2 is a component of myosin II that stabilizes the long α-helical neck of the myosin head and regulates its ATPase activity [44]. LOC91256 is a protein containing ankyrin repeats and shows similarity to a region of the cytoskeletal anchor protein ankyrin 1. Both proteins were confirmed to interact with USP6 by a GST (glutathione transferase)-pull-down assay in vitro and co-immunoprecipitation, and co-localization experiments in vivo [43]. Although the biological significance of these interactions remains unknown, the observation that USP6 appeared to cause redistribution of Myl2 and LOC91526 to the cell membrane led to the proposal that the binding of USP6 to Myl2 participates in RhoGTPase signalling [43,45].


USP7 is a deubiquitinating enzyme also known as HAUSP. USP7 contains four structural domains, at least three of which are responsible for binding to other proteins [46]. Like USP2a, USP7 is involved in the complex regulation of the p53 tumour suppressor through its interactions with p53, Mdm2 and Mdmx. USP7 also interacts with the viral proteins EBNA1 (Epstein–Barr nuclear antigen 1) and ICP0 (infected cell polypeptide 0).

p53, Mdm2 and Mdmx

USP7 was identified by MS analysis of affinity-purified p53-associated factors [30]. USP7 was found to bind directly to p53 in vitro as well as in vivo via co-immunoprecipitation assays [30]. USP7 was found to regulate p53 levels through its deubiquitinating activity as part of a feedback loop involving the E3 ligase enzyme Mdm2, which is also a substrate for USP7 deubiquitinase activity [47,48]. Although USP7 can directly regulate p53 levels, these levels can also be regulated by the DUB USP2a (Figure 3). By regulating p53 levels through their deubiquitinating activities (Figure 1), USP7 and USP2a may contribute to cancer pathogenesis, and therapeutic strategies that target these p53-specific DUBs may become important as cancer treatments [29].

USP7 can directly bind Mdm2 in vitro [48]. The DUB stabilizes Mdm2 in a p53-independent manner, providing an interesting feedback loop in p53 regulation [48,49]. Notably, USP7 is required for Mdm2 stability in normal cells and, in USP7-ablated cells, self-ubiquitinated Mdm2 becomes extremely unstable, leading indirectly to p53 activation [48].

New evidence supports a role for USP7 in the regulation of Mdmx stability as well [50]. Mdmx is an Mdm2 family member that is also involved in p53 degradation and contains a RING (really interesting new gene) domain, but does not appear to have any ubiquitin E3 ligase activity [51,52]. USP7 was shown to bind directly to and deubiquitinate Mdmx in vitro and in vivo [50].

EBNA1 and ICP0

EBNA1 interacts in the N-terminus of USP7, the same domain that interacts with p53, and the two proteins appear to compete for the same binding site [46]. By disrupting the p53–USP7 interaction, EBNA1 would be expected to promote cell-cycle progression and prevent apoptosis, which could be important for the host-cell immortalization typical of EBV (Epstein–Barr virus) [46]. Using an in vitro affinity-column assay and in vivo TAP (tandem affinity purification)-tagging to profile cellular protein interactions with EBNA1, USP7 was found to interact specifically with EBNA1 [53]. Furthermore, purified USP7 was shown to deconjugate ubiquitin from EBNA1 [46].

The C-terminus of USP7 binds another viral protein, the ICP0 protein of herpes simplex type 1 [46]. The ICP0 protein has ubiquitin E3 ligase activity in vitro and is important for induction of the lytic infectious cycle [54]. The interaction of viral proteins with USP7 suggests that some viruses may influence cellular events by sequestering or altering the activity of USP7 [46].


USP8 is an SH3 (Src homology 3)-binding protein [55]. It was first identified as a protein whose levels accumulate upon growth stimulation of human fibroblast cells and decrease in response to growth arrest [56]. USP8 is implicated in ubiquitin remodelling, regulation of epidermal-growth-factor receptors, clathrin-mediated internalization, endosomal sorting and the control of receptor tyrosine kinases in vivo [18,57]. The USP8 protein has several domains, including a RD (rhodanese homology domain) and the cysteine and histidine boxes of the catalytic core [55]. USP8 also interacts with the Hrs (hepatocyte receptor substrate)–STAM (signal transducing adaptor molecule) complex. The biological importance of USP8 was revealed by studies using Cre–loxP-mediated gene targeting in mice showing that lack of USP8 results in embryonic lethality [57].

SH3-domain-containing proteins

SH3-domains are protein-interaction modules that are involved in signal-transduction networks [58]. USP8 has been shown to interact with the SH3 domain of STAM2. Upon binding to STAM2, USP8's DUB activity appears to be activated, resulting in the deubiquitination of STAM2 and its protection from proteasomal-mediated degradation [55,59]. The crystal structure of the STAM2 SH3 domain in complex with the USP8 binding peptide was solved, and the interactions observed explained why the binding was tight, with a dissociation constant (Kd) of 27 μM [59]. STAM2, in co-operation with the protein Hrs, functions in the endocytic degradation pathway of growth-factor-receptor complexes, and USP8 may indirectly regulate this process [60].

Another Hrs-binding protein with an SH3 domain is Hbp (Hrs-binding protein). Using full-length Hbp as a probe in a far Western assay with a mouse liver cDNA-expression library, researchers identified a USP8 cDNA clone that encoded a Hbp-binding protein. Mouse USP8 shares approx. 80% amino-acid sequence identity with human USP8, and it is likely that human USP8 would also bind this protein [55].

Row et al. [61] identified and characterized a MIT (microtubule interacting and transport) domain at the N-terminus of USP8 and showed that, like other MIT-domain-containing proteins [such as AMSH (associated molecule with SH3 domain of STAM), which is discussed later in this review], it bound CHMP proteins (charged multivesicular body proteins). CHMP proteins are involved in the formation of multivesicular bodies and the degradation of internalized transmembrane receptor proteins [62]. There are several CHMP proteins, and the MIT domain of USP8 was found to interact with CHMP1A, CHMP1B, CHMP4C and CHMP7. The USP8–CHAMP1B interaction was later shown to be direct by using bacterially expressed proteins in vitro, and then confirmed in vivo by a co-immunoprecipitation assay [61].

Nrdp1 (neuregulin-receptor-degradation protein-1)

USP8 was first identified in an affinity-purification assay of proteins that bound to Nrdp1. Nrdp1 is a RING finger ubiquitin E3 ligase involved in ligand-stimulated epidermal-growth-factor receptor down-regulation [63] and has been implicated in the degradation of the inhibitor-of-apoptosis protein BRUCE (BIR repeat-containing ubiquitin-conjugating enzyme) [64]. USP8 and Nrdp1 were shown to interact in vitro via affinity-chromatography experiments with cell lysates from C2C12 myotubes, and the two proteins were also shown to interact in vivo by co-immunoprecipitation experiments [63]. The study found that the rhodanese and catalytic domains of USP8 mediate its interaction with Nrdp1 [63]. The crystal structure of the rhodanese domain of USP8 in complex with Nrdp1 was solved and revealed that the USP8–Nrdp1 interaction appeared to be dominated by salt bridges [18], which are more ‘drugable’ interactions than hydrophobic interactions. The biological significance of the USP8–Nrdp1 interaction is at least partially due to its effect on epidermal-growth-factor receptor regulation. Nrdp1 catalyses the ubiquitination of the epidermal-growth-factor receptor/ErbB family of receptor tyrosine kinases [65,66]. Nrdp1 not only associates with the ErbB3 receptor and stimulates its ubiquitination and degradation by proteasomes, but also promotes its own ubiquitination and subsequent proteasome-mediated degradation [63]. Deubiquitination activity of USP8 is required for Nrdp1 stability. By stabilizing Nrdp1, USP8 indirectly regulates ErbB3 stability and therefore may represent an attractive target for cancer therapy [66].


USP8 was identified by LC (liquid chromatography)–MS/MS (tandem MS) analysis of a pull-down assay designed to find 14-3-3ϵ-interacting partners from primary mouse tissue [67]. 14-3-3 protein family members execute diverse regulatory roles, primarily via interactions with proteins phosphorylated in a conserved sequence motif [68]. The study found that phosphorylation of USP8 at Ser680 was essential for its interaction with 14-3-3ϵ and for retaining/stabilizing USP8 in the cytosol [67]. The USP8–14-3-3 interaction was also confirmed in three other studies: one used TAP coupled with multidimensional protein-identification technology [69]; another used 14-3-3 affinity chromatography with HeLa cell extracts and found that the USP8–14-3-3 interaction appears to occur in a cell-cycle-dependent manner [70]; and, in a third study, 14-3-3ϵ, 14-3-3γ and 14-3-3ζ were identified as USP8-binding proteins using co-immunoprecipitation followed by MS analysis [71]. The last study showed that 14-3-3ϵ inhibited the activity of USP8 in vitro. Although the biological significance of the USP8–14-3-3ϵ interaction remains unknown, one hypothesis is that USP8 deubiquitinates and stabilizes negative regulators of the cell cycle that also bind 14-3-3 isoforms, such as the tumour suppressors NF1 (neurofibromin 1) and the TSC (tuberous sclerosis complex) proteins 1 and 2, which have been found to interact with 14-3-3ϵ and are known to undergo ubiquitin-dependent degradation [67]. Another hypothesis is that USP8 is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3 proteins during the interphase stage of the cell-cycle, and this regulation is reversed in the M-phase [71].


Fat facets/USP9 is a DUB that has been implicated in development, probably due to a specific role in regulating Delta and Notch signalling. The mouse and Drosophila orthologues of USP9 are essential for early embryonic development [72].

AMPK (AMP-activated protein kinase) kinases

USP9 was identified in a modified TAP strategy to find proteins that interact with AMPKs in HEK-293 cells. The study found that USP9 was associated with two AMPK kinases, MARK (microtubule-affinity-regulating kinase)-4 and NUAK1 (AMPK-related kinase 5) [73]. MARK proteins have an UBA (ubiquitin-associated) domain and regulate anterior–posterior cell polarity development at the one-cell stage of embryonic development in Caenorhabditis elegans and Drosophila [74,75].

Mib1 (Mind bomb 1)

Mib1 is an ubiquitin E3 ligase enzyme found in the post-synaptic density of neurons. Using a GST-affinity purification method with rat brain lysates followed by MS/MS, the USP9 orthologue was identified as an interacting protein [76].


Vasa (VAS) protein is a DEAD-box RNA helicase that is essential maternally for posterior patterning and germ-cell function in Drosophila [77,78]. VAS-containing complexes with USP9 were cross-linked, isolated by a tandem immunoprecipitation approach and identified by MS [77]. Using this method, the USP9 orthologue was identified as a major VAS-binding protein from both embryo and ovarian extracts.

Epsin/Lqf (liquid facets)

Lqf, a homologue of the vertebrate endocytic protein epsin, was found to associate with the Drosophila orthologue of USP9 [79]. In this study, the USP9 orthologue was identified in an anti-Lqf immunoprecipitate from embryo extracts. The study also showed that Lqf was ubiquitinated in vivo and deubiquitinated by Fat facets (the Drosophila orthologue of USP9). These results are consistent with previous genetic evidence implicating Lqf as a candidate for the key substrate of the USP9 orthologue [80].


Increased expression of USP10 has been associated with the disease glioblastoma multiforme [81]. At the cellular level, USP10 and its yeast homologue, UBP3, have been implicated in regulating anterograde and retrograde traffic between the Golgi and endoplasmic reticulum.

G3BP (RasGAP SH3 domain binding protein)/Bre5

A yeast two-hybrid assay using USP10 as bait isolated a cDNA clone encoding a protein called G3BP. This protein was shown to interact specifically with USP10, but not with other USP baits tested. The interaction was validated by performing the reverse two-hybrid assay, by in vitro binding and by in vivo co-immunoprecipitation assays in human cells. The two proteins appeared to co-purify as part of a large macromolecular complex that contains other proteins, perhaps including a USP10 substrate [82].

Interestingly, an interaction between the yeast homologues of USP10 and G3BP (UBP3 and Bre5, respectively) has also been detected in a high-throughput two-hybrid analysis of the yeast genome, thereby suggesting a role for the DUB in Golgi-to-endoplasmic-reticulum retrograde transport [83]. In yeast, UBP3 has been shown to regulate gene silencing through its interaction with SIR4 and to contribute to the cellular response to DNA damage [84,85].

The biological function of USP10 remains unknown. Soncini et al. [82] showed that although G3BP does not appear to be a substrate of USP10, it appears to inhibit the ability of USP10 to disassemble ubiquitin chains. Ubiquitin chain disassembly is important not only for the regeneration of free ubiquitin from chains released before proteasomal degradation, but also for generation of free ubiquitin subunits from synthesized linear polyubiquitin chains or from ubiquitin fusion proteins [86]. However, it appears that binding of the yeast homologues is required for the deubiquitination of Sec23, a substrate for Bre5–UBP3 [87].

AR (androgen receptor)

USP10 was identified as part of DNA-bound AR complexes purified from nuclear extracts of PC-3 cells stably expressing the AR in vivo [88]. This interaction was then confirmed in vitro by GST pull-down assays. USP10 appeared to modulate AR function. Cell-based trans-activation assays in PC-3 cells stably expressing the AR revealed that over-expression of wild-type USP10, but not of an enzymatically inactive form, stimulated AR activation of reporter constructs harbouring selective AREs (androgen response elements), SREs (non-selective steroid response elements) or the MMTV (mouse mammary tumour virus) promoter. Conversely, knockdown of USP10 expression was found to impair the MMTV response to androgen [88].


USP11 positively regulates IKKα [IκB (inhibitory κB) kinase α]. In response to TNF (tumour necrosis factor)α, USP11 functions as an upstream regulator of IKKα, which in turn stabilizes and activates p53 [89].

RelB complex

Using a TAP strategy to isolate complexes from control and TNFα-stimulated HEK-293 cells, USP11 was identified in a complex including components of the TNFα/NF-κB (nuclear factor κB) signalling pathway [90]. RelB is a transcription factor that forms a heterodimeric complex with p52 during NF-κB signalling [91,92]. Using TAP-tagged RelB, USP11 was specifically found in a complex that included the SWI/SNF chromatin-remodelling complex, HDAC (histone deacetylase) 6 and the lymphoid transcription-factor REQ [90].

BRCA (breast-cancer susceptibility gene)2

USP11 was identified using an immunopurification–mass-spectrometry approach to find novel proteins that associate with the BRCA2 gene product [93]. The study showed that in the cellular response to MMC (mitomycin)-induced DNA damage, BRCA2 appears to be regulated by ubiquitination that targets it for degradation at the proteasome. They showed that the two proteins interact in vivo, but whether this interaction was direct or mediated by other members of a multi-protein complex remains to be resolved. They found that overexpression of USP11, but not a catalytically inactive USP11 mutant, could deubiquitinate BRCA2. BRCA2 functions in the repair of DNA double-strand breaks by homologous recombination [94], and is an important protein in human disease because individuals carrying a germ-line mutation in the BRCA2 gene are predisposed to breast, ovarian and other types of cancer [93].


USP14 is a DUB that binds the Rpn1 subunit of the proteasome through its N-terminal ubiquitin-like domain [95]. Other studies have implicated USP14 in colorectal cancer [96] and ataxia in mice [97].

26S proteasome

Unlike the Rpn11 and UCH37 DUBs, which are two constituent proteasome subunits, the USP14 interaction with the proteasome is reversible [98]. The catalytic activity of USP14 is activated upon specific association with the 26S proteasome. The crystal structures of the 45 kDa catalytic domain of USP14 in isolation and in a complex with ubiquitin aldehyde revealed details about the significant conformational change that allows access of the ubiquitin C-terminus to the active site [17]. USP14 appears to function in the maintenance of cellular levels of monomeric ubiquitin in mammalian cells, and alterations in the levels of ubiquitin may contribute to neurological disease [99].


USP15 is a zinc-finger-containing DUB that has been shown to function in the cleavage of isopeptide bonds of polyubiquitin chains as well as in the cleavage of the ubiquitin–proline bond, a property previously thought to be unique to USP4, a protein with which USP15 shares significant sequence identity [100,101]

CSN (COP9 signalosome)

The CSN is a conserved protein complex that harbours deneddylating activity and contains subunits similar to the 26S proteasome lid [102]. It reverses the conjugation of the ubiquitin-like protein Nedd8 (neural-precursor-cell-expressed, developmentally down-regulated 8) to cullins, a modification necessary for optimal ubiquitination by the SCF (Skp1/cullin/F-box) ubiquitin ligases [103]. CSN activity is involved in the regulation of the ubiquitin–proteasome system, DNA repair, cell-cycle regulation and development [104106]. The biological role of the USP15/CSN complex may be in the protection of cullin–RING–ubiquitin ligases from auto-ubiquitination [102]. Interestingly, the USP15 orthologue in Saccharomyces pombe (UBP12) also binds the CSN [107].

USP20/VDU [VHL (von Hippel–Lindau protein)-interacting deubiquitinating enzyme]2

USP20 (also called VDU2) is a DUB that shares approx. 59% identity with USP33/VDU1 (see below) [108]. Both proteins have been shown to interact with the VHL tumour-suppressor ubiquitin E3 ligase enzyme [109,110].

VHL and HIF-1α (hypoxia-inducible factor-1α)

USP20 was shown to interact with the VHL β-domain and also to be ubiquitinated and degraded in a VHL-dependent manner. USP20 is unique from USP33 (another DUB that also interacts with VHL) in that it also interacts with HIF-1α. HIF-1α is the primary transcriptional response factor for cellular adaptation to hypoxic conditions and is ubiquitinated and degraded through the VHL pathway [111]. The action of USP20 may be to deubiquitinate HIF-1α, resulting in its stabilization [110].


USP22 is a DUB that has been postulated to play a critical role in cell-cycle progression [112]. USP22 is encoded by a gene in the eleven gene Polycomb/stem-cell signature, members of which play well-documented roles in cancer [113].

SAGA (Spt/Ada/Gcn5/acetyltransferase) complex

The SAGA complex is a multi-protein histone acetyltransferase transcriptional cofactor complex that is required for the function of sequence-specific transcription activators in eukaryotes [114]. USP22 was identified as a subunit of the human SAGA transcriptional cofactor complex [112]. In this study, USP22 was affinity-purified under non-denaturing conditions from nuclear extracts of H1299 human lung-cancer cells stably expressing FLAG epitope-tagged USP22. MS/MS analysis of the USP22 complexes revealed that most contained components of the SAGA complex. The yeast homologue of USP22 (UBP8) has also been shown to be a constitutive subunit of the yeast SAGA complex and to be required for SAGA-dependent transcription at some yeast genes [115,116]. USP22 appears to play a functional role in the human SAGA complex by deubiquitinating histone H2B, and as such may regulate gene transcription [112].


USP28 was found to regulate the Chk2 (checkpoint kinase 2)–p53–PUMA (p53 up-regulated modulator of apoptosis) pathway, a major regulator of DNA-damage-induced apoptosis in response to double-strand breaks in vivo [117]. The DUB is also required for maintaining stability of the Myc oncoprotein through its interaction with FBW7α [118].


USP28 was found to interact with FBW7α, an F-box protein that is part of an SCF-type ubiquitin ligase [117]. This ligase degrades proto-oncogenes in cellular growth and division pathways, including Myc, cyclin E, Notch and Jun. Using a co-immunoprecipitation assay, USP28 and FBW7α were shown to form a ternary complex with the Myc protein in vivo and to regulate Myc stability [119]. This study found that USP28 was required for Myc stability in human tumour cells and the stabilizing effect of USP28 was dependent on both its catalytic activity and its ability to reverse FBW7-mediated ubiquitination. Although it is not known if this mechanism pertains to all the FBW7α substrates, this interaction is clearly important in the regulation of Myc, an oncogenic transcription factor that is involved in many human tumours [120].


USP33 (also known as VDU1) was originally identified as a protein partner of VHL [109]. VHL is a tumour-suppressor ubiquitin E3 ligase enzyme, and mutations in the VHL gene result in the hereditary cancer syndrome called von Hippel–Lindau disease. The DUB interacts with both VHL as well as D2 (type-2 iodothyronine deiodinase).


In a study by Li et al. [109], USP33 was identified in a yeast two-hybrid assay using VHL as bait. The two proteins were later shown to interact directly in vitro and in vivo. The USP33–VHL interaction may have clinical significance, because naturally occurring mutations that have been found in the β-domain of USP33 disrupt the interaction between the two proteins. USP33 was found to be ubiquitinated in a VHL-dependent manner which targeted it for proteasomal degradation, and VHL mutations that disrupt the protein's interaction with the DUB abrogated USP33 ubiquitination. The findings from this study imply that USP33 is a downstream target for ubiquitination and degradation by the VHL E3 ligase.


USP33 was also identified in a yeast two-hybrid screen of a human brain library using D2 as bait [121]. The study found that D2 interacted with both USP33 and USP20/VDU2. The USP33–D2 interaction was then confirmed in mammalian cells. USP33 was found to co-localize with D2 in the endoplasmic reticulum, and co-expression prolonged the half-life and activity of D2 by deubiquitination [121]. USP33 is found to be increased in brown adipocytes by adrenaline (norepinephrine) or cold exposure, further amplifying the increase in D2 activity that results from catecholamine-stimulated de novo synthesis. Thus through its interaction with and subsequent degradation of D2, USP33 is believed to regulate thyroid hormone activation.


The CYLD gene encodes a tumour-suppressor protein that is mutated in familial cylindromatosis [122]. The CYLD DUB functions in the deconjugation of Lys63-linked polyubiquitin chains from target proteins and is involved in the negative regulation of NF-κB signalling [123127].

NFB pathway proteins

CYLD was found to interact with NEMO (NF-κB essential modulator; also known as IKKγ) via a yeast two-hybrid assay [124,126]. NEMO is a component of the IKK complex, which functions in the phosphorylation of IκB and subsequent release of the NF-κB transcription factor into the nucleus, where it acts as a transcription factor.

CYLD also interacts directly with TRAF [TNFR (TNF receptor)-associated factor]2, an adaptor molecule involved in signalling by members of the family of TNF/nerve-growth-factor receptors [124]. The inhibition of NF-κB activation by CYLD is mediated, at least in part, by the deubiquitination and inactivation of TRAF2 and, to a lesser extent, TRAF6 [126].

The clinical significance of the interactions with NEMO and TRAF2 and the subsequent regulation of the NF-κB pathway lie in the effect of CYLD on tumorigenesis. Mutations within the DUB domain of CYLD are found in cylindromatosis patients. Because they impair the deubiquitination of NEMO, TRAF2 and TRAF6, these mutations may lead to the enhanced activation of NF-κB, thereby contributing to tumour pathogenesis [127].

Full-length CYLD was also shown to interact the RING finger protein TRIP (TRAF-interacting protein) through a yeast two-hybrid screen using an HaCaT cDNA library [125]. This study confirmed the interaction of CYLD with the C-terminal domain of TRIP by far Western analysis and co-immunoprecipitations in mammalian cells.


More recently, CYLD has been shown to regulate Bcl-3 [128]. Bcl-3 is a constitutively nuclear protein, has transcriptional activation domains and can associate with p50 and p52 homodimers to act as a transcriptional co-activator [129]. Bcl-3 and CYLD were shown to interact specifically in vitro via a yeast two-hybrid assay and in vivo via co-immunoprecipitation analysis. This study showed that in response to PMA or UV light, CYLD translocates from the cytoplasm to the perinuclear region, where it binds to and deubiquitinates Bcl-3, thereby preventing nuclear accumulation of Bcl-3 and p50–Bcl-3- or p52–Bcl-3-dependent proliferation.



Mutations of the UCH-L1 gene and alterations of UCH-L1 protein activity have been found to be associated with several neurodegenerative disorders, including Parkinson's, Huntington's and Alzheimer's diseases [11]. UCH-L1 has also been found to be highly expressed in primary lung cancers and lung-cancer cell lines, suggesting that it may play a role in lung cancer tumorigenesis [130].

JAB1 (Jun activation domain-binding protein 1)/CSN subunit

Using UCH-L1 as bait in a yeast two-hybrid assay with an expression cDNA library derived from fetal brain, the DUB was shown to interact with JAB1 [131]. JAB1 is a subunit of the CSN complex and mediates numerous cellular processes, including the cytoplasmic transportation of cdk (cyclin-dependent kinase) inhibitor p27 (Kip1) and its subsequent proteasome-mediated degradation [103]. The study confirmed that the two proteins interact in vivo via co-immunoprecipitation. This interaction appeared to be functionally important, because both proteins appeared to be part of a heteromeric complex containing p27 in the nucleus of lung cancer cells. UCH-L1 may contribute to p27 degradation via its interaction and nuclear translocation with JAB1 [131].


UCH37 functions by processively removing ubiquitin from the distant end of polyubiquitin chains, and this has been suggested to serve as an ‘editing’ function that can rescue poorly ubiquitinated proteins from a proteolytic fate [98]. The best characterized role of UCH37 is through its interaction with the proteasome subunits S14 and Adrm1. It also interacts with UIP1 (UCH37 interacting-protein 1) and Smad proteins.

S14 and UIP1

By using a yeast two-hybrid screen, two proteins that interacted with UCH37 were identified: S14, a subunit of the 19S regulatory particle of the proteasome, and UIP1 [132]. These interactions were then confirmed by in vitro binding assays and in vivo co-immunoprecipitation analyses. The study found that the C-terminus of UCH37 is essential for the interaction with S14 or UIP1. Support for the fact that the two proteins occupy the same binding site came from the observation that UIP1 blocked the interaction between UCH37 and S14 in vitro [132].

Adrm1 (human Rpn13)

UCH37 was found to bind to the proteasome through Adrm1, a previously unrecognized orthologue of Saccharomyces cerevisiae Rpn13, which in turn is bound to the S1 (also known as Rpn2) subunit of the 19S complex [133]. Binding of Adrm1 to UCH37 relieved auto-inhibition by the UCH domain and activated its deubiquitination activity [133].


A novel specific interaction between Smad transcription factors and UCH37 was identified [134]. Using GST pull-down assays, UCH37 was shown to interact specifically with Smad7 in vitro and in vivo via co-immunoprecipitation with haemagglutinin-tagged UCH37 in transfected HEK-293 cells. UCH37 displayed weaker associations with Smad2 and Smad3. The UCH37-interaction region was distinct from the motif on Smad7 that interacts with Smurf (Smad–ubiquitin regulatory factor) ubiquitin ligases. This study demonstrated that UCH37 may have biological roles distinct from its role at the proteasome [135]. The study hypothesized that Smad7 could act as an adaptor to recruit UCH37 to the type I TGF-β (transforming growth factor-β) receptor and showed that UCH37 dramatically up-regulates TGF-β-dependent gene expression by deubiquitinating and stabilizing the type I TGF-β receptor [134].

BAP1 (BRCA1-associated protein 1)

The N-terminus of BAP1 has a UCH domain that shows high sequence homology to other members of the UCH family. BAP1 is unique, however, in that it also harbours a large C-terminal extension that appears to be important in substrate recognition.


BAP1 is a putative tumour suppressor that was first discovered in a yeast two-hybrid screen to identify proteins that interact with the RING finger domain of the BRCA product, BRCA1 [9,136]. In this study, BAP1 was shown to interact with the wild-type BRCA1 RING finger domain, but not with cancer-associated RING finger mutants. Furthermore, the two proteins appeared to interact in a biologically significant manner, because BAP1 augmented the growth-suppressive properties of BRCA1 in breast cancer cells.



A20 is a TNF- and IL-1 (interleukin 1)-inducible zinc-finger protein that has been characterized as an inhibitor of both NF-κB activation and apoptosis [137,138]. A20 is unique in that it contains both deubiquitinase activity as well as ubiquitin E3 ligase activity [139,140]. A20 inhibits NF-κB signalling through various pathways, including those involving TNFR-associated proteins, TRADD (TNFR-associated death domain protein), RIP (receptor-interacting protein) and TRAF2 [138].

NFB pathway proteins

The N-terminal region of A20 (that lacks the zinc-finger structures) binds to TRAF2 complexes [141]. A20 was identified in a yeast two-hybrid assay to search for TRAF2-interacting proteins, and this interaction was confirmed in vivo [141]. Mutational analysis revealed that the N-terminal half of A20 interacts with the conserved TRAF domain of both TRAF1 and TRAF2. TRAF2 functions in the activation of the NF-κB transcription factor triggered by TNF and the CD40 ligand [142]. Co-transfection experiments revealed that A20 blocked TRAF2-mediated NF-κB activation [141].

The C-terminal region (containing the functional zinc-finger structures) mediates A20 dimerization and binding to several other proteins that might be involved in the inhibition of NF-κB activation and apoptosis by A20 [141]. The C-terminal region has been found to interact with RIP and to a novel protein, ABIN (A20-binding inhibitor of NF-κB activation) [138].

Cezanne (cellular-zinc-finger anti-NF-κB)

Cezanne is an OTU-domain-containing protein that, like A20, is an inhibitor of the NF-κB signalling pathway [143]. Cezanne may have some functions that are distinct from A20. Although A20 does not have a global deubiquitinating activity and does not participate in ubiquitin recycling, expression of Cezanne has been shown to prevent the up-build of polyubiquitinated cellular proteins in cultured cells in response to the proteasome inhibitor MG132 [144].


Cezanne was found to interact with TRAF6 via co-immunoprecipitation studies in vivo. In contrast, reporter-gene experiments revealed a specific ability of Cezanne to down-regulate NF-κB. It is likely, therefore, that Cezanne participates in the regulation of inflammatory processes [143].

VCIP135 [VCP (valosin-containing protein) (p97)–p47 complex-interacting protein, p135]

VCIP135 is a deubiquitinating enzyme that was originally identified as an essential factor for p97–p47-mediated membrane fusion [145]. VCIP135 contains a region with homology to the catalytic domains of the deubiquitinating enzymes, Cezanne and A20 [146]. Cdc48/p97 (also called VCP) is an AAA (ATPase associated with various cellular activities) which, together with its adaptor p47, regulates several membrane fusion events, including reassembly of Golgi cisternae after mitosis [146].

Cdc48p97 complex

Using affinity chromatography, VCIP135 was found to bind to the p97–p47–syntaxin5 complex [145]. The study used a fragment of p47 [containing only one of the p97-binding sites (residues 171–270)] immobilized on to a bead, incubated it with rat liver cytosol and identified interacting proteins by microsequencing via electrospray MS. This study also found that VCIP135 dissociates the p97–p47–syntaxin5 complex via p97-catalysed ATP hydrolysis. In addition, by microinjection of antibodies against VCIP135 and p47 into living cells, they showed that VCIP135 and p47 are required for Golgi assembly and endoplasmic-reticulum network formation in vivo [145]. Other studies have confirmed that the p97–p47–VCIP135 complex is required for cell-cycle-dependent re-formation of the endoplasmic-reticulum network and that p97–p47-mediated reassembly of Golgi cisternae requires the deubiquitinating activity of VCIP135 [146,147].



Ataxin-3 is mutated by expansion of a polyglutamine tract in the human neurodegenerative disease spinocerebellar ataxia type 3 [148]. The protein contains an N-terminal Josephin domain followed by tandem UIMs (ubiquitin-interacting motifs) and a polyglutamine stretch.

PLIC-1 (protein linking inhibitor of apoptosis to the cytoskeleton-1)

Ataxin-3 was found to interact with the ubiquitin-like domain of PLIC-1. PLIC-1 is an ubiquitin-like protein that binds to several UIM-containing proteins, including Ataxin-3 [149]. In this study, GST pull-downs showed that Myc-tagged Ataxin-3 exogenously expressed in cultured cells interacted with GST-fused PLIC-1 ubiquitin-like domain in vitro. Furthermore, the study showed that the two proteins co-localize in small punctate structures in BHK (baby-hamster kidney) cells [149].


Another protein that interacts with Ataxin-3 is HDAC3. Ataxin-3 was found to bind to target DNA sequences in specific chromatin regions of the matrix metalloproteinase-2 gene promoter and repress transcription by recruitment of HDAC3 and the nuclear receptor co-repressor [150]. Both normal (Q23) and expanded (Q70) human full-length Ataxin-3 physiologically interacted with HDAC3 and nuclear receptor co-repressor in a SCA3 rat cell line and human pons tissue. The interaction between Ataxin-3 and HDAC3 is thought to result in the deacetylation of histones and reduce binding of the transcription factor GATA-2 to target regions of the matrix metalloproteinase-2 promoter.

Ataxin-3 has also been shown to interact with the major histone acetyltransferases CREB (cAMP-response-element binding protein)-binding protein, p300, and p300/CREB-binding protein-associated factor in vitro and in vivo, and inhibit transcription by these co-activators [151].



AMSH is a JAMM-motif endosomal DUB that can limit epidermal-growth-factor receptor down-regulation [152]. AMSH was found to interact with the SH3 domain of STAM, a component of the multivesicular body pathway [153], as well as components of the ESCRT-III (endosomal sorting complex required for transport-III) complex [154].

STAM and clathrin

STAM is a protein that regulates receptor sorting at the endosome [152]. Using an in vitro binding assay, GST–AMSH was found to interact with purified hexahistidine-tagged STAM, and this interaction required the SH3 domain of STAM, but not the UIM domain [153]. This study also identified clathrin as a novel binding partner of AMSH and confirmed the interaction in vivo via co-immunoprecipitation experiments from transiently transfected HEK-293T cells. Another study found that the AMSH–STAM interaction enhanced the deubiquitination of STAM-bound Lys63-linked ubiquitins. The study showed that STAM functions by binding to ubiquitinated substrates and holding the ubiquitin molecules in place for deubiquitination by AMSH [155].

ESCRT-III complex

AMSH has also been shown to interact with subunits of ESCRT-III. These include CHMP1A, CHMP1B, CHMP2A, CHMP3, CHMP4A and CHMP4B. These interactions have been observed via yeast two-hybrid assays; however, only the CHMP3, 1A, 1B and 2A interactions have been confirmed by alternative protein-interaction assays [62,153,154]. These interactions are consistent with a role of AMSH in the deubiquitination of endosomal cargo preceding lysosomal degradation. It has been suggest that both the DUB activity of AMSH and its CHMP3-binding ability are required to clear ubiquitinated cargo from endosomes [154,156].


Rpn11 is a JAMM metalloprotease that appears to be required for proper proteasomal processing of ubiquitinated substrates. Rpn11 (along with UCH37) is a constituent component of the proteasome [98]. Its enzymatic activity has only been detected in context with the 19S or 26S proteasome complexes.

26S proteasome

Rpn11 is one of three DUBs associated with the 19S regulatory core of the proteasome and is the most conserved lid component [157]. The biological significance of the interaction can be inferred from studies showing that RNA interference of Rpn11 decreases cellular proteasome activity via disrupted 26S proteasome assembly, and inhibits cellular protein degradation [98].


The association of DUBs with the proteasome offers specific examples of the roles of protein–protein interactions in regulating the activity and specificity of DUBs. The proteasome is composed of different sub-complexes (Figure 4); a 20S core proteasome harbouring the proteolytic active sites hidden within its barrel-like structure and two 19S caps that execute regulatory functions [158]. The 19S regulatory complex can be further subdivided into two sub-complexes, the base and the lid [159]. Cumulative evidence points to a partitioning of proteasome action between deubiquitination and proteolysis [160]. Ubiquitinated proteins that are targeted to the 26S proteasome for degradation first bind, possibly via the proteasome subunit Rpt2 or adaptors such as hRad23, Rpn10 or p97–AAA complexes, and are subsequently deubiquitinated, unfolded by the base and degraded inside the 20S core [157]. The 26S proteasome contains at least three DUBs. These include: (1) Rpn11/POH1, (2) UCH37/UCH2 and (3) USP14 [160]. Rpn11 and UCH37 are two constituent proteasome subunits, whereas the USP14 interaction is reversible [98]. The effect that proteasome binding has on these DUBs exemplifies the range of effects conferred by binding proteins. These effects include DUB structure, activity, specificity and/or regulation.

Figure 4 The association of DUBs with the proteasome offers specific examples of the roles of protein–protein interactions in regulating the activity and specificity of DUBs

The 26S proteasome is composed of different sub-complexes: a 20S core proteasome harbouring the proteolytic active sites hidden within its barrel-like structure and two 19S caps that execute regulatory functions. The 19S regulatory complex can be further subdivided into two sub-complexes, the base and the lid. The 26S proteasome contains at least three DUBs: (1) Rpn11/POH1, (2) UCH37/UCH2 and (3) USP14. Rpn11 and UCH37 are two constituent proteasome subunits, whereas the USP14 interaction is reversible.


The three main proteasome-associated DUBs (USP14, Rpn11 and UCH37) have all been shown to alter their conformation or enzyme activity upon association with their binding partner, in this case the 26S proteasome. DUB activity of USP14 was shown to increase by several orders of magnitude upon binding to the proteasome, suggesting that the proteasome is the physiological site of USP14 function [161]. Likewise, the enzymatic activity of Rpn11 has only been detected in the context of the 19S or 26S proteasome complexes [98]. Binding of UCH37 to Adrm1 [a previously unrecognized orthologue of S. cerevisiae Rpn13, which in turn is bound to the Rpn2 (also known as S1) subunit of the 19S complex] was found to relieve the auto-inhibition of UCH37 by its UCH domain and activate its DUB activity [133]. This ability of DUBs to be activated only when bound to a specific protein partner may have developed as a way to prevent non-specific deubiquitination of cellular proteins, which could in turn lead to cellular dysfunction.


Proteasome-bound UCH37 functions by processively removing ubiquitin from the distal end of polyubiquitin chains and has been suggested to have an ‘editing’ function [162]. The fact that UCH37 sequentially disassembles ubiquitin chains from their distal end may allow for another level of substrate regulation at the 26S proteasome. UCH37 may function to target specifically poorly ubiquitinated proteins or to clear slowly degraded proteins from a proteasome that may have ‘stalled’ [98,163]. Purified UCH37 exhibits slow non-processive cleavage of polyubiquitin chains at any position within the chain [133]. This suggests that the presentation of the substrate by its association with proteasome ubiquitin-binding sites enforces both the distal end specificity and its processivity.


The regulation of DUB activity by virtue of protein binding is exemplified again through the reversible USP14–proteasome interaction [98]. Therefore the reversible nature of the USP14 interaction may function as an important regulatory step in proteasomal-mediated degradation of certain substrates. Isolated USP14 exhibits a very sluggish catalysis, with little or no affinity for ubiquitin or polyubiquitin [164], suggesting that the substrate must be delivered by the action of the proteasome. Notably, neither USP14 nor Rpn11 exhibits much DUB activity in the absence of substrate engagement and the commencement of degradation.

Other roles of proteasomal DUBs

Unlike USP14, UCH37 is a stoichiometric constituent proteasome subunit. However, the binding of UCH37 to the proteasome may itself be regulated by its interaction with UIP1. A study by Li et al. [132] found that the C-terminus of UCH37 is essential for the interaction with both the proteasome and UIP1. The study showed that UIP1 can block the interaction between UCH37 and the proteasome subunit in vitro, thereby supporting the fact that the two proteins occupy the same binding site and that UIP1 may regulate UCH37's activities at the proteasome.

Other studies have shown that UCH37 may have biological roles distinct from its role at the proteasome [135]. In other words, there may be a non-proteasome-associated pool of UCH37. The study by Wicks et al. [135] hypothesized that Smad7 could act as an adaptor to recruit UCH37 to the type I TGF-β receptor and showed that UCH37 dramatically up-regulates TGF-β-dependent gene expression by deubiquitinating and stabilizing the type I TGF-β receptor [134].

Association with ligases

As mentioned above, USP14 regulates protein degradation at the proteasome by trimming the ubiquitin tag on the target substrate protein. Another protein that is recruited to proteasomes containing USP14, Hul5, may also regulate degradation when bound to the proteasome, but in a manner opposite to that of USP14. Hul5 is an ubiquitin-ligase enzyme that works by extending the number of ubiquitin moieties in the tag on substrates, thereby promoting proteasomal binding and subsequent degradation. As such, Hul5 associates with proteasomes containing USP14 and works antagonistically to USP14. The balance between these two opposing activities might control the substrate specificity of a sub-population of proteasomes, and adjusting the level of these specialized proteasomes would provide a new level of degradation control [165].


The examples discussed above demonstrate that a detailed understanding of DUB specificity and physiology will often require knowledge of associated substrates, adaptors and scaffolds. Such associations control specificity by delivering specific substrates, control activity by reversing mechanisms that maintain isolated DUBs in a cryptic state, and control the physiological roles of DUBs by affecting their stability/abundance and their cellular localization. The large number of DUBs and their binding partners suggests multiple roles for DUBs that are important in regulating numerous cellular pathways. These same features suggest that DUBs are attractive pharmacological targets. A truly specific inhibitor of an individual DUB should be limited in its action and largely restricted to the distinct pathway(s) regulated by that DUB. In addition, the fact that such inhibitors may only need to prevent the side-chain rotation of catalytic residue, the motion of an occluding loop or a single low-affinity interaction in the context of a larger macromolecular complex suggests that DUBs are attractive targets for drug development.

Abbreviations: AAA, ATPase associated with various cellular activities; AMPK, AMP-activated protein kinase; AR, androgen receptor; BRCA, breast-cancer susceptibility gene; BAP1, BRCA1-associated protein 1; Cezanne, cellular-zinc-finger anti-NF-κB; CHMP, charged multivesicular body protein; CREB, cAMP-response-element binding protein; CSN, COP9 signalosome; D2, type-2 iodothyronine deiodinase; DUB, deubiquitinating enzyme; EBNA1, Epstein–Barr nuclear antigen 1; ESCRT-III, endosomal sorting complex required for transport-III; FANCD2, Fanconi anaemia complementation group D; FAS, fatty acid synthase; GAP, GTPase-activating protein; GST, glutathione transferase; HAUSP, herpesvirus-associated USP; Hbp, Hrs-binding protein; HDAC, histone deacetylase; HEK-293, human embryonic kidney; HIF-1α, hypoxia-inducible factor-1α; Hrs, hepatocyte receptor substrate; IκB, inhibitory κB; ICP0, infected cell polypeptide 0; IKK, IκB kinase α; JAB, Jun activation domain-binding protein 1; JAMM, JAB1/MPN/Mov34 metalloenzyme; Lqf, liquid facets; MARK, microtubule-affinity-regulating kinase; Mdm2, murine double minute 2; Mib1, Mind bomb 1; MIT, microtubule interacting and transport; MJD, Machado–Joseph disease protease; MMTV, mouse mammary tumour virus; MS/MS, tandem MS; Myl2, light regulatory chain of myosin II; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; Nrdp1, neuregulin-receptor-degradation protein-1; OTU, otubain protease; PLIC-1, protein linking inhibitor of apoptosis to the cytoskeleton-1; RING, really interesting new gene; RIP, receptor-interacting protein; SAGA, Spt/Ada/Gcn5/acetyltransferase; SCF, Skp1/cullin/F-box; SH3, Src homology 3; G3BP, RasGAP SH3 domain binding protein; STAM, signal transducing adaptor molecule; AMSH, associated molecule with SH3 domain of STAM; TAP, tandem affinity purification; TGF-β, transforming growth factor-β; TNF, tumour necrosis factor; TNFR, TNF receptor; TRAF, TNFR-associated factor; TRIP, TRAF-interacting protein; UAF1, USP1-associated factor 1; UIP1, UCH37 interacting-protein 1; USP, ubiquitin-specific protease; UCH, ubiquitin C-terminal hydrolase; UIM, ubiqutin-interacting motif; VCP, valosin-containing protein; VCIP135, VCP(p97)–p47 complex-interacting protein, p135; VHL, von Hippel–Lindau protein; VDU, VHL-interacting deubiquitinating enzyme


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