Physiol Rev 93: 1289 –1315, 2013
doi:10.1152/physrev.00002.2013
DEUBIQUITYLASES FROM GENES TO ORGANISM
Michael J. Clague, Igor Barsukov, Judy M. Coulson, Han Liu, Daniel J. Rigden, and Sylvie Urbé
Cellular and Molecular Physiology, Institute of Translational Medicine, and Institute of Integrative Biology,
University of Liverpool, Liverpool, United Kingdom
Clague MJ, Barsukov I, Coulson JM, Liu H, Rigden DJ, Urbé S. Deubiquitylases
From Genes to Organism. Physiol Rev 93: 1289 –1315, 2013; doi:10.1152/
physrev.00002.2013.—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.
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
INTRODUCTION
CATALYTIC MECHANISM
DUBOMICS
CLEAVAGE SPECIFICITY
REGULATION OF DUB ACTIVITY
THE FIVE DUB FAMILIES: ROLES IN...
DUBs AS INTEGRAL COMPONENTS OF...
INFLUENCE ON CELL PHYSIOLOGY...
CONCLUSIONS
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I. INTRODUCTION
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 gen-
eration 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 Lys63linked 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 proteinprotein 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,
0031-9333/13 Copyright © 2013 the American Physiological Society
1289
CLAGUE ET AL.
A
B
K63
C
M1
lin
K48
K6
K29
K63
K33
K27
K11
K48
lin
K63
K6
ub
K11
C-term
K11
K6
K48
FIGURE 1. Structures of ubiquitin chains. A: structure of ubiquitin (PDB ID 1ubq) in cartoon representation
showing the side-chain positions of all seven Lys residues and Met1 (orange). B: superposition of the proximal
ubiquitin molecules of di-ubiqutin structures on the mono-ubiquitin oriented as in A. Chain types are represented by different colors: mono-ubiquitin, green; linear chain (PDB ID 2w9n), cyan; K6 (PDB ID 2xk5),
magenta; K11 (PDB ID 2xew), yellow; K48 (PDB ID 1aar), bronze; and K63 (PDB ID 2jf5), gray. C: surface
representation of di-ubiquitin and mono-ubiquitin structures in the same orientation as in B. Hydrophobic I44
patch is highlighted in blue, proximal ubiquitin is colored in green in each molecule and distal ubiquitin colored
as in B.
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 NF␬B 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).
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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 met-
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DUBs FROM GENES TO ORGANISM
alloproteases 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).
III. DUBOMICS
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 proteinprotein 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 comple-
ment 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 USP245, 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 Lys63linkages (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
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CLAGUE ET AL.
A
a
Proximal
Lys
Ubiquitin
H
O
N
C
Distal
Gly
Isopeptide Ubiquitin
bond
Catalytic triad
O
S1'
Asp
S1
S–
O–
Cys
NH+
HN
His
DUB (USP)
d
c
b
Attack
Lys
Ubiquitin
H
O
N
C
Tetrahedral
intermediate
Gly
Lys
Ubiquitin
O
Ubiquitin
S–
–
O
Asp
H
O–
N
C
O
O
Cys
NH+
HN
Oxyanion hole
Asp
NH3+
Lys
O
Ubiquitin
C
Gly
Ubiquitin
S
O
Release
–
S
O–
Cys
NH+
HN
His
Asp
HN
His
DUB (USP)
Gly
Ubiquitin
Cys
N
His
DUB (USP)
DUB (USP)
Acyl enzyme intermediate
e
f
g
O
Oxyanion hole
Deacylation
(water attack)
O
O
C
H
Gly
Ubiquitin
S
Tetrahedral
intermediate
O–
Asp
HO
Cys
N
Asp
HN
His
a
Ubiquitin
O
N
Isopeptide
bond
Release
S–
–
O
NH
Cys
+
Asp
Cys
NH+
HN
His
DUB (USP)
DUB (USP)
Distal
H
Proximal
Lys63
Catalytic triad
Gly
Ubiquitin
S
His
DUB (USP)
B
C
O
Gly
Ubiquitin
O–
HN
C
O–
O
H
HO
C
Gly
O
Ubiquitin
Active site
S1'
Glu
O– H
O
O
H
2+
S1
HO
Zn
His
Ser
His Asp
AMSH-LP
b
c
d
NH3+
Lys63
H
N
Lys63
Ubiquitin
Glu
–
O
O
His
O
H
H
2+
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Gly
O
Ubiquitin
HO
Zn
His Asp
AMSH-LP
Ubiquitin
H
C
Ser
Lys63
Ubiquitin
Glu
N
C
Gly
HO
O–
Ubiquitin
OH
O
His
2+
–
HO
Zn
Ser
Release
Glu
His Asp
AMSH-LP
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O
C
Gly
O
Ubiquitin
Release
O–
O
His
2+
HO
Zn
His Asp
AMSH-LP
Ser
DUBs FROM GENES TO ORGANISM
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 COOHterminal 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
FIGURE 2. Reaction mechanisms. Schematic overview of USP (A) and JAMM (B) action upon di-ubiquitin substrates. Covalent, isopeptide, and
noncovalent bonds are shown by black solid, red solid, and orange dashed lines, respectively. A, a: conserved residues form a catalytic triad.
Nearby S1= and S1 sites accommodate the proximal and distal ubiquitin units, respectively. b: The deprotonated thiol group carries out a
nucleophilic attack on the carbonyl carbon forming a negatively charged tetrahedral intermediate (c), with the negatively charged O⫺ occupying
the oxyanion hole where it is stabilized by hydrogen bonding (not shown). d: Collapse of the tetrahedral intermediate results in scission and
release of the distal ubiquitin leaving an acyl-enzyme intermediate. e: A water molecule attacks the acyl-enzyme intermediate, resulting in another
negatively charged intermediate shown in f. g: Proximal ubiquitin is released, and the original configuration of the catalytic triad is recovered.
B: metalloprotease activity of JAMM family members exemplified by AMSH-LP. a: Conserved residues coordinate zinc and water. b: AMSH-LP
specifically binds to lysine-63-linked ubiquitin chain, through S1 and S1= sites allowing the hydroxyl group of active site water molecule to carry
out nucleophilic attack on the carbonyl carbon between two ubiquitin molecules, forming a tetrahedral intermediate shown in c. d: Collapse of
intermediate leads to scission and product release.
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CLAGUE ET AL.
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
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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
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DUBs FROM GENES TO ORGANISM
FIGURE 3.—Continued
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
FIGURE 3. Schematic representation of domain architectures of human DUBs. Domains, drawn approximately to scale, were assigned by
HHsearch (240) analysis against the CDD (166), Pfam (202), and SMART (149) domain databases and against structures in the PDB (216).
Regions indicated in pink are those of greater than 20 residues predicted as intrinsically disordered by IUPRED (59). Since short UIMs were not
reliably predicted with HHsearch, consensus predictions for these were made considering Pfam search results and local searches of PROSITE
(238) with ScanProsite (78). Some homologs of zinc fingers and EF-hand structures are predicted to lack metal binding capacity due to their lack
of key ligating residues. Domains unique to PRPF8 are numbered as follows: 1) PRO8NT, 2) PROCN, 3) RRM_4, 4) U5_2-snRNA_bdg,
5) U6-snRNP_bdg, 6) PRP8_IV, 7) PROCT. For some sequences, certain stretches of amino acid (e.g., ⫹100) are not shown. Names used
correspond to NCBI-approved gene symbols except for those listed below. The expanded USP17 gene family is represented by one member,
DUB3, officially known as USP17-like protein 2. POH1 (PSMD14), AMSH (STAMBP), AMSH-LP (STAMBPL), YOD1 (OTU1), A20 (TNFAIP3),
TRABID (ZRANB1), Cezanne (OTUD7B), Cezanne 2 (OTUD7A). Enzymes indicated by (*) are predicted to be inactive based on sequence or
structural (PRPF8) analysis. Note that EIF3F has been proposed to deubiquitylate Notch (178) and USPL1 is a SUMO-specific protease (230).
**HIN1L is annotated as a hypothetical protein. DUBs are ordered according to bootstrapped neighbor-joining phylogenetic analysis of their
catalytic domains [using MEGA (249): see legend to Supplementary Figure 1 for detailed methods] shown as a tree to the left. Nodes supported
by bootstrap values of ⬎50% are indicated with a dot.
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Table 1. Proposed orthologs of human DUBs assigned in silico as bidirectional best hits (9, 280) against the species shown by
reciprocal BLAST analysis (10)
USP1
USP2
USP3
USP4
USP5
USP6
USP7
USP8
USP9Y
USP9X
USP10
USP11
USP12
USP13
USP14
USP15
USP16
USP17L2
USP18
USP19
USP20
USP21
USP22
USP24
USP25
USP26
USP27X
USP28
USP29
USP30
USP31
USP32
USP33
USP34
USP35
USP36
USP37
USP38
USP39
USP40
USP41
USP42
USP43
USP44
USP45
USP46
USP47
USP48
USP49
USP50
H. sapiens
D. rerio
C. elegans
S. pombe
S. cerevisiae
O94782
O75604
Q9Y6I4
Q13107
P45974
P35125
Q93009
P40818
O00507
Q93008
Q14694
P51784
O75317
Q92995
P54578
Q9Y4E8
Q9Y5T5
Q6R6M4
Q9UMW8
O94966
Q9Y2K6
Q9UK80
Q9UPT9
Q9UPU5
Q9UHP3
Q9BXU7
A6NNY8
Q96RU2
Q9HBJ7
Q70CQ3
Q70CQ4
Q8NFA0
Q8TEY7
Q70CQ2
Q9P2H5
Q9P275
Q86T82
Q8NB14
Q53GS9
Q9NVE5
Q3LFD5
Q9H9J4
Q70EL4
Q9H0E7
Q70EL2
P62068
Q96K76
Q86UV5
Q70CQ1
Q70EL3
F1QNF4
E7FEC1
E7FC36
Q8I077
Q9VR54
Q1MT86
Q9VZU7
P91502
Q11119
P38237
I3ISS5
I3IT16
Q9VYQ8
Q9VDD8
Q7JKC3
G5EBW2
Q9UTT1
P50101
P32571
Q4FE55
E7F2D1
F1QPF4
A4FUN7
F1QFS9
F1R4B7
E7EZD6
A8HAL1
P55824
Q9W0L7
O94269
Q01477
E7EYZ1
F1RCS0
A5PN09
E9QFQ5
A6H8I0
F1Q6K1
E7FFB3
A2BGT0
E7F6P1
E7FCR5
A5PMR2
A8WFZ5
E7F4C0
E7F6T8
F1RBA1
A3KQ59
F1Q7D2
F1QSB4
F1Q8C6
Q7ZUM8
E9QG68
A5WWB0
H9GYG8
E7EY58
D. melanogaster
I2HA92
Q9P7V9
Q9VKZ8
Q17361
Q92353
O60079
Q7JW61
P43593
P39538
P53874
Q9VVR1
Q09738
P50102
Q9P3U0
Q01476
G5EG81
Q09931
Q9W462
Q9BKQ6
Q9VW49
Q9VC56
Q7JQI1
Q9VRP5
Q9VWP1
O74442
O44787
Q9USR2
P43589
Q9W117
Q9W4C3
Q9VCT9
Q24574
Q9P7S5
P34547
Q22240
G5ECC7
P39967
Continued
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DUBs FROM GENES TO ORGANISM
Table 1.—Continued
H. sapiens
USP51
USP52
USP53
USP54
CYLD
USPL1
UCHL1
UCHL3
UCHL5
BAP1
ATXN3
ATXN3L
JOSD1
JOSD2
OTUB1
OTUB2
OTUD1
OTUD3
OTUD4
HIN1L
OTUD5
OTUD6A
OTUD6B
YOD1
A20
Cezanne 2
Cezanne
TRABID
VCPIP1
BRCC3
COPS5
COPS6
POH1
PSMD7
AMSH
AMSH-LP
MPND
MYSM1
PRPF8
EIF3F
EIF3H
Q70EK9
Q504Q3
Q70EK8
Q70EL1
Q9NQC7
Q5W0Q7
P09936
P15374
Q9Y5K5
Q92560
P54252
Q9H3M9
Q15040
Q8TAC2
Q96FW1
Q96DC9
Q5VV17
Q5T2D3
G3V0I6
Q7RTX8
Q96G74
Q7L8S5
Q8N6M0
Q5VVQ6
P21580
Q8TE49
Q6GQQ9
Q9UGI0
Q96JH7
P46736
Q92905
Q7L5N1
O00487
P51665
O95630
Q96FJ0
Q8N594
Q5VVJ2
Q6P2Q9
O00303
O15372
D. rerio
E7FG33
E7FGV9
E7F383
E7FEV5
Q6DRC5
Q6YI49
Q504C0
Q6NWL6
A1L2G3
Q7ZU73*
Q08C38
F1QCJ5
Q6DGP0
D. melanogaster
C. elegans
S. pombe
S. cerevisiae
A1Z7K9
P53015
Q09798
P53010
Q1W9Q0
Q8IPC5
Q7JMS4
Q10171
Q9UUB6
P35127
P35122
Q9XZ61
Q7K5N4
Q9UAV3
Q09444
O17850
Q9W422
Q9VL00
Q8IAA2
Q9XVR6
Q7JLI8
F1R341
F1QHE7
Q08BW0
Q9VTK7
O44438
Q7ZV00
Q567B1
E7F165
F1QRG1
Q9VUN9
Q9VRJ9
Q19681
A0JMQ9
F1QCM0
F1R395
Q6PC30
Q567F8*
F1QHE5
Q7ZYX7
Q6TH47
E7F3M4
Q08CH3
Q5RGA4
F1R7K1
E7F3G6
Q6AXJ2
Q9VH90
Q9UUK3
O13974
P38747
P43558
O94454
Q12468
P41878
O74440
P43588
Q08723
Q9N4T5
Q9XZ58
Q9VCY3
Q9V3H2
P26270
P91001
Q95PZ0
O76577
O61792
Q9VA71
Q9VKJ1
A1Z8U0
Q9VN50
Q9U9Q4
Q9P371
P34369
Q18967
O01974
O14187
O43060
Q9UT48
P33334
Species are represented by reference proteomes retrieved from UniProt. In a few italicized cases, the
reciprocal BLAST criterion was not satisfied for the human accession shown but for a different product of the
same gene. Emboldened accessions in other species are not those retrieved by reciprocal BLAST but instead
products of the same gene that are preferred for their greater length and/or reviewed status in UniProt.
Asterisks mark two similar cases where the preferred accession shown is not the UniProt reference genome
for D. rerio.
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
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CLAGUE ET AL.
A
PSMD7
OTUB1
EIF3F
POH1
EIF3H
CSN6
UCHL3
UCHL5
USP5
CSN5
PRPF8
USP14
USP9X
OTUD6B
USP7
USP47
USP15
BRCC3
OTUD4
AMSH
USP4
USP10
USP19
VCPIP1
USP3
CYLD
USP12
USP24
USP8
USP9Y
USP36
USP34
USP48
USP32
USP25
USP40
USP30
102
103
104
105
106
Copy number/cell
B
USP1
USP36
USP6
pm
USP3
USP39
USP19
USP7
er
USP11
USP22
nucleus
USP8
USP26
USP33
USP29
USP42
USP21
USP44
USP33
USP49
USPL1
USP21
mt
ee
mvb
1298
AMSH
AMSH-LP
golgi
mito
CYLD
USP33v3
BAP1
MYSM1
USP2a
USP30
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USP32
DUBs FROM GENES TO ORGANISM
Trachea
A20
Spinal cord
AMSH
Haemopoietic system
EIF3H, AMSHLP, PRPF8, OTUD5, A20
CYLD, USP3, USP4, USP7, USP8, USP9Y,
USP15, USP20, USP25, USP28, USP33,
USP34, USP36, USP39, USPL1, PARP11
Bone
MYSM1, POH1, USP9Y, USP16, USP36
Skin
VCPIP1, USP15, USP53, USP54
Brain
MYSM1, ATXN3L, OTUB1, Cezanne2,
TRABID, UCHL1, USP6, USP9X, USP11,
USP14, USP21, USP22, USP26, USP29,
USP32, USP33, USP35, USP42, USP46,
USP51, USP54
Lung
PRPF8, AMSH, OTUD1, OTUD6A, A20,
UCHL3, USP3
Heart
OTUB1, OTUD6A, USP13
Reproductive system
COPS5, COPS6, EIF3H, PRPF8, JOSD1,
OTUD1, OTUD5, Cezanne, Cezanne2,
TRABID, BAP1,UCHL3, USP1, USP4,
USP7, USP9X, USP10, USP13, USP18,
USP21, USP24, USP25, USP28, USP30
USP31, USP32, USP36, USP37, USP42,
USP44, USP45, USP47, USP48, USP51,
USPL1
Skeletal muscle
COPS5, EIF3F, OTUB2, OTUD1, USP2,
USP13, USP19, USP38, USP39, USP49,
USP54
Liver & biliary system
MPND, OTUB2, Cezanne, USP18, USP26,
USP29, USP30, USP31, USP35, USP40,
USP43, PARP11
Renal system
OTUD3, USP2, USP21, USP45
Pancreas
UCHL3, USP3, USP53
Digestive system
BRCC3, MPND, AMSHLP, YOD1, USP12
FIGURE 5. Differential DUB expression in human tissues. DUB transcript expression data were collated from
the EMBL gene atlas (112) and are shown for selected tissues. Inclusion criteria are that each DUB was
significantly overexpressed, relative to their mean expression in other tissues, in at least two studies, and
greater than 67% of all studies. Each DUB is shown for up to three tissues in which its expression was most
prevalent.
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
FIGURE 4. A: estimated cellular copy number for individual DUBs in Swiss 3T3 cells determined by quantitative mass spectrometry. Data
collated from Schwanhausser et al. (231) which provides estimates for copy number of 5,000 distinct proteins. Note that some further DUBs
may be present, but just not detected by this mass spectrometry experiment. B: subcellular localization of human DUBs. Only those DUBs which
have been localized to specific cytoplasmic organelles, the plasma membrane, or which exclusively localize to the nucleus are shown for
simplification. Source data are derived from Urbé et al. (261) supplemented by additional data for USP2A (159), USP3 (77), USP22 (244),
USP32 (4), USP33 (150, 254). pm, Plasma membrane; mito, mitochondria; mvb, multivesicular body; ee, early endosome; er, endoplasmic
reticulum; mt, microtubules. *Nucleolus.
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CLAGUE ET AL.
Table 2. Selectivity of DUBs between polyubiquitin chain types
USPs
CYLD
Ataxin 3
A20
OTUB1
OTUB2
OTUD3
OTUD5
Cezanne
Trabid
AMSH AMSH-LP Brcc36
Lys63
Lys48
Lys33
Lys29
Lys27
Lys11
Lys6
Linear
Reference Nos.
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫺
⫺
⫹⫹⫹
ND
⫹⫹⫹
⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫺
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹
⫺
ND
⫺
⫺
⫹⫹
ND
ND
ND
ND
ND
ND
ND
ND
⫹
⫺
⫹⫹
⫺
ND
⫺
⫺
ND
ND
ND
⫺
⫹⫹⫹
⫺
⫹
ND
ND
ND
ND
ND
ND
ND
ND
⫺
⫺
⫹⫹⫹
⫹
⫹
⫺
⫺
ND
ND
ND
⫹⫹⫹
⫺
⫺
⫹⫹⫹
⫺
ND
⫺
⫺
ND
⫹⫹⫹
ND
⫺
⫺
⫺
Variable
⫹⫹⫹
ND
⫺
ND
ND
ND
ND
ND
⫺
⫺
27, 67
27, 123, 268
27, 184, 279
27, 123, 268
60, 268, 270
60
95
114
27, 268
154, 268
27, 45, 123, 171, 225
Collation of data from published studies that inform on the discrimination of full-length or DUB catalytic domains
between polyubiquitin chain types. Most USPs are reported as relatively nonselective, with the exception of
CYLD and show a variable ability to hydrolyze linear chains. A (⫺) indicates where no activity was determined,
and number of (⫹) signs gives an indication of preference for that chain over others for a specific DUB and is
not an absolute indicator of activity. ND, not determined.
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
FIGURE 6. Association of DUBs with disease. Cancer expression data were accessed for each DUB from Oncomine (211), criteria used for
differential expression were P ⬍0.0001, 2-fold change and within top 10% of genes, for at least three studies. Cancer mutation data were taken
from COSMIC (74), with criteria that the DUB was mutated in at least 2% of all tumors; examples of tumor types are given, where two or more
samples were tested and greater than 10% showed mutations. Key to cancers: Ba, brain; Bl, bladder; Br, breast; Ce, cervical; Co, colorectal;
Es, esophagus; He, hemopoietic; Ki, Kidney; Le, leukemia; Li, liver; Lu, lung (NSCLC); LuS, lung (SCLC); Ly, lymphoma; LI, large intestine; Me,
melanoma; Ms, mesothelioma; Ov, ovary; UM, uveal melanoma; Pa, pancreatic; Pr, prostate; Sa, sarcoma; Se, seminoma; Sk, skin; St,
stomach; UA, upper aerodigestive tract; UT, urinary tract. Further information and full references for other expression, mutation, and disease
association data are given in Supplementary TABLE 2.
1300
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DUBs FROM GENES TO ORGANISM
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
Overexpressed
in cancer
BRCC3
COPS5
COPS6
EIF3H
MPND
MYSM1
PRPF8
POH1
AMSH
AMSH-LP
ATXN3
ATXN3L
JOSD1
OTUB1
OTUB2
OTUD1
OTUD3
OTUD4
OTUD6B
Cezanne2
Cezanne
A20
VCPIP1
BAP1
UCHL1
UCHL3
CYLD
DUB3
USP1
USP2
USP3
USP4
USP5
USP6
USP7
USP8
USP9X
USP9Y
USP10
USP11
USP13
USP14
USP15
USP16
USP17
USP19
USP21
USP22
USP24
USP25
USP26
USP28
USP29
USP31
USP32
USP33
USP34
USP36
USP37
USP38
USP40
USP42
USP44
USP45
USP46
USP47
USP48
USP49
USP51
USP53
USP54
USPL1
PARP11
Downregulated
in cancer
Ly, Se
Lu, Me
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 pro-
Mutated in cancer
Other disease association
Le
Moyamoya (cerebrovascular angiopathy )
Br
Br, Pr
Co
Co
LI, Sk
Ly, LI, Sk, UA
Retinitis pigmentosa
many
St
Se
Co, Ki
MIC-CAP syndrome
Machado Joseph disease
LI, Sk
Lu
Bl
Br, Co
Ly, Co
Ly, Co, Lu
Se
LI
LI, UA
Co
Ba
Co
Ly
many
Lu, Co, Pa
Co
LI, Sk
Li, UA
Ly, UA
LI, Sk, UT, UA
UM, Ki, Ms
Ba, Co, Ki
Se
many
Le, se
Co, Ki, Br
Ba, Sa, St
Pr, Ov
Ba
Lu, LuS, Bl, Pr
LuS
Ki
Pr
Lu
Pa
Me, Co
Me, Le, Sa
Le
Br, ki, Ov
Inflammatory conditions
Cancer predisposition syndrome
Neurodegenerative diseases
Sk
LI, Sk, UA
Familial cylindromatosis
LI, Sk, UT
LI
LI, Sk, UA, UT
LI, Sk, UT
LI, Sk, UA
LI, Sk, UA
LI, Sk, UA
He, Ly, LI, Sk, UA, Pa
LI, Sk, UA
Heart failure
Aneurysmal bone cyst
Male infertility, prostate cancer
Premature ovarian failure
Male infertility
Ba
LI
LI, Sk, UA
Ov
Br
Ba, Br, Ov
LI
Chronic myeloid leukemia
LI, Sk, UA
LI
copy number gain in cancer
Lu, Co, Es, Ce
Me, Co, Br
Ly, Br
Br
Br
Ly
Ly
Br
Se
Ki
Co
Ly
Ly
Se
Br
Le
Br, Es, Ki
Br, Se
Me
Br, Co, Lu, Ly
Co
amplification
overexpression
deletion
downregulation
Ly, Se
translocation
many
LI, Li, Sk
LI, Sk, UA, UT
LI, Sk, UA
LI, Sk, UA
LI, Sk
LI, Sk
LI
LI, Ov, Pr, Sk
LI, Sk, UA
Sk
Li
LI
LI, Sk
LI, Sk, UA
LI, UA
Parkinson's (late onset)
Crohn's disease
Male infertility
Parkinson's (late onset)
Depression
LI, Sk, UA
LI, Sk, UA, UT
LI, Sk
LI, Sk, UA
LI, Sk
LI
LI, Sk
LI
expansion
Cantu syndrome
SNP
association
somatic
mutation
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germline
mutation
1301
CLAGUE ET AL.
Table 3. Phenotypes associated with genetically modified DUB mouse models
DUB Family
DUB
Genotype
JAMM/MPN⫹
COPS5
JAMM/MPN⫹
COP6S
Reference
Nos.
Phenotype
Molecular Mechanism
Csn5Sfl/fl Lck-Cre
Targeted to thymocytes. Decreased
growth and development
Defective S-phase progression
and massive apoptosis,
dysregulated turnover of
P53, i␬B␣,ß-catenin
Cop6S⫺/⫺
Embryonic lethal (E7.5)
Cop6S⫹/⫺
Decreased growth, increased
sensitivity to gamma-radiation
and decreased radiation induced
tumors
Regulating DNA damageassociated apoptosis and
tumorigenesis through
MDM2-p53 pathway
294
Oxidative stress and genomic
instability
188
194
294
JAMM/MPN⫹
MYSM1
Mysm1tmla/tmla
Defects in bone marrow
hematopoiesis, with
lymphopenia, anemia,
thrombocytosis
JAMM/MPN⫹
PRPF8
Prpf8tm1.1Eap/⫹
Prpf8tm1.1Eap/⫹ tm1.1Eap
Knock-in of disease-associated
mutation. Retinal degeneration in
heterozygotes, more severe in
homozygotes
81
JAMM/MPN⫹
PSMD7
Mov34⫺/⫺
Embryonic lethal (develop to the
blastocyst stage and die shortly
after implantation)
243
JAMM/MPN⫹
STAMBP
Amsh⫺/⫺
Postnatal growth retardation and
lethality at P19-P23, Brain
atrophy and loss of
hippocampal/cerbral cortex
neurons
105
Amsh⫺/⫺
Josephin
ATXN3
Atxn3⫺/⫺
Atxn3
⫺/⫺
Failure to degrade proteins
including glutamate
receptors, leading to
accumulation of
ubiquitylated protein
aggregates
248
No overt phenotype, but heightened
anxiety
Increased levels of
ubiquitinated proteins
229
Defective cellular stress response
Decreased basal and stressinduced hsp70
transcription
205
OTU
OTUD5
Otud5Gt(A021B07)Wrst/Y
OTU
TNFAIP3
Tnfaip3⫺/⫺
Multi-organ inflammation
Increased NF␬B signaling
236
Tnfaip3fl/fl CD19-Cre
Targeted to B cells. Autoantibodies
and autoimmune disease similar
to SLE
Increased NF␬B signaling
236
Tnfaip3fl/fl CD11c-Cre
Targeted to dendritic cells.
Splenomegaly, lymphadenopathy
and autoimmune disease, e.g.,
colitis
Increased NF␬B signaling
236
UCH
BAP1
Abnormal embryo turning and
developmental patterning
Bap1⫺/⫺
Embryonic lethal (E9.5)
Bap1fl/fl ERT2⫹-Cre
Ubiquitous deletion in adult tissues.
Splenomegaly, myeloid
transformation.
63
57
Destabilization of epigenetic
regulators (HCF1 & OGT),
interaction ASXL1/ASXL2
57
UCH
UCHL1
Gad⫺/⫺ (lack Uchl1)
Defective fertilization and
preimplantation embryo
development
Gad⫺/⫺ (lack Uchl1)
Defective spermatozoa
Decreased apoptosis
134
UCH
UCHL3
Uchl3⫺/⫺
Testicular atrophy and germ cell
loss
Increased apoptosis
135
Uchl3⫺/⫺
Defective adipogenesis and
resistance to HFD-induced
obesity
Decreased IGF signaling,
increased muscle AMPK
activation
233, 247
Uch37⫺/⫺
Prenatal lethality, severely abnormal
brain development
UCH
USP
UCHL5
CYLD
CYLD
fl/fl
ALFP-Cre
179
8
Hepatic dysfunction, apoptosis and
subsequent cancer
Activation of TAK1 (TGFbeta
pathway) and JNK
signalling
189
Cyld⫺/⫺
Lung fibrosis in response to injury
Increased TGFbeta signalling
and smad3 stability
through loss of Akt
deubiquitylation
155
Elevated perinatal lethality, male
infertility. Fanconi anemia
Impaired Fancd2 foci during
DNA damage repair by
homologous recombination
116
Osteopeonia
Loss of ID proteins
277
USP
USP1
Usp1⫺/⫺
USP
USP2
Usp2⫺/⫺
Defective fertilization and sperm
motility
USP
USP4
Usp4⫺/⫺
Viable and developmentally normal.
Enhanced apoptosis in thymus
and spleen in response to
ionizing radiation
USP
USP5
Usp5⫺/⫺
Embryonic lethal (E7)
17
Hyperactive DNA damage
checkpoints and
upregulated levels and
activity of p53
292
63
Continued
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DUBs FROM GENES TO ORGANISM
Table 3.—Continued
DUB Family
USP
USP
DUB
USP7
USP8
Genotype
Phenotype
Molecular Mechanism
Reference
Nos.
Usp7⫺/⫺
Embryonic lethal (E6.5-E7.5)
Dysregulated Mdm2/p53
pathway
124
Hauspfl/fl nes-Cre
Brain targeted knockout. Neonatal
lethality, deficiencies in brain
development
Increased p53 levels,
transcriptional activity and
p53-mediated apoptosis
125
Ubpy⫺/⫺
Grossly disorganized embryogenesis
and complete embryonic lethality
(E9.5)
Ubpyfl/fl Mx1-Cre
Conditional knockout in adults. Liver
failure
Reduced levels of RTKs and
abnormal trafficking
185
No protein aggregates,
instead depletion of
synaptic ubiquitin pool,
increased GABA(A)
receptor turnover
14, 18, 32,
33, 49,
139,
278
USP
USP14
Usp14ax⫺J/ax⫺J
Mutation in intron leads to reduced
USP14 levels. Tremors,
abnormal brain morphology,
altered synaptic transmission
and increased apoptosis
USP
USP9x
Usp9xGt(XK141)Byg
Abnormal embrogenesis and
embryo size, open neural tube,
failure to form heart
USP
USP16
Usp16⫺/⫺
Compete embryonic lethality (E6)
USP
USP18
Usp18⫺/⫺
Tremors, seizures, abnormal
nervous system, death
Usp18⫹/⫺
Increased susceptibility to bacterial
infection
Usp18⫺/⫺
185
63
63
ISG15
214
Chemically induced mutation.
Increased inflammatory response
to Salmonella
Reduced IFN-gamma,
increased IL-6
212
USP
USP22
Usp22⫺/⫺
Embryonic lethal (E9.5-E10.5)
Sirt1 instability, increased
p53 transcriptional activity,
apoptosis
157
USP
USP25
USP25⫺/⫺
Enhanced inflammatory response to
IL-17
Loss of USP25 leads to
deubiquitylation of TRAF5 &
TRAF6, increased Il17dependent TRAF5
interaction with splicing
factor SF2, and
stabilization of
proinflammatory
transcripts CXCL1, IL-6
and TNF
295
USP
USP44
USP44⫺/⫺
Spontaneous tumor formation,
particularly in lung
Regulates mitotic checkpoint,
interacts with centrin to
control centriole positioning
293
USP
USP46
Usp46CS/CS
(mutation)
Low immobility in tail suspension
and forced swim tests
GABAergic transmission
256
USP
USP47
Usp47⫺/⫺
Usp46⫺/⫺
103
Increased sensitivity to ultraviolet
radiation
teins. 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 Alz-
63
heimer’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
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CLAGUE ET AL.
(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 ␣-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).
tin 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 NF␬B pathway (23,
239). In vitro data indicate DUB specificity for Lys48over Lys63-linked chains, but cellular models suggest a
critical role in cleaving activating Lys63 chains en bloc
from mediators of NF␬B signaling, such as receptor interacting protein 1 (RIP1) (123, 156, 271).
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). Germline 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 Olinked 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-␥ coactivator (PGC)-1␣,
are increased in euglycemic conditions. It is proposed
that N-acetylglucosylation of PGC-1␣ by HCF-1/OGT
promotes its stability, through the recruitment of BAP1
deubiquitylating activity (220).
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 NF␬B 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).
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 NF␬B pathway (88, 161,
236). The A20 gene can be induced by NF␬B 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 ubiqui-
1304
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
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DUBs FROM GENES TO ORGANISM
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.
However, these structures fail to clearly explain the Lys63
specificity of other JAMMs.
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).
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).
E. JAMM Family
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).
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[6]-S-x[2]-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 BRCA1RAP180 (45, 235), whilst MYSM1 expresses its histone
H2A deubiquitylating activity from within a complex containing the histone acetyltransferase (HAT) p300/CBPassociated 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).
VII. DUBs AS INTEGRAL COMPONENTS
OF PROTEIN MACHINERIES
ASSOCIATED WITH DEGRADATION
A. Proteasomal DUBs
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
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CLAGUE ET AL.
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.
1306
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)-␤ 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).
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DUBs FROM GENES TO ORGANISM
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-␤, 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-␤
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), ␤-catenin stability (99), and the NF␬B pathway (232). It is recruited to TGF-␤ 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-␤ 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-␤
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-␤ 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-␤ 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-␤ signaling (other hits included
USP11, USP15, USP19 but were not substantively followed
up). Depletion of USP4 inhibits TGF-␤ 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-␤
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
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CLAGUE ET AL.
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).
IX. CONCLUSIONS
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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.
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ACKNOWLEDGMENTS
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: clague@liv.ac.uk).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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