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Regulation of deubiquitinase proteolytic activityOscar W Huang and Andrea G Cochran
Available online at www.sciencedirect.com
Deubiquitinases (DUBs) are proteolytic enzymes whose
function is to oppose the process of the conjugation of ubiquitin
to a specific substrate. This task is accomplished through an
enzymatic cascade involving E1, E2, and E3 enzymes, which
collectively produce a product that is either monoubiquitinated,
or polyubiquitinated with multiple single ubiquitins or with
ubiquitin chains. The resulting modifications may impact
protein function or may lead to the degradation of the
ubiquitinated species, so the removal of such modifications
must be tightly regulated. On the basis of recent work featuring
crystal structures and detailed biochemical or biophysical
studies of DUBs, we will discuss here how posttranslational
modifications, protein binding partners, and reactive oxygen
species regulate their catalytic activity.
Addresses
Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San
Francisco, CA 94080, United States
Corresponding author: Huang, Oscar W (huang.oscar@gene.com)
Current Opinion in Structural Biology 2013, 23:xx–yy
This review comes from a themed issue on Catalysis and regulation
Edited by Alexander Wlodawer and Ben Dunn
0959-440X/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.sbi.2013.07.012
IntroductionThe conjugation of ubiquitin to a specific substrate is a
well-orchestrated event that starts with an ubiquitin-
activating enzyme, E1, transferring ubiquitin to an ubi-
quitin-conjugating enzyme, E2, which ultimately leads to
substrate ubiquitination through the action of an ubiqui-
tin ligase, E3 [1]. This posttranslational modification
creates a covalent isopeptide bond between the carboxyl
group of the ubiquitin C terminus and a lysine e-amino
group of a substrate protein or one of the seven lysines
(K6, K11, K27, K29, K33, K48, and K63) or the N
terminus of another ubiquitin. Therefore, substrates
can either be monoubiquitinated on one or multiple
lysines or be polyubiquitinated with one type or a variety
of chains. Given the large complexity of possible modi-
fications, the appropriate removal of ubiquitin presents a
significant problem to the cell. Enzymes called deubi-
quitinases (DUBs) carry out this function, and the activity
of these DUBs must be tightly regulated in order to
recognize both the correct substrate and the correct
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context in which to deubiquitinate target proteins. In
this review, we will limit our discussion to how posttran-
slational modifications (PTMs), protein binding partners,
and reactive oxygen species (ROS) regulate the proteo-
lytic activity of these enzymes. In addition, we will high-
light recent papers that include high-resolution structural
analyses (Table 1) or detailed mechanistic experiments
that help to explain the resulting effects at a molecular
level.
Structure and catalytic activity ofdeubiquitinasesDeubiquitinases (DUBs) are proteolytic enzymes whose
function is to cleave ubiquitin or ubiquitin-like proteins
from proproteins or ubiquitin(s) conjugated with target
substrate [2]. In the human genome, there are approxi-
mately 100 DUBs that can be divided into five families
based on their protease domains: the ubiquitin C-terminal
hydrolases (UCH), the ubiquitin-specific proteases (USP),
the ovarian tumor proteases (OTU), the Machado-Joseph
disease proteases (MJD), and the JAB1/MPN/Mov34 pro-
teases (JAMM) [3]. Together, these five families can be
further grouped into two classes based on their catalytic
mechanism. The UCH, USP, OTU, and MJD families are
cysteine proteases, whose enzymatic activity depends on
the thiol group of the cysteine in the active site. During
catalysis, the neighboring histidine residue, which is most
often polarized by asparagine or aspartate, accepts a proton
from the cysteine, allowing the resulting thiolate to make a
nucleophilic attack on the carbonyl of the scissile peptide
or isopeptide bond [3]. On the other hand, JAMM family
members belong to the class of zinc metalloproteases and
activate water for attack on the isopeptide bond [4]. In
order to monitor these DUB activities, the fluorogenic
substrate ubiquitin-7-amino-4-methylcoumarin (Ub-
AMC) and poly-ubiquitin chain substrates are commonly
used. Active site-targeting probes such as ubiquitin-alde-
hyde (Ub-al) and ubiquitin vinyl methyl ester (Ub-VME)
allow interrogation of the catalytically competent state [5].
Regulation by posttranslational modificationIt is hard to find a cellular protein that is not posttransla-
tionally modified, and this applies to DUBs as well.
Typical modifications reported on DUBs are phosphoryl-
ation, acetylation, ubiquitination, and sumoylation.
These PTMs have a wide range of consequences that
have been reviewed extensively [2,6�]. Interestingly, a
few examples suggest a direct effect, either positive or
negative, on DUB catalytic activity. One example is the
phosphorylation of CYLD on serine 418, which decreases
the rate of processing of poly-ubiquitinated forms of
TRAF2 and NEMO [7,8]. On the other hand, the
olytic activity, Curr Opin Struct Biol (2013), http://dx.doi.org/10.1016/j.sbi.2013.07.012
Current Opinion in Structural Biology 2013, 23:1–6
2 Catalysis and regulation
COSTBI-1154; NO. OF PAGES 6
Table 1
Structures discussed in the text
DUB Binding partner(s) Description PDB code Ref
OTUD5 Catalytic core of OTUD5/DUBA 3PFY n/a
OTUD5 Ub-al pSer177 DUBA Ub complex 3TMP [15��]
USP7 Ub-al USP7 catalytic core Ub complex 1NBF [26]
USP7 HUBL domain (non-catalytic) 2YLM [25��]
Ubp8 Sgf11, Sus1, Sgf73 SAGA DUB module 3M99 [32��]
Ubp8 Sgf11, Sus1, Sgf73 SAGA DUB module 3MHH [33��]
Ubp8 Sgf11, Sus1, Sgf73, Ub-al SAGA DUB module Ub complex 3MHS [33��]
A20 Active site Cys-SH 3ZJD [42�]
A20 Active site Cys-SOH 3ZJE [42�]
A20 Active site Cys-SO2H 3ZJG [42�]
A20 Active site Cys-SO3H 3ZJF [42�]
poly-ubiquitination of Ataxin-3 on lysine 117 is reported
to increase its ability to remove poly-ubiquitinated chains
[9,10]. Although the mechanisms by which these PTMs
regulate DUB catalytic activity remain largely unknown,
several recent detailed biochemical and structural studies
have revealed some aspects of these mechanisms.
For ubiquitin-specific protease 1 (USP1), the association
with UAF1 stimulates USP1 catalytic activity by 18–35-
fold toward Ub-AMC, largely through an increase in
catalytic turnover (kcat) [11,12]. The similar values of
substrate KM (within a factor of 2) for USP1 and the
USP1-UAF complex, as well as the closely similar Ub-al
Ki values for the two enzyme forms, show that the
formation of USP1-UAF1 complex does not affect the
binding to ubiquitin [12]. Surprisingly, formation of the
UAF1 complex is dependent on phosphorylation of USP1
at Ser313, as replacing Ser313 with alanine disrupts
complex formation [13�]. In contrast, replacement with
aspartic acid, a phosphomimetic residue, promotes for-
mation of a complex with similar kinetic properties as the
wild type USP1-UAF1 complex. Despite the lack of
structural data, the detailed biochemical data make clear
that phosphorylation on Ser313 of USP1 stimulates its
activity not through a direct influence on USP1 enzymatic
activity, but instead by the phosphorylation-dependent
recruitment of an activator [13�].
OTUD5, also known as DUBA, belongs to the OTU class
of DUBs and was discovered to be a negative regulator of
type I interferon production [14]. A detailed biochemical
study revealed that DUBA catalytic activity is strongly
and directly regulated by phosphorylation [15��]. Specifi-
cally, a single phosphorylation at Ser177 is both necessary
and sufficient to transform the completely inactive
enzyme to an active form capable of hydrolyzing Ub-
AMC, ubiquitin chain cleavage, and conjugation with Ub-
al [15��]. An intriguing finding is that substitution of
Ser177 with a phosphomimetic residue (Asp or Glu)
cannot activate the enzyme. NMR analysis and crystal
structures of apo-DUBA in both phosphorylated and
unphosphorylated forms and of phospho-DUBA in
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Current Opinion in Structural Biology 2013, 23:1–6
complex with ubiquitin-aldehyde reveal the role of the
phosphate in enzyme activation. In the Ub complex
(Figure 1a), the phosphate group interacts with and
structures the DUBA N terminus and an internal helix.
In addition, the phosphate interacts extensively with the
C-terminal tail of ubiquitin to promote productive for-
mation of the enzyme–substrate complex. Thus, DUBA
is a phospho-activated enzyme with a direct role for the
posttranslational modification in the catalytic mechanism.
Allosteric regulation by partner proteinsAn extensive profiling of DUB interactions has revealed
that most DUBs associate with other proteins [16].
Whether these are substrates or simply binding partners
of unknown function remains to be explored. However,
there have been many reports of allosteric regulation of
DUB activity by partner proteins. USP14, UCH37, and
POH1 are known to be largely inactive until they associ-
ate with the proteasome [2]. For example, the binding of
the WD40 protein ADRM1 to the auto-inhibitory C-
terminal extension of UCH37 stimulates activity toward
Ub-AMC, mainly through a 6-fold decrease in KM. How-
ever, when the substrate is diubiquitin, the stimulatory
effect of ADRM1 on UCH37 is dependent on recruit-
ment of the complex to the proteasome [17,18]. Another
WD40 family activator already described above is UAF1.
In addition to activating USP1 [11], largely through an
increase in kcat, UAF1 activates other DUBs in distinct
complexes, such as UAF1-USP12-WDR20 and UAF1-
USP46 [19,20]. This seems to be an evolutionarily con-
served mechanism of DUB activation because, in bud-
ding yeast, the WD40 protein DUF1 has stimulatory
activity toward UBP9 and UBP13 [21].
Another example of activation by a binding partner is
seen for the Polycomb repressive deubiquitinase (PR-
DUB). In Drosophila PR-DUB is composed of polycomb
group proteins ASX and the UCH-class DUB Calypso,
while the human complex includes ASXL1 and BAP1
[22]. In vitro, the removal of ubiquitin from mono-
ubiquitinated (mUb) histone H2A in nucleosomes, or
the release of AMC from Ub-AMC, is substantially
olytic activity, Curr Opin Struct Biol (2013), http://dx.doi.org/10.1016/j.sbi.2013.07.012
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Regulation of deubiquitinase proteolytic activity Huang and Cochran 3
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Figure 1
D100
G101N102
C103
3.1 Å
Ub
(a)
α6
α1
(b)
(c)
Current Opinion in Structural Biology
Structures showcasing how posttranslational modification, protein
binding partners, and reactive oxygen species regulate the catalytic
activity of DUBs. (a) Catalytic core of active, phosphorylated DUBA
(cyan) in complex with ubiquitin-aldehyde shown in orange (3TMP)
[15��]. The phosphate is shown as a black sphere, and two regions that
fold upon ubiquitin binding are shown in green (a1) and purple (a6). (b)
Yeast SAGA DUB module in complex with ubiquitin-aldehyde shown in
orange (3MHS) [33��]. The catalytic subunit Ubp8 is shown in cyan, with
Sgf11 in white, Sus1 in green, and Sgf73 in yellow. The 8 bound Zn
atoms are shown as spheres. (c) The catalytic cysteine 103 of A20 in the
reversibly oxidized sulfenic acid (SOH) state (3ZJE) [42�]. The oxygen
atom of the SOH group is surrounded by backbone amides from the
Cys-loop (residues 100–102). The NH group of Gly101 is within hydrogen
bonding distance (3.1 A); distances to the other amides are 3.6 and 4.1 A
for Asn102 and Asp100, respectively. The distance between the sulfur
and nitrogen of Asn102 is 4.9 A (orange dotted line) and is inconsistent
with the sulphenylamide formation indicated for USP-class DUBs [41].
higher in the presence of PR-DUB complex than for
Calypso/BAP1 alone [22]. However, as for the WD40
activators, it is not currently understood in structural
detail how ASX/ASXL1 binding activates the enzyme.
Recent structural papers mentioned below have shown
in greater detail how partner proteins can regulate DUB
activity.
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The association of guanosine 50 monophosphate synthe-
tase (GMPS) and USP7 was first documented in a puri-
fication from Drosophila embryo nuclear extract. The two
proteins were observed to form a tightly associated com-
plex, and GMPS was required for USP7-mediated deu-
biquitination of mUb histone H2B in vitro [23]. This
association and enhancement of USP7 catalytic activity
by GMPS was observed also for the human proteins [24].
A new structural and biophysical study clarified how
GMPS activates USP7 [25��]. USP7 contains a TRAF
substrate-binding domain, a catalytic domain, and five
ubiquitin-like (UBL) domains, collectively named the
HUBL domain. The HUBL domain was found to be
essential for USP7 activity against poly-ubiquitinated p53
in both biochemical and cellular assays. The crystal
structure of the HUBL domain reveals intimate packing
of the first two and the last two UBL units, with the third
UBL acting as a flexible tether. Comparable catalytic
activity of full-length USP7 versus a catalytic domain
linked directly to HUBL-45 (the fourth and fifth UBLs
only) indicates that HUBL-45 is the region required for
full USP7 activation. Small-angle X-ray scattering (SAXS)
data further suggest that the HUBL-45 region interacts
with the catalytic domain. Association with GMPS can
enhance USP7 activity through a 5.5-fold increase in kcat.
GMPS binds to HUBL-123 and may restrict the flexi-
bility of USP7, thereby promoting the activating inter-
action between HUBL-45 and the catalytic domain.
Although a structure showing this interaction is not yet
available, it appears that the interaction leads to pro-
ductive reorientation of certain active-site residues
(switching loop) [25��,26].
The SAGA complex is a multisubunit (and multifunc-
tional) histone acetyltransferase whose composition is
conserved from yeast to humans [27]. One of the 21
subunits of SAGA is the DUB Ubp8 (USP22 in human)
that deubiquitinates mUb histone H2B [28–30]. Ubp8
association with SAGA is dependent on Sgf11. A SAGA
DUB subcomplex (DUB module) composed of Ubp8,
Sgf11, Sus1, and Sgf73 exhibits Ub-AMC hydrolytic
activity, whereas Ubp8 alone does not [31]. Two groups
have now reported crystal structures of the yeast SAGA
DUB module [32��,33��]. These structures reveal how the
four proteins assemble into an intertwined complex
(Figure 1b) and suggest how complex formation activates
Ubp8. The DUB module consists of two functional lobes,
the assembly lobe and the catalytic lobe, that are struc-
turally coupled by Sgf73. In the assembly lobe, Sus1
clamps the Sgf11 N-terminal helix onto the non-catalytic
ZnF-UBP domain of Ubp8. In the catalytic lobe, the
Sgf11 C-terminal ZnF makes direct contact with a loop in
Ubp8 that contains the catalytic cysteine. In both apo and
ubiquitin aldehyde complex structures, the catalytic resi-
dues adopt a competent orientation [33��]. Thus it seems
likely that the activation mechanism involves stabiliz-
ation of this conformation by the partner proteins.
olytic activity, Curr Opin Struct Biol (2013), http://dx.doi.org/10.1016/j.sbi.2013.07.012
Current Opinion in Structural Biology 2013, 23:1–6
4 Catalysis and regulation
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Redox regulation by reactive oxygen speciesA newly reported mechanism of DUB catalytic regulation
is modification by reactive oxygen species [34]. ROS are
endogenous small molecules, such as hydrogen peroxide
(H2O2), that consist of radical and non-radical oxygen
species. ROS are generated either by normal mitochon-
drial oxidative metabolism or acutely by cellular response
to xenobiotics, cytokines, and bacterial invasion [35]. A
scavenging system exists to prevent cellular damage by
ROS, but when the cellular antioxidant defense system is
overwhelmed, ROS triggers oxidative stress, affecting
many cellular pathways, such as those involved in pro-
liferation and survival, ROS homeostasis and antioxidant
gene regulation, mitochondrial oxidative stress, apoptosis,
and the DNA damage response [35]. A mechanism by
which ROS affects a target protein is through oxidation of
reactive cysteine (Cys) residues. Oxidation of the sulfur
atom of Cys leads to the formation of reactive Cys sulfenic
acid (–SOH) that has the capability to form disulfide
bonds (–S–S–) with nearby thiols. Alternatively, Cys
sulfenic acid may undergo further oxidation to sulfinic
(–SO2H) or sulfonic (–SO3H) acids. The lower oxidized
states (–SOH and –S–S–) can be reversed to Cys by
reducing systems such as thioredoxin and peroxiredoxin,
but, in contrast, oxidation to sulfinic or sulfonic acids is
irreversible [36].
In mammalian cells, the E3 ligase Rad18 monoubiquiti-
nates PCNA, a DNA damage marker, in response to
blockage at a replication fork caused by DNA lesions
[37]. USP1, an important negative regulator of the DNA
damage tolerance pathway, deubiquitinates mono-ubi-
quitinated PCNA, (mUb-PCNA) [38]. The treatment
of human fibroblasts with oxidizing agents results in a
rapid and reversible increase in mUb-PCNA [39,40�,41].
Surprisingly, USP1 protein levels remained unchanged
during the rapid increase of mUb-PCNA after hydrogen
peroxide treatment; this differs from the USP1 degra-
dation normally observed in response to UV-mediated
DNA damage [38,40�]. Evidence that the DUB is the
direct target of oxidative stress is the observed labeling of
USP1 with a 1,3-cyclohexadione derivative, a sulfenic
acid-selective labeling reagent. Furthermore, the ROS-
induced labeling of USP1 is dependent on USP1 catalytic
activity and only indirectly (through the increase in
activity) on USP1 association with the activator UAF1
[40�]. USP1 isolated from cells treated with H2O2 is less
efficiently labeled with Ub-VME than protein from
untreated cells, confirming that the active-site cysteine
is the site of oxidation [40�]. In addition, DTT treatment
restores labeling by Ub-VME, consistent with reversible
sulfenic acid formation and little or no higher oxidation in
the treated cells. A possible explanation for the resistance
of DUBs to further oxidation is revealed by a new crystal
structure of the catalytic domain of the OTU-class DUB
A20 oxidized to the Cys sulfenic acid state [42�]. The
sulfenic acid OH forms a hydrogen bond with the
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backbone NH of Gly101 in the loop preceding the
catalytic Cys103 (Cys-loop; Figure 1c), which likely
stabilizes this reversible oxidation state and protects
the Cys from further, irreversible oxidization (sulfinic
and sulfonic acid states, also characterized structurally)
[42�]. A second mechanism protecting USP active-site
cysteines may be reversible formation of a sulphenyla-
mide [41]. Redox regulation seems to be a mechanism
broadly applicable to DUBs, including the majority of
OTU DUBs and many of the USP and UCH family
members [41,42�].
Conclusions and future directionOur current understanding of how the proteolytic activity
of DUBs can be regulated is aided by recent emergence
of structural and biochemical data. Whether the DUBs
are posttranslationally modified, bound to partner
protein(s), or modulated by reactive oxygen species,
the ultimate control of DUB catalytic activity is through
the control of the reactivity and conformation of its active
site residues. Ubiquitination is involved in many import-
ant biological processes, as well as in human diseases
such as cancer, inflammation, and infection. Therefore
the interest in DUBs will only increase, and the knowl-
edge of how cells effectively control them will continue
to advance.
References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:
� of special interest�� of outstanding interest
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32.��
Kohler A, Zimmerman E, Schneider M, Hurt E, Zheng N: Structuralbasis for assembly and activation of the heterotetramericSAGA histone H2B deubiquitinase module. Cell 2010,141:606-617.
Together with [33��], this study describes the structure of a distinct four-protein subcomplex (SAGA DUB module) within the larger 21-subunityeast SAGA complex. In addition, this study includes extensive muta-genesis evaluation (both biochemical and functional) of the interactionsseen in the crystal structure.
33.��
Samara NL, Datta AB, Berndsen CE, Zhang X, Yao T, Cohen RE,Wolberger C: Structural insights into the assembly andfunction of the SAGA deubiquitinating module. Science 2010,328:1025-1029.
Together with [32��], this study reports the structure of the yeast SAGADUB module, composed of the DUB Ubp8, Sgf11, Sus1, and Sgf73. Thecrystal structures of parent complex and of the ubiquitin aldehyde adductreveal how the four proteins assemble into an intertwined complex andsuggest that Ubp8 is activated through the stabilization of a catalyticallycompetent conformation by the partner proteins.
34. Clague MJ: Oxidation controls the DUB step. Nature 2013,497:49-50.
35. Ray PD, Huang BW, Tsuji Y: Reactive oxygen species (ROS)homeostasis and redox regulation in cellular signaling. CellSignal 2012, 24:981-990.
36. Roos G, Messens J: Protein sulfenic acid formation: fromcellular damage to redox regulation. Free Radic Biol Med 2011,51:314-326.
37. Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S:RAD6-dependent DNA repair is linked to modification of PCNAby ubiquitin and SUMO. Nature 2002, 419:135-141.
38. Huang TT, Nijman SM, Mirchandani KD, Galardy PJ, Cohn MA,Haas W, Gygi SP, Ploegh HL, Bernards R, D’Andrea AD:Regulation of monoubiquitinated PCNA by DUB autocleavage.Nat Cell Biol 2006, 8:339-347.
olytic activity, Curr Opin Struct Biol (2013), http://dx.doi.org/10.1016/j.sbi.2013.07.012
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6 Catalysis and regulation
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39. Zlatanou A, Despras E, Braz-Petta T, Boubakour-Azzouz I,Pouvelle C, Stewart GS, Nakajima S, Yasui A, Ishchenko AA,Kannouche PL: The hMsh2-hMsh6 complex acts in concert withmonoubiquitinated PCNA and Pol h in response to oxidativeDNA damage in human cells. Mol Cell 2011, 43:649-662.
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Cotto-Rios XM, Bekes M, Chapman J, Ueberheide B, Huang TT:Deubiquitinases as a signaling target of oxidative stress. CellRep 2012, 2:1475-1484.
This study provides the first evidence that ROS inactivation of DUBs(USP1 and USP7) is through reversible oxidation of the catalytic cysteineto sulfenic acid. In addition, these authors show that the sensitivity to ROSinactivation parallels DUB catalytic activity, indicating that an activatedthiol is required.
Please cite this article in press as: Huang OW, Cochran AG. Regulation of deubiquitinase prote
Current Opinion in Structural Biology 2013, 23:1–6
41. Lee JG, Baek K, Soetandyo N, Ye Y: Reversible inactivation ofdeubiquitinases by reactive oxygen species in vitro and incells. Nat Commun 2013, 4:1568.
42.�
Kulathu Y, Garcia FJ, Mevissen TE, Busch M, Arnaudo N,Carroll KS, Barford D, Komander D: Regulation of A20 and otherOTU deubiquitinases by reversible oxidation. Nat Commun2013, 4:1569.
This report provides the first crystal structures of a DUB (A20) in fourdifferent oxidized states (active-site thiol, plus sulfenic, sulfinic, andsulfonic acid states). Comparison of reduced and sulfenic acid statessuggests an explanation for the resistance of DUBs to oxidation beyondthe sulfenic acid state.
olytic activity, Curr Opin Struct Biol (2013), http://dx.doi.org/10.1016/j.sbi.2013.07.012
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