Regulation of deubiquitinase proteolytic activity

COSTBI-1154; NO. OF PAGES 6

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 ([email protected])

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

Please cite this article in press as: Huang OW, Cochran AG. Regulation of deubiquitinase prote

www.sciencedirect.com

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


[email protected]


http://www.sciencedirect.com/science/journal/0959440X

2 Catalysis and regulation


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



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




Regulation of deubiquitinase proteolytic activity Huang and Cochran 3


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.



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.






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



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

1. Dye BT, Schulman BA: Structural mechanisms underlyingposttranslational modification by ubiquitin-like proteins. AnnuRev Biophys Biomol Struct 2007, 36:131-150.

2. Reyes-Turcu FE, Ventii KH, Wilkinson KD: Regulation and cellularroles of ubiquitin-specific deubiquitinating enzymes. Annu RevBiochem 2009, 78:363-397.

3. Nijman SM, Luna-Vargas MP, Velds A, Brummelkamp TR,Dirac AM, Sixma TK, Bernards R: A genomic and functionalinventory of deubiquitinating enzymes. Cell 2005, 123:773-786.

4. Amerik AY, Hochstrasser M: Mechanism and function ofdeubiquitinating enzymes. Biochim Biophys Acta 2004,1695:189-207.

5. Borodovsky A, Ovaa H, Kolli N, Gan-Erdene T, Wilkinson KD,Ploegh HL, Kessler BM: Chemistry-based functionalproteomics reveals novel members of the deubiquitinatingenzyme family. Chem Biol 2002, 9:1149-1159.

6.�

Kessler BM, Edelmann MJ: PTMs in conversation: activity andfunction of deubiquitinating enzymes regulated via post-translational modifications. Cell Biochem Biophys 2011, 60:21-38.

This review focuses on posttranslationally modified DUBs whose activityand function have been characterized. These authors also include anextensive list of all PTMs of DUBs known to date.

7. Reiley W, Zhang M, Wu X, Granger E, Sun SC: Regulation of thedeubiquitinating enzyme CYLD by IkappaB kinase gamma-dependent phosphorylation. Mol Cell Biol 2005, 25:3886-3895.

8. Hutti JE, Shen RR, Abbott DW, Zhou AY, Sprott KM, Asara JM,Hahn WC, Cantley LC: Phosphorylation of the tumorsuppressor CYLD by the breast cancer oncogene IKKepsilonpromotes cell transformation. Mol Cell 2009, 34:461-472.



http://refhub.elsevier.com/S0959-440X(13)00145-0/sbref0005




























Regulation of deubiquitinase proteolytic activity Huang and Cochran 5


9. Todi SV, Winborn BJ, Scaglione KM, Blount JR, Travis SM,Paulson HL: Ubiquitination directly enhances activity of thedeubiquitinating enzyme ataxin-3. EMBO J 2009,28:372-382.

10. Todi SV, Scaglione KM, Blount JR, Basrur V, Conlon KP,Pastore A, Elenitoba-Johnson K, Paulson HL: Activity and cellularfunctions of the deubiquitinating enzyme and polyglutaminedisease protein ataxin-3 are regulated by ubiquitination atlysine 117. J Biol Chem 2010, 285:39303-39313.

11. Cohn MA, Kowal P, Yang K, Haas W, Huang TT, Gygi SP,D’Andrea AD: A UAF1-containing multisubunit protein complexregulates the Fanconi anemia pathway. Mol Cell 2007,28:786-797.

12. Villamil MA, Chen J, Liang Q, Zhuang Z: A noncanonical cysteineprotease USP1 is activated through active site modulation byUSP1-associated factor 1. Biochemistry 2012,51:2829-2839.

13.�

Villamil MA, Liang Q, Chen J, Choi YS, Hou S, Lee KH, Zhuang Z:Serine phosphorylation is critical for the activation ofubiquitin-specific protease 1 and its interaction with WD40-repeat protein UAF1. Biochemistry 2012, 51:9112-9123.

This study shows that phosphorylation at Ser313 of USP1 is required torecruit the activator UAF1. Ser313 is located in an inserted domain of theUSP1 catalytic core, suggesting a long-range allosteric activationmechanism.

14. Kayagaki N, Phung Q, Chan S, Chaudhari R, Quan C,O’Rourke KM, Eby M, Pietras E, Cheng G, Bazan JF et al.: DUBA: adeubiquitinase that regulates type I interferon production.Science 2007, 318:1628-1632.

15.��

Huang OW, Ma X, Yin J, Flinders J, Maurer T, Kayagaki N,Phung Q, Bosanac I, Arnott D, Dixit VM et al.: Phosphorylation-dependent activity of the deubiquitinase DUBA. Nat Struct MolBiol 2012, 19:171-175.

This work shows phosphorylation at DUBA Ser177 is required for enzymeactivity. A crystal structure of the active OTU domain ubiquitin aldehydeadduct shows the phosphate buried in the interior of the complex. Thephosphate interacts with two different regions of the enzyme and theubiquitin C-terminal tail to promote a productive enzyme-substrate inter-action. This is an unusual example of how phosphorylation can controlenzyme activity.

16. Sowa ME, Bennett EJ, Gygi SP, Harper JW: Defining the humandeubiquitinating enzyme interaction landscape. Cell 2009,138:389-403.

17. Yao T, Song L, Xu W, DeMartino GN, Florens L, Swanson SK,Washburn MP, Conaway RC, Conaway JW, Cohen RE:Proteasome recruitment and activation of the Uch37deubiquitinating enzyme by Adrm1. Nat Cell Biol 2006, 8:994-1002.

18. Yao T, Song L, Jin J, Cai Y, Takahashi H, Swanson SK,Washburn MP, Florens L, Conaway RC, Cohen RE et al.: Distinctmodes of regulation of the Uch37 deubiquitinating enzyme inthe proteasome and in the Ino80 chromatin-remodelingcomplex. Mol Cell 2008, 31:909-917.

19. Cohn MA, Kee Y, Haas W, Gygi SP, D’Andrea AD: UAF1 is asubunit of multiple deubiquitinating enzyme complexes. J BiolChem 2009, 284:5343-5351.

20. Kee Y, Yang K, Cohn MA, Haas W, Gygi SP, D’Andrea AD: WDR20regulates activity of the USP12 x UAF1 deubiquitinatingenzyme complex. J Biol Chem 2010, 285:11252-11257.

21. Kanga S, Bernard D, Mager-Heckel AM, Erpapazoglou Z,Mattiroli F, Sixma TK, Leon S, Urban-Grimal D, Tarassov I,Haguenauer-Tsapis R: A deubiquitylating complex required forneosynthesis of a yeast mitochondrial ATP synthase subunit.PLoS ONE 2012, 7:e38071.

22. Scheuermann JC, de Ayala Alonso AG, Oktaba K, Ly-Hartig N,McGinty RK, Fraterman S, Wilm M, Muir TW, Muller J: HistoneH2A deubiquitinase activity of the Polycomb repressivecomplex PR-DUB. Nature 2010, 465:243-247.

23. van der Knaap JA, Kumar BR, Moshkin YM, Langenberg K,Krijgsveld J, Heck AJ, Karch F, Verrijzer CP: GMP synthetasestimulates histone H2B deubiquitylation by the epigeneticsilencer USP7. Mol Cell 2005, 17:695-707.



24. Sarkari F, Sanchez-Alcaraz T, Wang S, Holowaty MN, Sheng Y,Frappier L: EBNA1-mediated recruitment of a histone H2Bdeubiquitylating complex to the Epstein–Barr viruslatent origin of DNA replication. PLoS Pathog 2009,5:e1000624.

25.��

Faesen AC, Dirac AM, Shanmugham A, Ovaa H, Perrakis A,Sixma TK: Mechanism of USP7/HAUSP activation by its C-terminal ubiquitin-like domain and allosteric regulation byGMP-synthetase. Mol Cell 2011, 44:147-159.

These authors report the first structure of USP7 HUBL domain, a five-UBLdomain C-terminal tail that is required for full activity of the enzyme. UBLs4 and 5 (HUBL-45) are identified as critical for activation, most likelythrough direct interaction with the catalytic domain. The authors extendthe study to explore the mechanism of a second activator GMPS,providing evidence that GMPS activates USP7 by binding to HUBL-123 to further promote HUBL-45 interaction with the catalytic domain.

26. Hu M, Li P, Li M, Li W, Yao T, Wu JW, Gu W, Cohen RE, Shi Y:Crystal structure of a UBP-family deubiquitinating enzyme inisolation and in complex with ubiquitin aldehyde. Cell 2002,111:1041-1154.

27. Spedale G, Timmers HT, Pijnappel WW: ATAC-king thecomplexity of SAGA during evolution. Genes Dev 2012, 25:527-541.

28. Henry KW, Wyce A, Lo WS, Duggan LJ, Emre NC, Kao CF, Pillus L,Shilatifard A, Osley MA, Berger SL: Transcriptional activation viasequential histone H2B ubiquitylation and deubiquitylation,mediated by SAGA-associated Ubp8. Genes Dev 2003,17:2648-2663.

29. Lee KK, Florens L, Swanson SK, Washburn MP, Workman JL: Thedeubiquitylation activity of Ubp8 is dependent upon Sgf11 andits association with the SAGA complex. Mol Cell Biol 2005,25:1173-1182.

30. Daniel JA, Grant PA: Multi-tasking on chromatin with the SAGAcoactivator complexes. Mutat Res 2007:618.

31. Kohler A, Schneider M, Cabal GG, Nehrbass U, Hurt E: YeastAtaxin-7 links histone deubiquitination with genegating and mRNA export. Nat Cell Biol 2008,10:707-715.

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.

























































































































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.

40.�

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.



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.



















Regulation of deubiquitinase proteolytic activity

Documents

Transcript of Regulation of deubiquitinase proteolytic activity