Ubiquitylation is a reversible post-translational modifi-cation with key roles in various signal transduction cas-cades and in determining protein stability. In humans, around 600 ubiquitin E3 ligases ensure the specificity of substrate selection. Ubiquitylation is then diversified by the generation of chains, which are assembled through isopeptide bond formation between the carboxy-terminal Gly and any one of seven internal Lys residues of ubiq-uitin (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 or Lys63). Alternatively, the amino terminus can be used to extend ubiquitin chains (known as linear chains). All forms of chain linkage exist in eukaryotic cells1,2, but only Lys48- and Lys63-linked chains have been extensively studied3.

The human genome encodes approximately 79 de ubiquitinases (DUBs; also known as deubiquitylating or deubiquitinating enzymes) that are predicted to be active and which oppose the function of E3 ligases4,5. They can be subdivided into five families: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumour proteases (OTUs), Josephins and JAB1/MPN/MOV34 metalloenzymes (JAMMs; also known as MPN+ and hereafter referred to as JAMM/MPN+). The UCH, USP, OTU and Josephin families are Cys proteases, whereas the JAMM/MPN+ family members are zinc metalloproteases.

The domain architectures of USPs and other DUB family members are shown in FIG. 1 and FIG. 2, respec-tively. This cornucopia contains multiple domains that probably mediate protein–protein interactions. One striking feature is the abundance of predicted ubiquitin- binding domains (UBDs), including the zinc finger ubiquitin-specific protease domain (ZnF-UBP domain),

the ubiquitin-interacting motif (UIM) and the ubiquitin-associated domain (UBA domain), which typically binds monoubiquitin with low affinity6. The presence of one, or multiple, ubiquitin-like folds (UBL folds) is also widely predicted7. These domains have low sequence homol-ogy but adopt a similar three-dimensional structure to ubiquitin; however, the lack of the crucial C-terminal Gly-Gly motif renders them refractory to cleavage by DUB activities.

DUB activities fall into three major functional catego-ries (FIG. 3). First, ubiquitin can be transcribed from several genes as a linear fusion of multiple ubiquitin molecules or with ribosomal proteins, such that the generation of free ubiquitin requires DUB activity. Second, DUBs can remove ubiquitin chains from post-translationally modi-fied proteins, leading to reversal of ubiquitin signalling or to protein stabilization by rescue from either protea-somal (for example, cytosolic proteins) or lysosomal (for example, internalized receptors) degradation. However, once a commitment to these degradative machines has been made, associated DUB activities can recycle ubiq-uitin, thereby contributing to ubiquitin homeostasis. Third, DUBs can be used to edit the form of ubiquitin modification by trimming ubiquitin chains.

It has become clear that DUBs can display specificity for both substrates and particular ubiquitin chain types. Here, we discuss recent data pertaining to how this spe-cificity might be achieved, for which structural studies have been particularly informative. We also cover the means by which DUB activities can be regulated, through post-translational modifications, interacting proteins and subcellular localization, illustrating these points

*Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK.‡Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, L69 3BX, UK.e-mails: dk@mrc-lmb.cam.ac.uk; clague@liv.ac.uk; urbe@liv.ac.ukdoi:10.1038/nrm2731

MetalloenzymeAn enzyme that requires a metal ion, such as zinc, for its activity and catalytic mechanism. The positive charge of the metal is used to position components of the reaction cycles.

Zinc finger ubiquitin-specific protease domain(ZnF-UBP domain). A zinc finger that is present in histone deacetylase 6 and several ubiquitin-specific proteases, and which in some but not all cases has been shown to bind ubiquitin.

Breaking the chains: structure and function of the deubiquitinasesDavid Komander*, Michael J. Clague‡ and Sylvie Urbé‡

Abstract | Ubiquitylation is a reversible protein modification that is implicated in many cellular functions. Recently, much progress has been made in the characterization of a superfamily of isopeptidases that remove ubiquitin: the deubiquitinases (DUBs; also known as deubiquitylating or deubiquitinating enzymes). Far from being uniform in structure and function, these enzymes display a myriad of distinct mechanistic features. The small number (<100) of DUBs might at first suggest a low degree of selectivity; however, DUBs are subject to multiple layers of regulation that modulate both their activity and their specificity. Due to their wide-ranging involvement in key regulatory processes, these enzymes might provide new therapeutic targets.

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USP

ZnF-MYNDZnF-UBP

M

Z

DUSPD

UBL Ubiquitin-like

UIMUBA

Ataxin 2C

MITMIT

EF-hand

Rhodanese, exonuclease,TBC-RABGAP,CAP-Gly domain, MATH domain, CS domainTransmembrane

Coiled coilCAAX CAAX-box

785 aa605 aa520 aa Z963 aa D UBL UBL858 aa Z

1,406 aa UBLTBC1,102 aa UBLMATH UBL UBL UBL

RhodMIT1,118 aa2,547 aa UBL +700 and +3002,555 aa UBL +700 and +300

798 aa963 aa D UBL UBL370 aa

Z863 aa494 aa UBL

D UBL UBL981 aa823 aa Z530 aa372 aa

UBL M1,318 aaD DZ914 aa

565 aaZ525 aa

+600 and +400UBL2,620 aa1,087 aa

913 aa438 aa

1,077 aa922 aa517 aa

UBL1,352 aa1,604 aa D UBL UBL CAAX

Z D D942 aa+1,000 and +1,000UBL3,546 aa

1,017 aa1,121 aa979 aa

1,042 aaZ565 aa

UBLUBL1,235 aa358 aa

1,325 aaUBL1,124 aa

Z712 aaZ814 aa

366 aaUBLUBLUBL UBL1,375 aa

UBLD D D1,035 aaZ688 aa

339 aaZ711 aa

1,202 aa UBL Exo1,073 aa

+400MIT1,684 aaCAPCAPCAP956 aa

1,092 aa

B B-box

B

CSCS

0 200 400 600 800 1,000 1,200 1,400 1,600

USP1USP2USP3USP4USP5USP6USP7USP8USP9XUSP9YUSP10USP11USP12USP13USP14USP15USP16DUB3USP18USP19USP20USP21USP22USP24USP25USP26USP27XUSP28USP29USP30USP31USP32USP33USP34USP35USP36USP37USP38USP39*USP40USP41USP42USP43USP44USP45USP46USP47USP48USP49USP50*USP51USP52*USP53*USP54*CYLDUSPL1*

Ubiquitin-associated domain(UBA domain). A short (40 amino acid) sequence motif, first found in proteins associated with the ubiquitylation pathway, that mediates (poly)ubiquitin binding.

Ubiquitin-like fold (UBL fold). Ubiquitin contains a distinct three-dimensional fold, which has been used in many proteins related to the ubiquitin system and also in unrelated proteins.

MITA domain found in micro-tubule-interacting and trafficking proteins that forms a three-helix bundle. Some MIT domains, including those of AMSH and USP8, bind to charged multi-vesicular body proteins.

Figure 1 | Domain structure of ubiquitin-specific proteases. The domain architecture of ubiquitin-specific proteases (USPs) reveals an abundance of predicted ubiquitin-binding domains, including the zinc finger ubiquitin-specific protease (ZnF-UBP) domain, the ubiquitin-interacting motif (UIM) and the ubiquitin-associated (UBA) domain. Also common are potential ubiquitin-like (UBL) domains, although most of these have not been validated at a structural level. Crystal structures are currently available for the catalytic domains of USP2, USP7, USP8 (also known as UBPY), USP14 and the cylindromatosis-associated deubiquitinase (DUB, also known as deubiquitylating or deubiquitinating enzyme), CYLD. The domain architecture and other features shown relate to the primary UniProt sequence entry and are based on combined analysis of each RefSeq-associated transcript using the following databases: Pfam, PROSITE, SMART, Interpro and HHPRED. The annotation of the UBL domains is based on REF. 8, the annotation of the USP54 domain found in microtubule-interacting and trafficking proteins (MIT) is based on REF. 133 and the annotation of the B-box in CYLD is based on REF. 15. USP17-like protein 2 (also known as DUB3) belongs to the USP17 gene family, members of which form part of highly polymorphic tandem repeat sequences on chromosomes 4 and 8 that encompass many genes134. Enzymes that are predicted to be inactive based on sequence analysis are indicated (*), although experimental verification remains incomplete135. Numbers with a ‘+’ indicate the number of amino acids in the sequence that are not shown. Ataxin 2C, ataxin 2-like carboxy-terminal domain; CAP, CAP-Gly domain; CS, CHORD-SGT1 domain; DUSP, domain in USPs; EF-hand, Ca2+ binding motif; MATH, meprin and TRAF homology domain; TBC/RABGAP, domain in Tre-2, Bub2 and Cdc16/RAB GTPase activating protein; ZnF-MYND, MYND (myeloid, nervy and DEAF1)-type zinc fingers.

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0 200 400 600 800 1,000 1,200 1,400 1,600

UCH

NZF

Ubiqutin-like

UIMUBA

MIT

SWIRM domain

Coiled coil

SANT domain

JosephinOTU

ZnF-C2H2

ZnF-A20

JAMM/MPN+

271 aaOTUB1234 aaOTUB2

Cezanne 843 aa A

Cezanne 2 926 aa A

708 aaTRABID N N N1,222 aaVCPIP1

348 aaOTU1 C790 aaA20 A A A A A A A

NC

A

1,114 aaOTUD4

293 aaOTUD6B288 aaOTUD6A571 aaOTUD5

481 aaOTUD1398 aaOTUD3

223 aaUCHL1UCHL3 230 aaUCHL5 329 aaBAP1 729 aa

376 aaATXN3ATXN3L 355 aaJOSD1 202 aaJOSD2 188 aa

BRCC36 316 aaCSN5 334 aaPOH1 310 aaAMSH 424 aa MIT

MIT

AMSH-LP 436 aa MITMPND 471 aaMYSM1 828 aaPRPF8* 2,335 aa

SW

SW

S

S

443 aaHIN1L**

+800

UCH

Josephin

OTU

JAMM/MPN+

UBLUBL

UBL

Otubains

OTUs

A20-like OTUs

Catalytic domains:

pKaThe log10 acid dissociation constant. The pKa of a given molecule corresponds to the pH value at which its acid and conjugate base forms are balanced.

with examples drawn from key physiological pro cesses. Although our focus is on the DUBs of eukaryotic, prin-cipally mammalian, cells, the reader is reminded of the fascinating incidence of DUBs in pathogenic organisms (for a review see REF. 8).

Structure and catalytic activity of DUBsMost DUBs catalyse a proteolytic reaction between a Lys ε-amino group and a carboxyl group corresponding to the C terminus of ubiquitin. The Cys protease DUB

families (USPs, UCHs, OTUs and Josephins) rely on two or three crucial amino acid residues, constituting a catalytic diad or triad, respectively. In these enzymes, a nearby His side chain lowers the pKa of a catalytic Cys, enabling a nucleo philic attack on isopeptide linkages. A third residue (usually Asn or Asp) aligns and polar-izes the catalytic His, however this is not always essential for activity, as exemplified in the OTU family member tumour necrosis factor-α-induced protein 3 (TNFAIP3; also known as A20)9.

Figure 2 | Domain structure of ucH, Josephin, oTu and JaMM/MPN+ DuBs. Accessory ubiquitin-binding domains (ubiquitin-interacting motifs (UIMs), ubiquitin-associated (UBA) domains, NPl4-type zinc fingers (NZFs) and A20-type zinc fingers (ZnF-A20s)) are not unique to ubiquitin-specific proteases (USPs; see also FIG. 1) but also occur in at least two of the other four deubiquitinase (DUB, also known as deubiquitylating or deubiquitinating enzyme) families (the Josephins and the ovarian tumour proteases (OTUs)). Only a subset of these have been experimentally verified. The annotation of ubiquitin-binding domains in OTUs is based on REF. 49. Intriguingly, Pfam and HHPRED analysis also indicate the potential existence of a ubiquitin-like (UBL) fold in two OTUs. Crystal structures are available for the catalytic domains of ataxin 3 (ATXN3), OTUB1, OTUB2, A20 (also known as tumour necrosis factor-α-induced protein 3 (TNFAIP3)), OTU1 (also known as YOD1) and associated molecule with the SH3 domain of STAM (AMSH)-like protease (AMSH-LP; also known as STAMBPL1). The domain architecture and other features shown relate to the primary UniProt sequence entry and are based on combined analysis of each RefSeq-associated transcript using the following databases and tools: Pfam, PROSITE, SMART, PSORTII, Interpro and HHPRED. Numbers with a ‘+’ indicate the number of amino acids in the sequence that are not shown. Pre-mRNA-processing splicing factor 8 (PRPF8) is predicted to be inactive based on structural analysis (*)136. PRPF8 contains several unique domains in addition to those annotated here. ** indicates a hypothetical protein. BAP1, ubiquitin carboxy-terminal hydrolase BAP1; BRCC36, BRCA1/BRCA2-containing complex subunit 36; CSN5, COP9 signalosome subunit 5; HIN1L, putative HIN1-like protein; JAMM/MPN+, JAB1/MPN/MOV34 metalloenzyme catalytic domain; MIT, domain found in microtubule-interacting and trafficking proteins; MPND, MPN domain-containing protein; POH1, 26S proteasome-associated PAD1 homologue 1 (also known as PSMD14); SWIRM, SWI3, RSC8 and moira domain; SANT, SWI-SNF, ADAN-CoR, TFIIIB/Myb domain; UCH, ubiquitin carboxy-terminal hydrolase; ZnF-C2H2, C2H2-type zinc finger.

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Ub

UBB

UBC Ub Ub Ub Ub Ub Ub Ub Ub

Ub Ub Ub

UBA52 Ub

UBA80 Ub

L

S

Ub

Ub

a Precursor processing

d Recycling

b Rescue from degradation

c Removal ofnon-degradativeubiquitin signal

e

f Editing

Protein

Protein

UbUb

UbUb

UbUb

UbUb

Degradativesignal

Degradation

Non-degradativesignal

Protein

Protein

Protein

Ub

UbUbUbUbUbUbUbUb

Ub

UbUb

UbUb

UbUb

Free ubiquitin pool

DUB UbiquitinUb

Acyl intermediateAn intermediate in the Cys DUB reaction mechanism, in which the DUB is covalently bound to the C terminus of the distal ubiquitin. A sulphur acyl bond is formed between the C-terminal Gly of ubiquitin and the catalytic Cys of the DUB.

Oxy-anion holeFound next to the catalytic Cys of a DUB, this environment stabilizes the negative charge that is created during the transition state before the formation of the acyl intermediate, by supplying hydrogen-donating amide groups, for example on Asn or Gln.

A detailed understanding of the reaction mechanism of Cys proteases has been gained from studies of the plant protease papain10. A feature of this mechanism is a catalytic acyl intermediate, in which the carboxyl group is covalently bound to the catalytic Cys after the amino group has been cleaved. The negatively charged trans-ition state is stabilized by hydrogen-donating residues, which form an oxy-anion hole nearby. In a second step, a water molecule hydrolyses the acyl-Cys intermediate to complete the catalytic cycle (see Supplementary inform-ation S1 (figure)). Strikingly, all Cys protease DUB families have divergent folds, but their catalytic residues superpose with only small deviations when bound to the ubiquitin C terminus9. The binding of ubiquitin to DUBs also imparts conformational order to otherwise unstructured loops.

Structural dynamics of USP domains. USP domains consist of three subdomains that have been likened to the palm, thumb and fingers of a hand11 (FIG. 4). The catalytic centre lies at the interface between the Palm and Thumb subdomains, and the Fingers subdomain grips the distal ubiquitin. Note that distal refers here and hereafter to the relative position of ubiquitin moieties in

a ubiquitin chain; in a ubiquitin dimer this corresponds to the ubiquitin molecule that is conjugated through its C-terminal Gly.

Some apo-USP domains — that is, USP domains not bound to a substrate — are predominantly in non-productive catalytic configurations, but undergo con-formational changes when ubiquitin binds. This can be considered as shifting an equilibrium between active and inactive conformations, towards the active state. This is well characterized for USP7 (also known as Herpes virus-associated USP (HAUSP)), for which ubiquitin binding is required to bring the catalytic Cys in range of the His residue11,12 (FIG. 4a). By contrast, the catalytic triads of USP14 and USP8 (also known as UBPY) are properly aligned for catalysis in the absence of ubiquitin, but ubiquitin-binding surface loops occlude the active site13,14. For USP14, ubiquitin binding leads to translocation of these loops, allowing access of the ubiquitin C terminus to the active site13. In USP8, which has so far only been crystallized in the apo form, the Fingers subdomain is retracted, further blocking the ubiquitin-binding site14 (see Supplementary information S2 (figure)). However, inactive conformations are not a global feature of USPs: the USP domain of the cylindromatosis-associated DUB, CYLD, is both poised for catalysis and accessible15 (see Supplementary information S2 (figure)).

The linear amino acid sequence of USP domains can be disrupted by large polypeptide insertions138, which can fold into independent domains, such as the B-box domain in CYLD15 (FIG. 1; see Supplementary information S2 (figure)) and the UBA domains in USP5 (also known as ISOT) and USP13 (also known as ISOT3)12. The UBA domains of USP5 constitute additional ubiquitin-binding sites and affect enzyme activity12, whereas the B-box in CYLD influences subcellular localization15. Several USP insertions are predicted to adopt UBL folds, although no clear function has been assigned to these8 (FIG. 1).

OTU domains — variations of the catalytic triad. OTU domains can be phylogenetically divided into three sub-classes: the Otubains (present in OTUB1 and OTUB2), the A20-like OTUs (present in A20, valosin-containing protein p97/p47 complex-interacting protein p135 (VCIP135; also known as VCPIP1), OTU domain- containing protein 7B (OTUD7B; also known as Cezanne) and ubiquitin thioesterase ZrANB1 (also known as TrABID)) and the OTUs5 (FIG. 2). representative structures for each class are available (FIG. 4b; see Supplementary information S3 (figure)).

The structure of yeast Otu1 covalently bound to ubiquitin has revealed that a large surface loop, which is disordered in the apo-structures of OTUB2, OTUB1 and A20 (REFS 9,16–18), forms the bulk of ubiquitin interactions19. Superposition of the ubiquitin-bound structure of Otu1 onto the structure of A20 suggests that the binding site for a distal ubiquitin moiety must have diverged in A20, because a helical domain blocks access to this site. As with USPs, the catalytically inactive conformations have been determined; for example, in the apo-form of OTUB1, the catalytic His residue is not productively aligned with the Cys residue16.

Figure 3 | general roles of DuBs. a | Ubiquitin is encoded by four genes (UBC, UBB, UBA52 and UBA80) and is transcribed and translated as a linear fusion consisting of multiple copies of ubiquitin or ubiquitin fused to the amino terminus of two ribosomal proteins, 40S ribosomal protein L40 (L) and 60S ribosomal protein S27a (S). Note that the polyubiquitin genes encode an extension one or several amino acids long at their carboxyl terminus (shown in yellow). Generation of free ubiquitin from these precursors is a key function of deubiquitinases (DUBs; also known as deubiquitylating or deubiquitinating enzymes). b | Deubiquitylation can rescue proteins from degradation. c | Alternatively, deubiquitylation can remove a non-degradative ubiquitin signal. d | DUBs have a crucial role in maintaining ubiquitin homeostasis and preventing degradation of ubiquitin together with substrates of the 26S proteasome and lysosomal pathways (recycling of ubiquitin). e | Disassembly of ubiquitin chains generated by en bloc removal from substrates ensures that recycled ubiquitin re-enters the free ubiquitin pool. f | Some DUBs might function to edit ubiquitin chains and thereby help to exchange one type of ubiquitin signal for another.

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Ub interaction(Ile44)

Disorderedactive sitecrossover loop

Catalyticcentre

Ub interaction(Ile44)

Formed uponUb interaction

Ub Ile44patch

Catalyticcentre

b Otu1 (OTU family)

a USP7 (USP family)

c UCH-L3 (UCH family)

Ub Ile44patch

Catalyticcentre

Active sitecrossover loop

Catalyticcentre(active)

Ub interaction(Ile44)

Catalyticcentre(inactive)

Formed uponUb interaction

Catalyticcentre(inactive)

Helical arm‘closed’

Helical arm‘open’

d ATXN3 (MJD family)

Ub Ile44patch

Ub Ile44patch

Thumb

PalmFingers

Thumb

Palm

Fingers

(NMR model ofubiquitin interaction)

Catalyticcentre

Structural features of UCH enzymes. The UCH DUB family was the first to be structurally characterized. In the apo-enzyme structure of ubiquitin C-terminal hydrolase isozyme L3 (UCHL3), a prominent loop cov-ers the active site20 (FIG. 4c). Surprisingly, in the ubiquitin aldehyde-bound structure, this active-site crossover loop straddles the C-terminal residues of the bound ubiqui-tin21. Such an arrangement suggests that substrates must fit through this confined loop, preventing binding of large, folded ubiquitin conjugates. Even poly ubiquitin chains would be too big to be threaded through, which is consistent with the failure to observe UCHL1- or UCHL3-mediated hydrolysis of tetra-ubiquitin chains22. An elegant study, in which the crossover loop was extended systematically, showed that only significant extension made UCHL3 catalytically active against ubiquitin polymers23. Consequently, it was suggested that UCH enzymes could act merely on small peptide conjugates, for example those that may be produced as by-products of proteasomal or lysosomal degradation. UCH enzymes may also process the short C-terminal extensions of polymeric ubiquitin precursors24 (FIG. 3). However, UCH enzymes do act on unfolded larger sub-strates and could potentially deubiquitylate protein ter-mini, which might be able to thread into the active-site crossover loop21.

Distinct conformations of the Josephins. Ataxin 3 (ATXN3) is the best-studied member of the Josephin family. It contains a poly-Gln stretch, the extension of which leads to the neurodegenerative disorder Machado–Joseph disease (MJD)25. Several solution structures of the Josephin domain of ATXN3 (REFS 26–28), and a recent solution complex structure with two bound ubiquitin molecules28, reveal yet another fold for Cys DUBs, in which an extended helical arm is proposed to regulate access to the active site (FIG. 4d; see Supplementary infor-mation S4 (figure)). A second ubiquitin-binding site maps to the back of this arm, remote from the active site. Hence, ATXN3 may interact with two distal ubiquitins in a polymer to stabilize an open conformation.

Structures of the JAMM/MPN+ metalloproteases. The catalytic mechanism of action of the JAMM/MPN+ family of metalloenzymes has been elucidated in the recent structures of associated molecule with the SH3 domain of STAM (AMSH)-like protease (AMSH-LP, also known as STAMBPL1) bound to a Lys63-linked diubiquitin molecule (see Supplementary information S5 (figure))29. This structure is the only current example in which a DUB has been crystallized with a ubiquitin chain bound across its active site (FIG. 5). The JAMM/MPN+ motif coordinates two zinc ions, one of which activates a water molecule to attack the isopeptide bond. The amino group is subsequently released from the charged catalytic intermediate, by a mechanism similar to that of cytidine deaminase30,31.

Interestingly, JAMM/MPN+ DUBs are commonly found in association with large protein complexes, for example the 26S proteasome-associated PAD1 homologue 1 (also known as POH1 and PSMD14,

Figure 4 | Structures of cys DuBs. Inactive (green) and active (blue) structures of the catalytic domains of deubiquitinases (DUBs; also known as deubiquitylating or deubiquitinating enzymes) in cartoon representation, with ubiquitin (Ub) shown in yellow. Ile44-interacting motifs are shown in dark blue, and elements that become ordered on ubiquitin binding are shown in orange. More detailed information can be found in Supplementary information S1–S4 (figures). a | Structures of inactive (Protein Data Bank (PDB) identifier 1nb8) and active (PDB identifier 1nbf) ubiquitin-specific protease 7 (USP7)11. The Thumb, Palm and Fingers subdomains of the USP domain are indicated. b | Structure of yeast ovarian tumour-domain containing protein 1 (Otu1; the yeast orthologue of human OTU1 (also known as YOD1)) bound to ubiquitin (PDB identifier 3by4)19, with ubiquitin excluded (left) and shown (right). c | Structure of ubiquitin carboxy-terminal hydrolase L3 (UCHL3) in the absence (PDB identifier 1uch)20 and presence (PDB identifier 1xd3) of ubiquitin137. The active site crossover loop is highlighted. d | Solution structures of ataxin 3 (ATXN3) in the inactive, closed conformation (PDB identifier 2aga)27 and a nuclear magnetic resonance (NMR) model of the open conformation in which the distal ubiquitin is shown (PDB identifier 2jri)28. All ATXN3 structures are derived from NMR analysis and the ubiquitin complex was modelled from chemical shift analysis28. Therefore, whereas the ubiquitin position can be derived, the C terminus of the bound ubiquitin is not defined in the structure and was excluded from the model.

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Zinc ions

Catalyticcentre

Ile44interaction

Distal Ub

Proximal Ub(free C terminus)

Lys63 linkage

Proximal Ub

Gln62

Glu64

Lys63

Gly76

Glu

Ser

His

HisAsp

ZnGly75

Specificity-determiningcontacts

Ub Ile44patch

Distal Ub

a b c

DistalUsed here in the context of DUB cleavage to refer to the relative position of ubiquitin moieties in a ubiquitin chain; in a ubiquitin dimer, distal corresponds to the ubiquitin molecule that is conjugated through its C-terminal Gly.

B-boxA small zinc-binding fold resembling a RING domain, but lacking E3 ligase activity. It is frequently found in the tripartite motif (TRIM) ubiquitin E3 ligases in a conserved array consisting of RING, B-box and coiled coil domains. The function is unknown.

Machado–Joseph disease(MJD). A rare hereditary ataxia — that is, a disease characterized by lack of muscle control — also called spinocerebrellar ataxia type 3. The name derives from two families of Portuguese and Azorean descent, who were among the first patients described.

26S proteasomeA large multisubunit protease complex that selectively degrades multi-ubiquitylated proteins. It contains a 20S particle, which incorporates three distinct proteolytic activities, and one or two regulatory 19S particles.

and homologous to yeast rpn11)32, COP9 signalosome subunit 5 (CSN5)33 and the ESCRT machinery-associated DUB AMSH (also known as STAMBP)34. The lys63-specific deubiquitinase BrCC36 is a component of at least two different complexes: the BrCA1 A complex, which is implicated in the DNA damage response, and the recently identified BrCC36 isopeptidase complex (BrISC)35–38.

Specificity of DUBsDUBs can display specificity at multiple levels to dis-tinguish between the many ubiquitin-like molecules, isopeptides (using an ε-amino group) and linear pep-tides (using an α-amino group), and between different types of ubiquitin linkage and chain structure (BOX 1). We are lacking much mechanistic detail about how DUBs exercise specificity; however, a few common principles can be derived from recent structural and functional analysis.

Ubiquitin versus ubiquitin-like protein recognition. All DUB catalytic domains have a primary ubiquitin-binding site that encompasses extensive interactions with the distal ubiquitin in a polyubiquitin chain (see Supplementary information S6 (figure)). The main inter-actions are formed through the ubiquitin Ile44 patch, its canonical protein interaction site7 and additional sur-faces that depend on the DUB family (see Supplementary information S6 (figure)). Ubiquitin–DUB interactions cover 20–40% of the total ubiquitin surface, far in excess of most characterized UBDs (usually ~10%)7.

The C terminus of the distal ubiquitin extends from its binding site towards the DUB catalytic centre and is held firmly in place for catalysis. It is this stretch of amino acids that allows DUBs to distinguish between

ubiquitin and the many other ubiquitin-like molecules. The C-terminal sequence of ubiquitin, Leu71-Arg72-Leu73-Arg74-Gly75-Gly76, is different from those of ubiquitin-like molecules, such as neuronal precursor cell expressed developmentally downregulated pro-tein 8 (NEDD8), in which the C-terminal sequence is Leu-Ala-Leu-Arg-Gly-Gly, and small ubiquitin-like modifier (SUMO), in which the C-terminal sequence is Gln-Gln/Glu-Gln-Thr-Gly-Gly) (see Supplementary information S7 (table)). Interferon-stimulated gene 15 (ISG15), a ubiquitin-like molecule with two ubiquitin fold repeats, contains the same C-terminal sequence as ubiquitin.

Elegant biochemical work has revealed specifi-city determinants for individual DUB families in this C-terminal sequence, showing that Arg74 and Gly75 are crucial for ubiquitin recognition by DUBs39. A number of ISG15 cross-reactive DUBs have been identified, as well as a family of viral OTU domain-containing DUBs that have been shown to disassemble both ISG15 and ubiquitin from cellular proteins40,41. USP18 can cleave a linear fusion of ISG15 but not of ubiquitin42. Dual spe-cificity for NEDD8 and ubiquitin has also been reported for USP21 (REF. 43). To date, there are no structures of ISG15- or NEDD8-bound DUBs.

Isopeptide versus peptide cleavage. The chemical fea-tures of an isopeptide bond differ significantly from those of a peptide bond. Whereas the Lys side chain in an isopeptide bond has no conformational restraint and has five freely rotatable bonds, the peptide linkage has reduced rotational freedom22. Furthermore, the follow-ing side chain and carboxyl group in the peptide bond demand a more spacious environment around the DUB active site.

Figure 5 | Structures of a JaMM/MPN+ metalloprotease. Structure of the catalytic domain of AMSH-LP (associated molecule with the SH3 domain of STAM (AMSH)- like protease; also known as STAMBPL1), a JAMM/MPN+ (JAB1/MPN/MOV34 metalloenzyme) deubiquitinase (DUB; also known as deubiquitylating or deubiquitinating enzyme). a | AMSH-LP (shown in green) in isolation (Protein Data Bank identifier 2znr). b | AMSH-LP in complex with Lys63-linked diubiquitin (PDB identifier 2znv)29, with the protein shown in blue and ubiquitin (Ub) shown in yellow. c | A close-up view of the active site. The complex structure was obtained from a catalytic mutant (Glu292Ala) and hence the active apo-structure is shown with Lys63-linked diubiquitin derived from this complex29. The specificity-determining interactions of the Gln62 and Glu64 of the proximal ubiquitin with AMSH-LP are shown as grey dotted lines. Active site residues are shown in orange and zinc ions are shown as light green spheres and interactions in the active centre are indicated by yellow dotted lines.

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S

Lys

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Ub

S

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LysUb

Ub UbUb

Ub

S

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UbUb

a Linkage specificity

b Exo- versus endo-deubiquitylationExo Endo

Distal S

Lys Ub Ub Ub UbDistal

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c Substrate-specific DUBs

d Mono-deubiquitylation

e Recycling DUBs – UCH and ISOT

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GG Ub Ub Ub UbDistal

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LysTa

il

Tail

63 4848

48 1111

11

6363

DUB

Substrate

UbiquitinUb

SUb

Crossover loop

COP9 signalosomeAn eight-subunit protein complex that regulates protein ubiquitylation and turnover in various developmental and physiological contexts. Extensively characterized in plants but fundamental to all eukaryotes, this complex post-translationally modifies the cullin subunit of E3 ubiquitin ligases by cleaving off the covalently coupled peptide NEDD8.

ESCRT machinery(Endosomal sorting complex required for transport). A multimeric protein complex that was first identified biochemically in yeast. The ESCRT machinery controls the sorting of endosomal cargo proteins into internal vesicles of multivesicular bodies.

Linear ubiquitin chains, which are linked by peptide bonds, provide a precursor to ubiquitin but also have physio-logical roles in nuclear factor-κB (NF-κB) signalling44,45. Structural analysis shows that these chains have a topol-ogy similar to that of Lys63-linked chains22. We recently tested the ability of a number of DUBs to pro cess linear chains, and found that most USP domains could cleave these chains with low efficiency, whereas members of the OTU, JAMM/MPN+ and UCH domain-containing DUB families were in active and hence might be true isopeptide- specific proteases22. USP domain-containing DUBs can potentially cleave peptide bonds that are unconnected to ubiquitin. For example, it has been proposed that USP1 undergoes inactivation by auto-proteolysis at an intrinsic Leu-Leu-Gly-Gly motif46.

Specificity for ubiquitin chain linkages. We currently have an incomplete picture of DUB specificity, owing to the unavailability of well-defined ubiquitin chain

preparations, other than linear, Lys63- or Lys48-linked chains. It is, however, interesting to note that each Lys in ubiquitin has a unique sequence context, which might be used for specific recognition by DUBs (see Supplementary information S8,S9 (figure, table)). As highly specific DUBs exist, it is possible that new DUB families will be discovered once appropriate reagents are available (see Supplementary information S10 (box) for currently available substrates and assays).

Nevertheless, bearing this caveat in mind, the ability of DUBs to discriminate particular chain linkages is strik-ing. For USP and OTU domain-containing DUBs, both Lys48 and Lys63 linkage-specific members have been described. The 26S proteasome-associated USP14 shows specificity for Lys48-linked ubiquitin chains13, whereas CYLD cannot cleave this chain type efficiently but hydro-lyses Lys63-linked and linear chains15,22. However, most USPs that have been analysed are promiscuous22. The OTU family has evolved Lys48 linkage-specific members,

Box 1 | Layers of DUB specificity

Deubiquitylation, with its intrinsic complexity in dealing with polymers, can provide specificity on a number of levels, and many different ways to hydrolyse ubiquitin have been reported.

The existence of eight topologically different ubiquitin chains allows for differential recognition by the deubiquitinases (DUBs, also known as deubiquitylating or deubiquitinating enzymes). In addition to DUBs that do not discriminate between chain linkages, many chain type-specific DUBs exist (see the figure, part a). Importantly, this level of specificity is not restricted to particular DUB families, and the ubiquitin-specific proteases (USPs), the ovarian tumour proteases (OTUs) and the JAB1/MPN/MOV34 metalloenzymes (JAMMs, also known as MPN+) all include linkage-specific members.

In ubiquitin chains, cleavage (shown by the arrows) can occur from the ends (exo) or within a chain (endo). Endo-DUBs must accommodate ubiquitin molecules on either side of the cleavage site, whereas exo-DUBs only need to bind to a single ubiquitin (see the figure, part b).

DUBs may be targeted to their substrates directly. Ubiquitylated target sequences in substrates may be recognized by these DUBs, leading to a single-step chain amputation (see the figure, part c, left). Promiscuous DUBs might remove ubiquitin completely from substrates. Alternatively, they might leave the substrate monoubiquitylated, especially if the DUB is linkage-specific (see the figure, part c, right). The monoubiquitin could then be extended again with a different linkage (ubiquitin chain editing).

Many proteins are monoubiquitylated on one or multiple sites, and DUBs might exist that specifically recognize their cognate protein substrate to remove monoubiquitin (see the figure, part d).

Ubiquitin carboxy-terminal hydrolase (UCH) family members are designed to effectively remove small disordered sequences from the C terminus of ubiquitin, such as peptide remnants after proteasomal degradation and C-terminal extensions of polyubiquitin precursors. A ‘crossover’ loop restricts access to the active site and might prevent binding of large, fully folded ubiquitin conjugates (see the figure, part e, left). DUBs are also responsible for the recycling of free ubiquitin from unattached ubiquitin chains. USP5 (also known as ISOT) is especially well equipped for this task by virtue of a zinc finger ubiquitin-specific protease (ZnF-UBP) domain that recognizes the free C terminus of ubiquitin (see the figure, part e, right).

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14-3-3 proteinsA family of regulatory proteins that bind to phosphorylated forms of various proteins, which are involved in signal transduction and cell cycle control.

such as OTUB1 (REFS 16,47), but also Lys63 linkage- specific DUBs, including TrABID and de ubiquitinating enzyme A (DUBA; also known as OTUD5)48,49. Six human JAMM/MPN+ domain-containing DUBs (AMSH, AMSH-LP, CSN5, BrCA1/BrCA2-containing complex subunit 36 (BrCC36), POH1 and histone H2A deubiquitinase MYSM1) show Lys63 linkage specificity (REFS 38,50,51 and M.C. and S.U., unpublished observa-tions), suggesting that JAMM/MPN+ enzymes have a common mechanism of achieving such specificity. The Josephin DUB ATXN3 has activity directed towards Lys63 linkage but might favour mixed linkage chains52.

Proximal ubiquitin: determining linkage specificity. Structural analyses of two Lys63 linkage-specific DUBs have indicated mechanisms that might be used to achieve linkage specificity. A structure of AMSH-LP in complex with Lys63-linked diubiquitin revealed that it exploited the open conformation of this ubiquitin chain, contact-ing all the residues involved in the linkage, including all atoms of the Lys63 side chain29 (FIG. 5). Compared with the extensive binding site for the distal ubiquitin in the dimer, AMSH-LP makes few contacts with the proximal ubiquitin29. Importantly, however, AMSH-LP specifically interacts with residues Gln62 and Glu64 in the proximal ubiquitin, a combination of neighbouring amino acids that is unique to Lys63 (see Supplementary information S9 (table)). The specific recognition of the Lys63 sequence context allows AMSH-LP to select this chain type over other linkages.

The crystal structure of the catalytic domain of CYLD also suggested determinants for specificity, although a complex with ubiquitin is not available15. The predicted proximal ubiquitin-binding site is highly conserved between CYLD homologues, but diverges from other USP domains. This indicates that proximal ubiquitin recog nition is important, as seen with AMSH-LP. Another CYLD-specific feature is an extended loop near the active site, which might select against Lys48-linked ubiquitin chains. Other USP domains, which have a significantly shorter loop at this position, cleave Lys48- and Lys63-linked ubiquitin chains equally well, and shortening of this loop in CYLD makes the enzyme less specific15. Interestingly, CYLD, but not AMSH, also hydrolyses linear ubiquitin chains that have an open structure equivalent to that of Lys63-linked chains22.

Positioning DUBs on a ubiquitin chain. Ubiquitin chains can be cleaved from the end (exo) or internally (endo) (BOX 1). The 26S proteasome-associated DUB USP14 has exo-activity, as it cleaves Lys48-linked chains from the distal end only, exclusively gener-ating monoubiquitin13. By contrast, DUBs that regu-late ubiquitin-mediated signalling (CYLD, USP9X, AMSH-LP and A20) were shown to have endo-activity, releasing longer chains from their substrates by internal cleavage15,17,29,53.

The molecular basis for the different points of attack in CYLD and USP14 became clear from their structures. USP14, like most USP domain-containing proteins, con-tains a Fingers subdomain, which contacts up to 40% of

the distal ubiquitin and blocks access to Lys48 or Lys63 (FIG. 4a; see Supplementary information S2,S6 (figures)). This allows USP14 to bind to the distal end of an ubiq-uitin chain, but not to internal linkages13. By contrast, CYLD lacks the Fingers subdomain, allowing access to Lys63. Hence, CYLD can bind to internal ubiquitins in Lys63-linked chains, as ubiquitin chains can extend distally from the bound ubiquitin15.

Direct and specific recognition of the ubiquitin–ubiquitin linkage would seem to disallow removal of the most proximal (or first) ubiquitin attached to the protein substrate. How then is this monoubiquitin processed? Details are scarce, as it is a major challenge to produce monoubiquitylated substrates in a defined manner. Monoubiquitin processing might require substrate-directed, nonspecific DUBs that accommodate a wider range of sequences in their proximal binding site. Alternatively, UCH enzymes might be ideally suited to remove the proximal ubiquitin, if it occupies a non-structured region. Ubiquitin chain editing — that is, the change of one chain type (for example, a ‘signalling’ Lys63-linked chain) to another type (for example, a ‘degrading’ Lys48-linked chain) — would benefit from substrates that are not fully deubiquitylated, but that have left the proximal ubiquitin on the substrate.

DUBs for unattached ubiquitin chains. USP5 and USP13 harbour a specialized ZnF-UBP domain, which recognizes the C-terminal Gly-Gly motif in unattached chains54. These dedicated ubiquitin recycling DUBs are highly active against unattached chains regardless of the linkage12, and efficiently replenish the cellular monoubiquitin pool55.

Regulation of DUBsThe in vitro activity of DUBs is often low, hinting at widespread activation mechanisms imposed by cellular context. Furthermore, the substrate-binding site can be occluded and the catalytic triad of some DUBs seems to be in an inactive conformation, suggesting that a con-formational change must be necessary for activity. We discuss below three major regulatory mechanisms: post-translational modifications, allosteric interactions and subcellular localization.

Post-translational modifications. There is extensive crosstalk between phospho and ubiquitin signalling networks56. For example, inducible phosphorylation of CYLD inhibits its capacity to deubiquitylate TNF receptor-associated factor 2 (TrAF2) and suppress NF-κB signalling57. Constitutive phosphorylation of USP8 in interphase cells might inhibit or dampen its isopeptidase activity directly and through the recruit-ment of 14-3-3 protein family members58. In mitosis, this phosphorylation is lost and the resulting enhanced activity of USP8 might relate to its recruitment to the midbody during cytokinesis59,60. Ubiquitin-like modifications can themselves regulate DUB activity. Ubiquitylation of ATXN3 activates its catalytic activity61, whereas sumoylation of USP25 inhibits its activity by steric hindrance62.

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Catalytic rateThe number of substrate molecules that are converted into a product by an enzyme molecule in a unit of time, when the enzyme is fully saturated with substrate.

WD40 domainA domain consisting of 4–10 WD40 repeats of 44–60 amino acids, which assemble into a propeller-shaped scaffold. Many distinct protein- and peptide-binding sites have been described in these adaptor domains.

NucleolusA subnuclear electron-dense structure composed of protein and nucleic acids that has a key role in the biogenesis of ribosomal RNA.

Early endosome(Also known as sorting endosome). A tubular, vesicular structure that receives material directly from the plasma membrane and is a precursor of the mature (late) endosome. Early endosomes have a key role in sorting material for recycling or degradation in lysosomes.

LysosomeA membrane-bound organelle in higher eukaryotic cells that has an acidic interior and is the major storage site of the degradative enzymes (acidic hydrolases) that are responsible for the breakdown of internalized proteins and many membrane proteins. It is functionally equivalent to the yeast vacuole.

As Cys proteases, some DUBs might be sensitive to redox regulation by reactive oxygen or nitrogen species, similar to the reactive Cys residues of phosphatases63. It has been proposed that hydrogen peroxide inhibits nega-tive regulation of NF-κB signalling by modifying Cezanne and thereby impairing its capacity to deubiquitylate recep-tor interacting protein 1 (rIP1)64. Both DUB activity and oxidation require a low pKa Cys residue that is achieved through charge withdrawal by a nearby His residue of the catalytic triad. The observation that several DUBs (for example, USP7), in the absence of bound ubiquitin, main-tain this essential His residue at a distance that is beyond the range for effective charge withdrawal suggests that this may be an acquired resistance to oxidative stress.

Some DUBs are subject to post-translational proteo-lytic cleavage. These include USP1, which undergoes inactivating auto-proteolysis46, and A20, which is pro-teolytically inactivated by mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1)65.

Protein interactions and allosteric activation. Many DUBs contain additional ubiquitin-binding domains or motifs, which might directly regulate their activ-ity or specificity. USP25, OTUD5 and ATXN3 require intact UIMs to efficiently hydrolyse polyubiquitin chains27,49,62. UIMs can also impart linkage specificity — for example the UIM of ATXN3 is necessary for Lys63 selectivity52. The endosomal DUBs AMSH and USP8 are activated by association with the UIM-containing signal transducing adaptor molecule 2 (STAM2)34,66, presum-ably through STAM2 increasing the efficiency of sub-strate capture. This principle is likely to be used by many DUBs. For example, A20 binds to the ubiquitin-binding proteins TAX1-binding protein 1 (TAX1BP1) and the A20-binding inhibitor of NFκB activation proteins (ABINs), which may affect catalysis or substrate target-ing67,68. Interacting proteins can also inhibit DUB activity: UCH37 (also known as UCHL5) activity is reduced by association with the INO80 chromatin-remodelling com-plex69, and the yeast enzyme Doa4 is directly inhibited by regulator of free ubiquitin chains 1 (rfu1)70.

In the case of USP5, binding of free ubiquitin to the N-terminal ZnF-UBP domain induces an allosteric conformational change leading to an increased catalytic rate for ubiquitin chain processing54. Several alternative examples of allosteric activation by interacting proteins have been described (see also REF. 71). The WD40 domain-containing protein USP1-associated factor 1 (UAF1; also known as WDr48) is found in a heterodimeric com-plex with at least three USPs (USP1, USP12 and USP46). In vitro analysis shows a major increase in the catalytic rate of all three DUBs caused by WD40 domain-dependent binding of UAF1, but not for several other USP family members72,73. Interestingly, WD40 domains are highly enriched in a global survey of proteins that interact with 75 DUBs74. Drosophila melanogaster USP7 is found in a heteromeric complex with guanosine 5′ monophosphate synthase (GMPS), which stimulates its in vitro activity towards ubiquitylated histones and the tumour suppres-sor protein p53 (REF. 75). Other DUBs require incorpora-tion into large macromolecular complexes, such as the

26S proteasome (see below) or the COP9 signalosome (for example, CSN5), for activity. Such large complexes might serve merely as scaffolds to present DUBs with their physiological substrates, or might regulate cata-lytic activity directly, for example through allosteric activation.

Regulation by subcellular localization. Several DUBs, such as USP8 and CYLD, have been shown to undergo epidermal growth factor (EGF)-dependent translocation to endosomes or association with a phosphotyrosine-associated protein interaction network (defined by anti-phosphotyrosine immunoprecipitation)76,77.

Subcellular localization will determine the palette of substrates available for processing. Only USP19 and USP30 have predicted transmembrane domains. USP30 has been localized to mitochondria and implicated in the regulation of mitochondrial morphology78. Several DUBs show nuclear accumulation, and USP36 specifi-cally localizes to the nucleolus and regulates its structure and function79. Coupling of localization and activation is an important principle of cellular biology. This is exem-plified by the recruitment of AMSH to early endosomes by its activator STAM34.

Physiological roles of DUBsThe physiological roles of DUBs are as pervasive as the ubiquitin–proteasome system itself. Here we use selected examples to illustrate broad functional categorization.

Regulation of protein stability. Both lysosomal and pro-teasomal degradation can be elicited by ubiquitylation, although the nature of the chain linkage is thought to dif-fer. Lys63-linked polyubiquitin chains or multiple mono-ubiquitin specifies sorting of receptors to the lysosome through engagement with the ESCrT machinery80, whereas Lys48-linked polyubiquitin chains and probably most other ubiquitin linkages can target the protein for proteasomal degradation2,81. It follows that deubiquityla-tion will rescue proteins from degradation and enhance their stability.

If accumulation of the substrate protein is deleterious (for example, if the protein is the product of a proto-oncogene), then the relevant DUB may be an attractive drug target through which to destabilize the substrate (see Supplementary information S11 (box) for a brief description of currently available inhibitors)82–84. For example, high fatty acid synthase levels, which are asso-ciated with poor prognosis in prostate cancer, can be reduced by enhanced proteasomal degradation follow-ing depletion of USP2 by small interfering rNA (sirNA) or antisense oligonucleotides85. Other prominent exam-ples include stabilization of the proto-oncogene MYC by USP28 (REF. 86) or of p53 and its E3 ubiquitin ligase MDM2 by USP7 (REF. 87).

USP44 deubiquitylates CDC20, a co-activator of the E3 ubiquitin ligase anaphase promoting complex or cyclosome (APC/C) that promotes the degradation of many key cell cycle regulators. USP44 activity is crucial for the regulation of the spindle checkpoint and con-sequently for the correct execution of chromosome

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Inactivation

Degradation

Inactivation

Chain editingStabilizationDegradation

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StabilizationDegradation

ERBB3

InactivationActivation

NF-κB signalling

a

c d

b

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NRDP1 USP8

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TRAF2 CYLD

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NF-κB signalling

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–

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–+

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+ –

–+

UbiquitinUb

segregation during the final stages of mitosis. As long as there are unattached kinetochores present, CDC20 is held in complex with mitotic spindle assembly check-point protein MAD2, which prevents it from activat-ing the APC/C. release of CDC20 from this complex requires its ubiquitylation by the APC/C itself. USP44 is proposed to counteract this activity, thereby prevent-ing premature checkpoint inactivation88. The balance between the APC/C ubiquitylating activity and the USP44 deubiquitylating activity can therefore control cell cycle progression.

Internalized ubiquitylated receptors are subject to deubiquitylation in early or sorting endosomes, which can promote receptor recycling at the expense of lyso-somal sorting. This seems to be the mode of action of AMSH, as its depletion by sirNA enhances downregula-tion of activated EGF receptor (EGFr), which is princi-pally modified with monoubiquitin and Lys63-linked ubiquitin chains50,89,90. Similarly, the epithelial Na+ chan-nel ENaC is returned to the apical surface of kidney cells

after deubiquitylation by endosome-localized UCHL3 (REF. 91). The lysosomal sorting machinery itself can also be targeted for proteasomal degradation; the ESCrT0 complex, which engages ubiquitylated EGFr, is stabi-lized by USP8 (REFS 76,92). Similarly, another endocytic adaptor, sorting nexin 3 (SNX3), which governs ENaC trafficking, is stabilized by the vasopressin (anti diuretic hormone)-inducible DUB USP10 (REF. 93).

Many DUBs are found in association with E3 ligases, which have an intrinsic tendency to self-ubiquitylate (FIG. 6). Hence, stabilization of E3 ligases by deubiquityl-ation is a major aspect of DUB physiology and, recipro-cally, E3s may destabilize their cognate DUB through ubiquitylation94. For example, USP19 supports cell pro-liferation by stabilizing KPC1 (also known as rNF123), a ubiquitin ligase for the cyclin-dependent kinase inhibi-tor p27Kip1 (also known as CDKN1B)95, and the activity of USP8 stabilizes neuregulin receptor degradation pro-tein 1 (NrDP1; also known as rNF41), an E3 ligase that negatively regulates the stability of the receptor tyrosine kinase ErBB3 (REF. 96). One of the functions of CYLD in the NF-κB signalling pathway (see below) is to oppose the E3 ligase activity of its interacting partner TrAF2 (REFS 97–99).

E3–DUB interactions may also allow fine-tuning of the ubiquitylation status of a common substrate. In the case of the DUB USP7, both the E3 ligase MDM2 and its substrate p53 have been proposed as relevant USP7 substrates100. Extreme coupling of DUB and E3 ligase activities is seen with A20, which combines DUB activ-ity (through the OTU domain) with E3 ligase activity (through the A20-type zinc fingers (ZnF-A20)) in one single polypeptide chain to modulate the ubiquitylation status of key adaptors in the NF-κB signalling cascade (for example, rIP1 (see below)), providing a streamlined device for ubiquitin chain editing101. A recent proteomic survey of the interaction profile of 75 DUBs by Sowa et al. also identified multiple E3 ligase-containing complexes, highlighting the prevalence of the coupling of positive and negative regulators in the ubiquitin system74.

Ubiquitin homeostasis and ‘proofreading’. Once the fate of a protein is sealed as degradation in the lysosome or proteasome, it is economical to recycle the ubiquitin. Proteasomal degradation of ubiquitylated substrates is proposed to be coupled to the recycling activity of the JAMM/MPN+ DUB POH1 in mammals (rpn11 in yeast)32,102, one of three 26S proteasomal DUBs. POH1 is a core structural component of the 19S proteasome lid that recognizes proteasomal substrates and governs entry into the central proteolytic chamber of the 26S proteasome. Like other JAMM/MPN+ proteins, POH1 shows specificity for Lys63-linked polyubiquitin chains, but can also cleave the first isopeptide bond that links the substrate and the proximal ubiquitin32. The other two proteasomal DUBs, UCH37 and USP14, might be redundant: ubiquitylated proteins accumulate only when both are knocked down, indicating a defect in the ubiquitin–proteasome system. Nevertheless, the proteo-lytic activity of the 26S proteasome towards a peptide substrate is unimpaired103.

Figure 6 | Diverse roles for DuB–e3 ligase interactions. Many deubiquitinases (DUBs; also known as deubiquitylating or deubiquitinating enzymes) interact with E3 ubiquitin ligases. In principle, such coupling between opposing catalytic activities may serve various purposes, as indicated in the central diagram. a | Many E3 ligases (for example, neuregulin receptor degradation protein 1 (NRDP1; also known as RNF41), a negative regulator of the receptor tyrosine kinase ERBB3) undergo auto-ubiquitylation and may be stabilized by an associated DUB (for example, ubiquitin-specific protease 8 (USP8; also known as UBPY))96. b | Ubiquitylation has also been associated with the activation of signalling pathways, for example in the nuclear factor-κB (NF-κB) pathway, in which the cylindromatosis-associated DUB, CYLD, opposes the E3 ligase tumour necrosis factor receptor-associated factor 2 (TRAF2)97–99. Note that TRAF2 is not the sole substrate of CYLD in this pathway. c | Interactions between E3 ligases and DUBs might also allow fine-tuning of the ubiquitylation status of a common substrate: both the E3 ubiquitin ligase MDM2 and its substrate p53 have been proposed to be USP7 substrates100. d | The ovarian tumour protease (OTU) A20 (also known as TNFAIP3) combines DUB activity (through the OTU domain) with E3 ligase activity (through the A20-type zinc fingers (ZnF-A20)) in one single polypeptide chain to modulate the ubiquitylation status of key adaptors in the NF-κB signalling cascade, for example receptor interacting protein 1 (RIP1). + and – refer to the addition and removal of ubiquitin by the E3 ligase and DUB activities, respectively.

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An emerging view is that the 26S proteasome can extensively remodel ubiquitin chains on bound substrates through opposing ubiquitin ligase (HECT ubiquitin ligase 5 (Hul5)) and deubiquitylating activities104. This suggests that the fate of a proteasome-bound substrate might not be definitively sealed and that remodelling of its ubiquitin chain, during a kinetically defined time window, can exert an influence. The yeast homologue of USP14, Ubp6, becomes ~300-fold more active on bind-ing to the base of the 19S lid of the proteasome105, whereas UCH37 is activated by direct binding of the adhesion regulating molecule 1 (ADrM1; the homologue of yeast rpn13) subunit to its auto-inhibitory C-terminal tail106. Ubp6 is upregulated following depletion of free ubiquitin levels, and has been proposed to be a component of a cellular response to ‘ubiquitin stress’ that can be induced by many insults107. Ubp6 delays substrate degradation by a non-catalytic mechanism and also switches the mode of chain degradation from rpn11-mediated en bloc removal to a processive reaction108.

Deubiquitylation is not necessary for lysosomal sort-ing, other than to maintain free ubiquitin levels. In yeast, the relevant DUB has been identified as Doa4 (REF. 109), and in mammalian cells USP8 (also known as UBPY) has been linked with this activity76. As for the 26S protea-some, a balance of additional endosome-associated DUB activities, for example by AMSH, and ligase activities, such as by the E3 ubiquitin ligase CBL might determine protein fate90.

Unanchored isopeptide-linked ubiquitin chains can be processed to free ubiquitin. USP5 (also known as ISOT) is ideally suited to this task by virtue of its ability to recognize a free C terminus on the proximal ubiquitin (see above)54. Loss of USP5 or its yeast homologue, Ubp14, leads to accumulation of free ubiquitin chains and inhibition of protein degradation owing to competi-tion with substrates for 26S proteasome binding55,110. In yeast, the processing of unanchored chains is shared with other DUBs, notably Doa4, which is upregulated during heat shock to maintain the free ubiquitin pool70,111.

Negative regulation of ubiquitin signals. The identifica-tion of more than 15 UBDs has strengthened an emerg-ing view that reversible ubiquitylation might have key signalling roles akin to phosphorylation, independently of protein degradation112. Dynamic ubiquitylation of histones regulates chromatin structure, and the role of ubiquitin in DNA damage repair pathways is also firmly established. Condensation of chromosomes in metaphase is accompanied by deubiquitylation of his-tones H2A and H2B113. Four enzymes (MYSM1, USP3, USP16 and USP22) have been proposed to directly remove ubiquitin from ubiquitylated histone H2A114–117

. However, one difficulty in reconciling these papers is that each claims a significant effect on global ubiquityl-ated H2A levels following individual gene knockdown. The sum of their proposed action is far greater than the total ubiquitylated H2A pool.

MYSM1, USP16 and USP22 might share a common role in the transcriptional control of potentially dis-tinct gene cohorts, and USP22, USP16 and USP3 DUB

activities have been implicated in cell cycle progres-sion114–117. USP3, which can remove ubiquitin from both H2A and H2B histones, might indirectly influence the cell cycle through effects on DNA damage repair path-ways, leading to DNA damage checkpoint activation116.

Monoubiquitylation of Fanconi anaemia complemen-tation group D2 protein (FANCD2) and proliferating cell nuclear antigen (PCNA) promotes DNA repair through chromatin association and the enhanced recruitment of enzymes involved in the translesion synthesis repair pathway, respectively. Both of these proteins are sub-strates of USP1 (REFS 46,118), but only deubiquitylation of FANCD2 is necessary for DNA repair, reflecting the need for a dynamic population of FANCD2 (REF. 119).

The major inflammatory pathway leading to acti-vation of the transcription factor NF-κB is regulated by reversible ubiquitylation120. Here, the role of two DUBs, CYLD and A20, is fairly well understood. CYLD was originally identified as a tumour suppressor and is mutated in cylindromatosis, a condition resulting in the development of benign tumours on the scalp. CYLD DUB activity was shown to negatively regulate NF-κB activa-tion by processing Lys63-linked polyubiquitin chains on TrAF2, which are required for activation of inhibitor of NF-κB kinase (IKK) by TrAF2 (REFS 97–99). As CYLD gene expression is upregulated by NF-κB, it provides a conduit for negative-feedback regulation of this pathway. Furthermore, CYLD has been linked to a wide variety of substrates and cellular processes, including cell cycle progression (reviewed in REF. 121).

Deficiency of A20 in mice causes persistent activa-tion of NF-κB by Toll-like and TNF receptors122,123. A20 also acts as a tumour suppressor in human B cell lym-phoma124–126. A20 negatively regulates activation of IKK through the removal of Lys63-linked ubiquitin chains from TrAF6 and rIP1 (REFS 122,127). In vitro, A20 pro-cesses Lys63-linked chains with low efficiency compared with Lys48-linked chains, but effectively removes Lys63-linked chains from TrAF6 en bloc9,17. Two E3 ligases, atrophin 1-interacting protein 4 (AIP4; also known as ITCH) and rING finger protein 11 (rNF11), have also been reported to associate with A20 and might have key roles in the Lys48-linked ubiquitylation of rIP1 (REFS 128,129).

The NF-κB pathway is under exquisite control by reversible ubiquitylation, and regulatory roles for another DUB, Cezanne, have also been shown130. It is likely that other major signal transduction cascades will prove to be crucially regulated by reversible ubiquityla-tion, but details are more fragmented and much remains to be unravelled. OTUD5 negatively regulates the type I interferon response by deubiquitylating TrAF3 (REF. 49). USP9X can remove non-canonical Lys33- and Lys29-linked inhibitory polyubiquitin chains from cer-tain 5′ AMP-activated protein kinase-related kinases involved in cellular energy homeostasis, and has also been implicated in the transforming growth factor-β pathway by deubiquitylating SMAD4 (REFS 53,131). The Lys63-linked ubiquitin chain-specific OTU DUB TrABID was recently shown to act as a positive regu-lator for Wnt signalling48. Finally, mitogen activated

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1. Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nature Biotech. 21, 921–926 (2003).

2. Meierhofer, D., Wang, X., Huang, L. & Kaiser, P. Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. J. Proteome Res. 10, 4566–4576 (2008).

3. Ikeda, F. & Dikic, I. Atypical ubiquitin chains: new molecular signals. ‘Protein modifications: beyond the usual suspects’ review series. EMBO Rep. 9, 536–542 (2008).

4. Scheel, H. Comparative Analysis of the Ubiquitin-Proteasome System in Homo sapiens and Saccharomyces cerevisiae. Thesis, Univ. Cologne (2005).Highly comprehensive bioinformatic study of the ubiquitin–proteasome system, which deserves a wide readership.

5. Nijman, S. M. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005).

6. Hurley, J. H., Lee, S. & Prag, G. Ubiquitin-binding domains. Biochem. J. 399, 361–372 (2006).

7. Zhu, X., Menard, R. & Sulea, T. High incidence of ubiquitin-like domains in human ubiquitin-specific proteases. Proteins 69, 1–7 (2007).

8. Edelmann, M. J. & Kessler, B. M. Ubiquitin and ubiquitin-like specific proteases targeted by infectious pathogens: emerging patterns and molecular principles. Biochim. Biophys. Acta 1782, 809–816 (2008).

9. Komander, D. & Barford, D. Structure of the A20 OTU domain and mechanistic insights into deubiquitination. Biochem. J. 409, 77–85 (2008).

10. Storer, A. C. & Menard, R. Catalytic mechanism in papain family of cysteine peptidases. Methods Enzymol. 244, 486–500 (1994).

11. Hu, M. et al. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041–1054 (2002).The first USP domain structure, in the presence and absence of ubiquitin. It defined the USP fold and revealed conformational changes on ubiquitin binding.

12. Reyes-Turcu, F. E., Shanks, J. R., Komander, D. & Wilkinson, K. D. Recognition of polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase T. J. Biol. Chem. 283, 19581–19592 (2008).

13. Hu, M. et al. Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J. 24, 3747–3756 (2005).

14. Avvakumov, G. V. et al. Amino-terminal dimerization, NRDP1–rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitin-specific protease 8 (USP8). J. Biol. Chem. 281, 38061–38070 (2006).

15. Komander, D. et al. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module. Mol. Cell 29, 451–464 (2008).Provides a rationale for the Lys63-linked ubiquitin chain specificity of CYLD.

16. Edelmann, M. J. et al. Structural basis and specificity of human otubain 1-mediated deubiquitination. Biochem. J. 418, 379–390 (2009).

17. Lin, S. C. et al. Molecular basis for the unique deubiquitinating activity of the NF-κB inhibitor A20. J. Mol. Biol. 376, 526–540 (2008).

18. Nanao, M. H. et al. Crystal structure of human otubain 2. EMBO Rep. 5, 783–788 (2004).

19. Messick, T. E. et al. Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein. J. Biol. Chem. 283, 11038–11049 (2008).

20. Johnston, S. C., Larsen, C. N., Cook, W. J., Wilkinson, K. D. & Hill, C. P. Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution. EMBO J. 16, 3787–3796 (1997).

21. Johnston, S. C., Riddle, S. M., Cohen, R. E. & Hill, C. P. Structural basis for the specificity of ubiquitin C-terminal hydrolases. EMBO J. 18, 3877–3887 (1999).

22. Komander, D. et al. Molecular discrimination of structurally equivalent Lys63-linked and linear polyubiquitin chains. EMBO Rep. 5, 466–473 (2009).First survey of ubiquitin chain linkage specificities across DUB families.

23. Popp, M. W., Artavanis-Tsakonas, K. & Ploegh, H. L. Substrate filtering by the active site crossover loop in UCHL3 revealed by sortagging and gain-of-function mutations. J. Biol. Chem. 284, 3593–3602 (2009).The active site crossover loop in UCHL3 requires extension to allow polyubiquitin chain cleavage.

24. Larsen, C. N., Krantz, B. A. & Wilkinson, K. D. Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases. Biochemistry 37, 3358–3368 (1998).

25. Riess, O., Rub, U., Pastore, A., Bauer, P. & Schols, L. SCA3: neurological features, pathogenesis and animal models. Cerebellum 7, 125–137 (2008).

26. Nicastro, G. et al. The solution structure of the Josephin domain of ataxin-3: structural determinants for molecular recognition. Proc. Natl Acad. Sci. USA 102, 10493–10498 (2005).Together with references 27 and 28, this paper reveals the large conformational changes exhibited by the Josephin family of DUBs.

27. Mao, Y. et al. Deubiquitinating function of ataxin-3: insights from the solution structure of the Josephin domain. Proc. Natl Acad. Sci. USA 102, 12700–12705 (2005).

28. Nicastro, G. et al. The josephin domain of ataxin-3 contains two distinct ubiquitin binding motifs. Biopolymers 20 Apr 2009 (doi:10.1002/bip.21210).

29. Sato, Y. et al. Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains. Nature 455, 358–362 (2008).The first structure of a DUB with a diubiquitin bound across the active site. It revealed the mechanism of action of JAMM/MPN+ proteases and suggested a rationale for the Lys63-linked ubiquitin chain specificity of AMSH-LP.

30. Tran, H. J., Allen, M. D., Lowe, J. & Bycroft, M. Structure of the Jab1/MPN domain and its implications for proteasome function. Biochemistry 42, 11460–11465 (2003).

31. Maytal-Kivity, V., Reis, N., Hofmann, K. & Glickman, M. H. MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function. BMC Biochem. 3, 28 (2002).

32. Yao, T. & Cohen, R. E. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407 (2002).

33. Cope, G. A. et al. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 298, 608–611 (2002).

34. McCullough, J. et al. Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery. Curr. Biol. 16, 160–165 (2006).

35. Dong, Y. et al. Regulation of BRCC, a holoenzyme complex containing BRCA1 and BRCA2, by a signalosome-like subunit and its role in DNA repair. Mol. Cell 12, 1087–1099 (2003).

36. Wang, B. & Elledge, S. J. Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage. Proc. Natl Acad. Sci. USA 104, 20759–20763 (2007).

37. Shao, G. et al. The Rap80–BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8–Ubc13-dependent ubiquitination events at DNA double strand breaks. Proc. Natl Acad. Sci. USA 106, 3166–3171 (2009).

38. Cooper, E. M. et al. K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1. EMBO J. 28, 621–631 (2009).

39. Drag, M. et al. Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes). Biochem. J. 415, 367–375 (2008).

40. Catic, A. et al. Screen for ISG15-crossreactive deubiquitinases. PLoS ONE 2, e679 (2007).

41. Frias-Staheli, N. et al. Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses. Cell Host Microbe 2, 404–416 (2007).

42. Malakhov, M. P., Malakhova, O. A., Kim, K. I., Ritchie, K. J. & Zhang, D. E. UBP43 (USP18) specifically removes ISG15 from conjugated proteins. J. Biol. Chem. 277, 9976–9981 (2002).

43. Gong, L., Kamitani, T., Millas, S. & Yeh, E. T. Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDD8-conjugated proteins. J. Biol. Chem. 275, 14212–14216 (2000).

44. Tokunaga, F. et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nature Cell Biol. 11, 123–132 (2009).

45. Rahighi, S. et al. Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation. Cell 136, 1098–1109 (2009).

46. Huang, T. T. et al. Regulation of monoubiquitinated PCNA by DUB autocleavage. Nature Cell Biol. 8, 339–347 (2006).

47. Wang, T. et al. Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1. J. Mol. Biol. 386, 1011–1023 (2009).

48. Tran, H., Hamada, F., Schwarz-Romond, T. & Bienz, M. Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains. Genes Dev. 22, 528–542 (2008).

49. Kayagaki, N. et al. DUBA: a deubiquitinase that regulates type I interferon production. Science 318, 1628–1632 (2007).

50. McCullough, J., Clague, M. J. & Urbe, S. AMSH is an endosome-associated ubiquitin isopeptidase. J. Cell Biol. 166, 487–492 (2004).First direct demonstration of in vitro chain linkage specificity and proposed function in regulating receptor fate.

protein kinase signalling is negatively regulated by the activity of USP17-like protein 2 (also known as DUB3), apparently by deubiquitylating and thereby inhibiting ras converting enzyme 1 (rCE1)132.

Concluding remarksUbiquitin is a key regulator of many cellular physio-logical processes. The influence of DUBs is correspond-ingly broad. Structural studies across all DUB families have provided insight into the means by which selectivity is achieved for different classes of ubiquitin chain sub-strate. Many DUBs need to undergo structural rearrange-ments to adopt an active conformation, suggesting a high degree of regulation. Functional studies are providing an

inventory of physiologically relevant substrates and link-ing them to many pathological conditions. As enzymes, DUBs are attractive drug targets that are likely to provide a major focus for future pharmacological intervention strategies82–84.

Important issues to be addressed in future studies include broadening the characterization of chain spe-cificity beyond Lys48- and Lys63-linked polyubiquitin, understanding the interplay between multiple DUBs that associate with specific macromolecular complexes (for example, the proteasome or the ESCrT machinery) and defining the means by which the role of DUBs in highly choreographed events (for example, the cell cycle) is coordinated.

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51. Nakamura, M., Tanaka, N., Kitamura, N. & Komada, M. Clathrin anchors deubiquitinating enzymes, AMSH and AMSH-like protein, on early endosomes. Genes Cells 11, 593–606 (2006).

52. Winborn, B. J. et al. The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains. J. Biol. Chem. 283, 26436–26443 (2008).

53. Al-Hakim, A. K. et al. Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-linked polyubiquitin chains. Biochem. J. 411, 249–260 (2008).

54. Reyes-Turcu, F. E. et al. The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 124, 1197–1208 (2006).Defines a new mode of ubiquitin recognition and allosteric regulation of DUB activity.

55. Amerik, A., Swaminathan, S., Krantz, B. A., Wilkinson, K. D. & Hochstrasser, M. In vivo disassembly of free polyubiquitin chains by yeast Ubp14 modulates rates of protein degradation by the proteasome. EMBO J. 16, 4826–4838 (1997).

56. Hunter, T. The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol. Cell 28, 730–738 (2007).

57. Reiley, W., Zhang, M., Wu, X., Granger, E. & Sun, S. C. Regulation of the deubiquitinating enzyme CYLD by IκB kinase gamma-dependent phosphorylation. Mol. Cell. Biol. 25, 3886–3895 (2005).

58. Mizuno, E., Kitamura, N. & Komada, M. 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase. Exp. Cell Res. 313, 3624–3634 (2007).

59. Pohl, C. & Jentsch, S. Final stages of cytokinesis and midbody ring formation are controlled by BRUCE. Cell 132, 832–845 (2008).

60. Mukai, A. et al. Dynamic regulation of ubiquitylation and deubiquitylation at the central spindle during cytokinesis. J. Cell Sci. 121, 1325–1333 (2008).

61. Todi, S. V. et al. Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3. EMBO J. 28, 372–382 (2009).

62. Meulmeester, E., Kunze, M., Hsiao, H. H., Urlaub, H. & Melchior, F. Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25. Mol. Cell 30, 610–619 (2008).

63. Ross, S. H. et al. Differential redox regulation within the PTP superfamily. Cell Signal. 19, 1521–1530 (2007).

64. Enesa, K. et al. Hydrogen peroxide prolongs nuclear localization of NF-κB in activated cells by suppressing negative regulatory mechanisms. J. Biol. Chem. 283, 18582–18590 (2008).Suggests that DUBs of the NF-κB pathway might be targets of reactive oxygen species.

65. Coornaert, B. et al. T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-κB inhibitor A20. Nature Immunol. 9, 263–271 (2008).

66. Row, P. E. et al. The MIT domain of UBPY constitutes a CHMP binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation. J. Biol. Chem. 282, 30929–30937 (2007).

67. Iha, H. et al. Inflammatory cardiac valvulitis in TAX1BP1-deficient mice through selective NF-κB activation. EMBO J. 27, 629–641 (2008).

68. Wagner, S. et al. Ubiquitin binding mediates the NF-κB inhibitory potential of ABIN proteins. Oncogene 27, 3739–3745 (2008).

69. Yao, T. et al. Distinct modes of regulation of the Uch37 deubiquitinating enzyme in the proteasome and in the Ino80 chromatin-remodeling complex. Mol. Cell 31, 909–917 (2008).

70. Kimura, Y. et al. An inhibitor of a deubiquitinating enzyme regulates ubiquitin homeostasis. Cell 137, 549–559 (2009).

71. Ventii, K. H. & Wilkinson, K. D. Protein partners of deubiquitinating enzymes. Biochem. J. 414, 161–175 (2008).

72. Cohn, M. A. et al. A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway. Mol. Cell 28, 786–797 (2007).Together with reference 73, this paper shows that a WD40 protein can allosterically activate three DUBs of the USP family.

73. Cohn, M. A., Kee, Y., Haas, W., Gygi, S. P. & D’Andrea, A. D. UAF1 is a subunit of multiple deubiquitinating enzyme complexes. J. Biol. Chem. 8, 5343–5351 (2008).

74. Sowa, M. E., Bennett, E. J., Gygi, S. P. & Harper, J. W. Defining the human deubiquitinating enzyme interaction landscape. Cell 16 Jul 2009 (doi: 10.1016/j.cell.2009.04.042).Comprehensive proteomic study of the interaction profiles of 75 human DUBs.

75. van der Knaap, J. A. et al. GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7. Mol. Cell 17, 695–707 (2005).

76. Row, P. E., Prior, I. A., McCullough, J., Clague, M. J. & Urbe, S. The ubiquitin isopeptidase UBPY regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation. J. Biol. Chem. 281, 12618–12624 (2006).

77. Blagoev, B., Ong, S. E., Kratchmarova, I. & Mann, M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nature Biotech. 22, 1139–1145 (2004).

78. Nakamura, N. & Hirose, S. Regulation of mitochondrial morphology by USP30, a deubiquitinating enzyme present in the mitochondrial outer membrane. Mol. Biol. Cell 19, 1903–1911 (2008).

79. Endo, A. et al. Nucleolar structure and function are regulated by the deubiquitylating enzyme USP36. J. Cell Sci. 122, 678–686 (2009).

80. Lauwers, E., Jacob, C. & Andre, B. K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway. J. Cell Biol. 3, 493–502 (2009).

81. Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).

82. Daviet, L. & Colland, F. Targeting ubiquitin specific proteases for drug discovery. Biochimie 90, 270–283 (2008).

83. Nicholson, B., Marblestone, J. G., Butt, T. R. & Mattern, M. R. Deubiquitinating enzymes as novel anticancer targets. Future Oncol. 3, 191–199 (2007).

84. Hoeller, D. & Dikic, I. Targeting the ubiquitin system in cancer therapy. Nature 458, 438–444 (2009).

85. Graner, E. et al. The isopeptidase USP2a regulates the stability of fatty acid synthase in prostate cancer. Cancer Cell 5, 253–261 (2004).This paper, together with references 86 and 87, highlights the potential relevance of DUBs as therapeutic drug targets.

86. Popov, N. et al. The ubiquitin-specific protease USP28 is required for MYC stability. Nature Cell Biol. 9, 765–774 (2007).

87. Cummins, J. M. & Vogelstein, B. HAUSP is required for p53 destabilization. Cell Cycle 3, 689–692 (2004).

88. Stegmeier, F. et al. Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 446, 876–881 (2007).Highlights the crucial role for USP44 in the progression of the cell cycle.

89. Huang, F., Kirkpatrick, D., Jiang, X., Gygi, S. & Sorkin, A. Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol. Cell 21, 737–748 (2006).

90. Clague, M. J. & Urbe, S. Endocytosis: the DUB version. Trends Cell Biol. 16, 551–559 (2006).

91. Butterworth, M. B. et al. The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel. J. Biol. Chem. 282, 37885–37893 (2007).

92. Mizuno, E., Kobayashi, K., Yamamoto, A., Kitamura, N. & Komada, M. A deubiquitinating enzyme UBPY regulates the level of protein ubiquitination on endosomes. Traffic 7, 1017–1031 (2006).

93. Boulkroun, S. et al. Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3. Am. J. Physiol. Renal Physiol. 295, F889–F900 (2008).

94. Li, Z. et al. Ubiquitination of a novel deubiquitinating enzyme requires direct binding to von Hippel-Lindau tumor suppressor protein. J. Biol. Chem. 277, 4656–4662 (2002).

95. Lu, Y. et al. USP19 deubiquitinating enzyme supports cell proliferation by stabilizing KPC1, a ubiquitin ligase for p27Kip1. Mol. Cell Biol. 29, 547–558 (2009).

96. Cao, Z., Wu, X., Yen, L., Sweeney, C. & Carraway, K. L. Neuregulin-induced ErbB3 downregulation is mediated by a protein stability cascade involving the E3 ubiquitin ligase Nrdp1. Mol. Cell. Biol. 27, 2180–2188 (2007).

97. Brummelkamp, T. R., Nijman, S. M., Dirac, A. M. & Bernards, R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature 424, 797–801 (2003).Together with references 98 and 99, this study put the spotlight on the tumour suppressor function of a DUB.

98. Trompouki, E. et al. CYLD is a deubiquitinating enzyme that negatively regulates NF-κB activation by TNFR family members. Nature 424, 793–796 (2003).

99. Kovalenko, A. et al. The tumour suppressor CYLD negatively regulates NF-κB signalling by deubiquitination. Nature 424, 801–805 (2003).

100. Brooks, C. L., Li, M., Hu, M., Shi, Y. & Gu, W. The p53–Mdm2–HAUSP complex is involved in p53 stabilization by HAUSP. Oncogene 26, 7262–7266 (2007).

101. Heyninck, K. & Beyaert, R. A20 inhibits NF-κB activation by dual ubiquitin-editing functions. Trends Biochem. Sci. 30, 1–4 (2005).

102. Verma, R. et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615 (2002).

103. Koulich, E., Li, X. & Demartino, G. N. Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome. Mol. Biol. Cell 19, 1072–1082 (2008).

104. Crosas, B. et al. Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities. Cell 127, 1401–1413 (2006).

105. Leggett, D. S. et al. Multiple associated proteins regulate proteasome structure and function. Mol. Cell 10, 495–507 (2002).

106. Yao, T. et al. Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1. Nature Cell Biol. 8, 994–1002 (2006).

107. Hanna, J., Meides, A., Zhang, D. P. & Finley, D. A ubiquitin stress response induces altered proteasome composition. Cell 129, 747–759 (2007).

108. Hanna, J. et al. Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell 127, 99–111 (2006).

109. Amerik, A. Y., Nowak, J., Swaminathan, S. & Hochstrasser, M. The DoA4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways. Mol. Biol. Cell 11, 3365–3380 (2000).

110. Dayal, S. et al. Suppression of the deubiquitinating enzyme USP5 causes the accumulation of unanchored polyubiquitin and the activation of p53. J. Biol. Chem. 8, 5030–5041 (2008).

111. Amerik, A. Y., Li, S. J. & Hochstrasser, M. Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae. Biol. Chem. 381, 981–992 (2000).

112. Kirkin, V. & Dikic, I. Role of ubiquitin- and Ubl-binding proteins in cell signaling. Curr. Opin. Cell Biol. 19, 199–205 (2007).

113. Mueller, R. D., Yasuda, H., Hatch, C. L., Bonner, W. M. & Bradbury, E. M. Identification of ubiquitinated histones 2A and 2B in Physarum polycephalum. Disappearance of these proteins at metaphase and reappearance at anaphase. J. Biol. Chem. 260, 5147–5153 (1985).

114. Zhu, P. et al. A histone H2A deubiquitinase complex coordinating histone acetylation and H1 dissociation in transcriptional regulation. Mol. Cell 27, 609–621 (2007).

115. Joo, H. Y. et al. Regulation of cell cycle progression and gene expression by H2A deubiquitination. Nature 449, 1068–1072 (2007).

116. Nicassio, F. et al. Human USP3 is a chromatin modifier required for S phase progression and genome stability. Curr. Biol. 22, 1972–1977 (2007).

117. Zhang, X. Y. et al. The putative cancer stem cell marker USP22 is a subunit of the human SAGA complex required for activated transcription and cell-cycle progression. Mol. Cell 29, 102–111 (2008).

118. Nijman, S. M. et al. The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway. Mol. Cell 17, 331–339 (2005).A nice example of the application of an siRNA library screen to identify USP1 as a regulator of FANCD2.

119. Oestergaard, V. H. et al. Deubiquitination of FANCD2 is required for DNA crosslink repair. Mol. Cell 28, 798–809 (2007).

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120. Chiu, Y. H., Zhao, M. & Chen, Z. J. Ubiquitin in NF-κB signaling. Chem. Rev. 4, 1549–1560 (2009).

121. Sun, S. C. Deubiquitylation and regulation of the immune response. Nature Rev. Immunol. 8, 501–511 (2008).

122. Boone, D. L. et al. The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses. Nature Immunol. 5, 1052–1060 (2004).

123. Lee, E. G. et al. Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice. Science 289, 2350–2354 (2000).

124. Compagno, M. et al. Mutations of multiple genes cause deregulation of NF-κB in diffuse large B-cell lymphoma. Nature 459, 717–721 (2009).

125. Kato, M. et al. Frequent inactivation of A20 in B-cell lymphomas. Nature 459, 712–716 (2009).

126. Schmitz, R. et al. TNFAIP3 (A20) is a tumor suppressor gene in Hodgkin lymphoma and primary mediastinal B cell lymphoma. J. Exp. Med. 5, 981–989 (2009).

127. Wertz, I. E. et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430, 694–699 (2004).First description of ubiquitin chain editing by a DUB.

128. Shembade, N. et al. The E3 ligase Itch negatively regulates inflammatory signaling pathways by controlling the function of the ubiquitin-editing enzyme A20. Nature Immunol. 9, 254–262 (2008).

129. Shembade, N., Parvatiyar, K., Harhaj, N. S. & Harhaj, E. W. The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling. EMBO J. 5, 513–522 (2009).

130. Enesa, K. et al. NF-κB suppression by the deubiquitinating enzyme Cezanne: a novel negative feedback loop in pro-inflammatory signaling. J. Biol. Chem. 283, 7036–7045 (2008).

131. Dupont, S. et al. FAM/USP9x, a deubiquitinating enzyme essential for TGFβ signaling, controls Smad4 monoubiquitination. Cell 136, 123–135 (2009).

132. Burrows, J. F. et al. USP17 regulates Ras activation and cell proliferation by blocking RCE 1 activity. J. Biol. Chem. 14, 9587–9595 (2009).

133. Rigden, D. J., Liu, H., Hayes, S. D., Urbé, S. & Clague, M. J. Ab initio protein modelling reveals novel human MIT domains. FEBS Lett. 583, 872–878 (2009).

134. Burrows, J. F., McGrattan, M. J. & Johnston, J. A. The DUB/USP17 deubiquitinating enzymes, a multigene family within a tandemly repeated sequence. Genomics 85, 524–529 (2005).

135. Quesada, V. et al. Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases. Biochem. Biophys. Res. Commun. 314, 54–62 (2004).

136. Pena, V., Liu, S., Bujnicki, J. M., Luhrmann, R. & Wahl, M. C. Structure of a multipartite protein-protein interaction domain in splicing factor prp8 and its link to retinitis pigmentosa. Mol. Cell 25, 615–624 (2007).

137. Misaghi, S. et al. Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate. J. Biol. Chem. 280, 1512–1520 (2005).

138. Ye, Y., Scheel, H., Hofmann, K. & Komander, D. Dissection of USP catalytic domains reveals five common insertion points. Mol. Biosyst. 17 Jul 2009 (doi:10.1039/b907669g)

AcknowledgementsWe thank K. Hofmann and D. Rigden for bioinformatic dis-cussions and advice. S.U. is a Cancer Research UK Senior Research Fellow.

DATABASESUniProtKB: http://www.uniprot.orgA20 | AMSH | AMSH-LP | ATXN3 | CDC20 | Cezanne | CSN5 | CYLD | Doa4 | DUB3 | FANCD2 | ISG15 | MALT1 | NEDD8 | Otu1 | OTUB1 | OTUB2 | OTUD5 | PCNA | POH1 | SNX3 | STAM2 | TAX1BP1 | TRABID | TRAF2 | UCH37 | UCHL1 | UCHL3 | USP1 | USP2 | USP3 | USP5 | USP7 | USP8 | USP9X | USP10 | USP12 | USP13 | USP14 | USP16 | USP18 | USP19 | USP21 | USP22 | USP25 | USP28 | USP30 | USP36 | USP44 | USP46 | VCPIP1

FURTHER INFORMATIONDavid Komander’s homepage: http://www2.mrc-lmb.cam.ac.uk/PNAC/Komander_DMichael J. Clague’s homepage: http://pcwww.liv.ac.uk/~clagueSylvie Urbé’s homepage: http://pcwww.liv.ac.uk/~urbeHHPRED: http://toolkit.tuebingen.mpg.de/hhpredInterpro: http://www.ebi.ac.uk/interpro Pfam: http://www.sanger.ac.uk/Software/PfamPROSITE: http://www.expasy.ch/prosite PSORTII: http://psort.hgc.jp/form2.htmlSMART: http://smart-heidelberg.de RefSeq: http://www.ncbi.nlm.nih.gov/RefSeq

SUPPLEMENTARY INFORMATIONSee online article: S1 (figure) | S2 (figure) | S3 (figure) | S4 (figure) | S5 (figure) | S6 (figure) | S7 (table) | S8 (figure) | S9 (table) | S10 (box) | S11 (box)

all liNkS are acTive iN THe oNliNe PDf

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NATUrE rEVIEWS | Molecular cell Biology VOLUME 10 | AUGUST 2009 | 563

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