Cell Science at a Glance 2325

DUBs at a glance Rose the Nobel Prize in Chemistry in 2004 (DUBs) (Amerik and Hochstrasser, 2004; (Wilkinson, 2004). This early work D’Andrea and Pellman, 1998; Wilkinson, Keith D. Wilkinson spawned a large number of studies that 1997). Enzymes that reverse the Department of Biochemistry, Emory University investigated the role of and modification by UBLs are similarly School of Medicine, Atlanta, GA 30322, USA several other ubiquitin-like named: desumoylating enzymes remove e-mail: [email protected] (UBLs) as targeting signals in virtually all SUMO (small ubiquitin-related modifier), Journal of Cell Science 122, 2325-2329 aspects of cellular metabolism deneddylating enzymes remove NEDD8 Published by The Company of Biologists 2009 (Chen, 2005; Cohn and D’Andrea, 2008; (neural precursor cell expressed, doi:10.1242/jcs.041046 Saksena et al., 2007; Weake and Workman, developmentally downregulated 8) and de- 2008). The best understood example of ISGylating enzymes remove ISG15 ubiquitylation is the marking of proteins (interferon-stimulated product 15). Introduction for delivery to the 26S proteasome, The discovery of protein ubiquitylation resulting in their degradation (Chiba and There are many fine reviews describing the three decades ago was the beginning of our Tanaka, 2004; Guo et al., 2007; Hershko complex enzymatic mechanisms that understanding of a new mechanism by and Ciechanover, 1998; Schwartz and regulate the conjugation of ubiquitin and which proteins are marked for assembly Hochstrasser, 2003; Varshavsky et al., UBLs to target proteins; the reader is into macromolecular complexes or 1989). referred to these for details of the movement between cellular compartments. ubiquitylation machinery (Belgareh-Touze The finding that ubiquitylation (the Similar to all regulated targeting pathways, et al., 2008; Dye and Schulman, 2007; covalent attachment of the small protein the process of ubiquitylation is reversible. Hochstrasser, 2007; Starita and Parvin, ubiquitin to other proteins) targeted The enzymes that reverse the modification 2006). Briefly, ubiquitin is first thiol- proteins for degradation earned Avram of proteins by ubiquitin are collectively esterified at its C-terminus by the action of Hershko, Aaron Ciechanover and Irwin known as deubiquitylating enzymes the E1 ubiquitin-activating enzyme and DUBs at a Glance Keith D. Wilkinson

Substrate-specificity of protease families DUBs both stimulate K63-specific DUBs K63-linked ubiquitin used in Deubiquitylation of histones regulates acting on ubiquitin-like proteins and inhibit proteolysis in NF-␬B signaling endocytosis and cargo sorting transcription and chromatin dynamics

DUB protein family Ligand L Activity L USP UCH OTU JAMM MJD SENP Receptor L Substrate Elongation and Initiation of Deubiquitylating Ub Ub Ub Multiple DUBs? E1, Ubc13/Uev1A AMSH termination transcription (isopeptidase) PolyUb PolyUb PolyUb PolyUb ProUb TRAF TRAF AMSH? complex A20, CYLD complex Cbl E1,E2,E3 Deneddylating Nedd8 Nedd8 Nedd8 Nedd8 USP5 T β N A20 A T E Multiple B A USP14 2/3 αM SAGA DeISGylating ISG15 K O L UCH37 DUBs? 1 USP22•SAGA K4-Me3 RIP1 USP8 Desumoylating SUMO Substrate ATP Early endosome H3 H2B TF- H2B POH1 CYLD, A20 assoc. Ub ligases MLL3 AMSH? N Substrate POH1 β E USP3 DUBs remodel and disassemble αM UCH37 O H3 polyubiquitin chains Proteasome USP21 β ATP Journal of Cell Science Lid TrCP Corepressor- Aurora B Receptor 19S Peptides κ κ H2A H2A Protein E1,E2,E3 I B I B assoc. Ub ligases regulator NF-κB NF-κB S10-P Base κ USP16 ProUb Ub Ubiquitylated NF- B Lysosome H3 protein α Multivesicular body USP14 β 20S 2A-DUB•PCAF β protease Altered KEY PolyUb α Silencing and chromatin localization, K48-linked polyubiquitin Reaction USP7•GMP synthetase binding, Proteasome condensation K63-linked polyubiquitin Influence stability or activity Monoubiquitin DUB activity

DUBs regulate DNA repair and the stability of multiple proteins at cell-cycle checkpoints

κ Spindle checkpoint A20 Ub ligase and DUB for RIP1 Deletion causes prolonged NF- B Cdc25-mediated checkpoint arrest responses and inflammation Cdh1 attachment APC/C Polyglutamine repeat expansion causes Cdc14B Cdc20 Ataxin-3 K63-linked polyubiquitin APC/C MAD2 p31 type 3 Spinal Cerebellar Ataxia Ub- Ub- MAD2• Plk1 Claspin Ub- Ub- APC/CCdc20 Ubiquitylated Cyclin E Myc Cdc20 Cdc20 BAP1, USP11 BRCA1/2 DNA-repair pathways; cancer Cdh1 USP28 APC/C APC/C ? APC/C Cdc25 ? (Inactive) USP44 ? Claspin phosphatase (Active) Fbw7α USP28 M K63-ubiquitylated TRAFs, Plk1 SCF CYLD Mutation causes benign tumors and failure to Wee1 Cdk1- pCdk1- pCdk2- Cdk2- Myc G1 κ Claspin cyclin B1 cyclin B1 cyclin E cyclin E RIP1, others downregulate NF- B and JNK signaling p-Wee1 βTrcpp pCdh1 G2 p53-mediated arrest ? SCF USP28 APC/C Common cytokine receptor, Overexpression increases substrate half-life, DUB1, DUB2 gamma chain prolongs cytokine response Ub- Ub- G2/M S-phase Proliferation ATM, CHK2 transition G2 block S block progression and apoptosis Wee1 Claspin MDMX pMDMX Mutation causes gracile axonal dystrophy in mice; UCH-L1 ? S P53 MDMX/ accumulated in neuronal inclusion bodies in humans MDM2 pCdh1 APC/C CIP/WAF1 USP7 p21 USP7 USP1 PCNA, FANCD2 Mutated in Fanconi Anemia; involved in DNA repair USP7 Cdk2/ Ub- Fatty acid synthesase? Involved in prostate cancer; APC/C Cyclin E protein USP2A PCNA-mediated DNA repair MDM2? protects from apoptosis BAP1 Cell-cycle arrest Crosslink repair FANCD2 FANCD2 USP3 USP4 Rb? Ro52 Oncoprotein linked to lung cancer and Sjogren’s syndrome USP1/UAF1 Pol η Double-strand-break DNA repair Oncogenic when overexpressed from USP6/TRE17 ? Translesion DNA damage 17p13 translocations synthesis Brcc36 H2AX H2AX RAD18 Role in DNA repair and oxidative PCNA- ABRA1 USP7/HAUSP p53, MDM2, FOXO RAD6 Checkpoint RAD5, Ubc13/Uev1 bound stress response ? leision X RAP 80 Proteasome-bound Mutated in ataxic mouse; UBP14 Error-free synthesis USP1 polyubiquitin ubiquitin depletion phenotype degradation Proteolysis USP3 Double-strand-break BRCA1/BARD1 USP33/VDU1, Regulation of HIF-1α; role in angiogenesis repair pVHL ubiquitin ligase USP20/VDU2 and metastasis

Abbreviations: 2A-DUB, histone H2A deubiquitinase; ABRA1, abraxas protein forming a complex required for double- factor that is a homolog of the Myelocytomatosis viral oncogene; Nedd8, neural precursor cell expressed, developmentally strand-break DNA repair; AMSH, associated molecule with the SH3 domain of STAM; APC/C, anaphase-promoting downregulated 8; NEMO, NF-κB essential modulator; NF-␬B, nuclear factor-κB; OTU, a DUB family of proteins with the ovarian complex/cyclosome [activity requires co-activators Cdc20 (cell division cycle 20) or Cdh1 (Cdc20 homolog 1) whose identity tumor domain (Pfam PF02338, EC 3.1.2.-); PCAF, p300/CBP-associated factor; PCNA, proliferating cell nuclear antigen; Plk1, is shown as a superscript]; ATM, ataxia telangiectasia, mutated homolog; BARD1, BRCA1-associated RING domain 1 polo-like kinase 1; POH1, DUB of the 19S lid complex; Pol η, DNA polymerase eta; ProUb, the pro-protein gene products of (heterodimerizes with BRCA1 to form an active ubiquitin ligase); BRCA1, breast cancer 1 tumor suppressor; BRCC36, ubiquitin-encoding ; pVHL, von Hippel-Lindau tumor suppressor; RAD, radiation sensitive (genes required for DNA repair BRCA1/BRCA2-containing complex subunit 36; βTrCP, β-transducin repeat-containing homolog protein; Cbl, Casitas functions); RAP80, required for double-strand-break DNA repair; Rb, retinablastoma tumor suppressor; RIP1, receptor-interacting B-lineage lymphoma; Cdk, cyclin-dependent kinase; Chk2, checkpoint kinase 2; DUB, deubiquitylating enzyme; E1, a protein 1; SAGA, Spt-Ada-Gcn5-acetyltransferase complex; SCF, a class of ubiquitin ligases consisting of Skp1, a and an ubiquitin-activating enzyme; E2, a ubiquitin-conjugating enzyme; E3, a ubiquitin ligase; FANCD2, product of the Fanconi F-box protein whose identity is indicated by a superscript; SENP, a family of human SUMO- or Nedd8-specific proteses with the anemia, complementation group D2 gene; FOXO, forkhead transcription factor O; H2A, histone H2A; H2B, histone H2B; ubiquitin-like protease domain (Pfam PF02902, EC 3.4.22); SUMO, small ubiquitin-related modifier; TAB2/3, TAK1-binding protein H2AX, histone H2AX; H3, histone H3; HIF-1α, hypoxia-inducible factor 1α; IκB, inhibitor of NF-κB; ISG15, product of the 2/3; TAK1, TGFβ-activated kinase 1; TF, transcription factor; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; interferon-stimulated gene 15; JAMM, a DUB family of proteins with the Jab1/MPN metalloenzyme domain (Pfam PF01398, UAF1, USP1-associated factor 1; Ub, ubiquitin; Ubc13/Uev1, heterodmeric E2 ubiquitin-conjugating enzyme that synthesizes EC 3.1.2.15); L, ligand; MAD2, mitotic arrest-deficient 2 protein inhibitor of APC/CCdc20; MDM2, murine double minute 2; K63-linked polyubiquitin chains; UCH, a DUB family of proteins with the ubiquitin C-terminal hydrolase domain (Pfam PF01088, MDMX, MDM2-like p53-binding protein; MJD, a DUB family of proteins with the Machado-Joseph Disease protein domain EC,3.4.19.12); USP, a DUB family of proteins with the ubiquitin-specific protease domain (Pfam PF00443, EC 3.1.2.15). © (Pfam PF02099, EC 3.4.22.-); MLL3, myeloid/lymphoid or mixed-lineage leukemia protein 3 complex; Myc, a transcripion Journal of Cell Science 2009 (122, pp. 2325-2329)

(See poster insert) 2326 Journal of Cell Science 122 (14)

then is subsequently transferred to one of et al., 2007; Kirkpatrick et al., 2005; of specific DUBs induces very limited several E2 ubiquitin-conjugating enzymes Mayor et al., 2005; Xu and Peng, 2008), and specific cellular phenotypes and that act as carrier proteins. Finally, although the specific pathways in which pathologies (Shanmugham and Ovaa, ubiquitin is transferred to a residue these more complex polyubiquitin chains 2008; Singhal et al., 2008). For example, of the target protein through the combined are involved remain poorly understood. the mutation or deletion of the major action of the E2-ubiquitin thiol ester and neuronal DUB in mammals, UCH-L1 one of hundreds of E3 ubiquitin ligases. In this article and its accompanying poster, (ubiquitin C-terminal hydrolase L1), The ubiquitylation signal that is attached I summarize our understanding of the causes a localized axonal dystrophy but can consist of a single ubiquitin, multiple metabolic function of DUBs and discuss few other overt effects (Setsuie and Wada, or a polyubiquitin chain in their roles in regulating several ubiquitin- 2007). A benign tumor syndrome of hair which successive ubiquitin molecules are dependent processes. Here, I use the term follicles known as cylindromatosis is assembled by the ubiquitylation of DUBs to refer only to those enzymes that caused by the mutation of CYLD, a USP- ubiquitin itself (Baboshina and Haas, 1996; act on ubiquitin. Much less is known family DUB named after the disease it Chau et al., 1989; Hofmann and Pickart, about the enzymes that act on UBLs (Hay, causes. Although the major defects caused 1999; Koegl et al., 1999; Tokunaga et al., 2007; Love et al., 2007; Mikolajczyk by mutation of CYLD are limited to the 2009; Wu-Baer et al., 2003). Because any et al., 2007; Reverter et al., 2005; nuclear factor-κB (NF-κB) pathway, of the seven lysine residues of ubiquitin, or Sulea et al., 2006) and they will not be (Courtois, 2008) this DUB has also been its amino terminus, can be modified by discussed here. Although much of what is shown to have important roles in cell-cycle a subsequent ubiquitin to form a known about DUBs was first observed in regulation (Stegmeier et al., 2007). polyubiquitin chain, there is a huge yeast, the yeast pathways or enzyme names Interfering with the function of USP1 variation in the structure of polyubiquitin are not emphasized. The poster illustrates mainly causes DNA-repair defects (Cohn signals that can be attached. A the role of over 20 of nearly 100 and D’Andrea, 2008), whereas deleting polyubiquitin chain can involve linkages to mammalian DUBs that act on ubiquitin USP14 in mice results in ataxia, a the same lysine residue on each ubiquitin (Nijman et al., 2005), and concentrates on movement disorder characterized by moiety to yield a homogeneous chain, or it the DUBs about which something is uncoordinated motions (Crimmins et al., can involve linkages to different lysine known regarding their physiology or 2006). residues on different ubiquitin moeities, pathology. For pathways where the which results in a heterogeneous linear or substrate or the process regulated is known A significant aspect of specificity is the branched chain. in some detail, specific examples are ability of DUBs to recognize and act on provided. It should be noted that a role for different types of polyubiquitin. The Ubiquitylation is a versatile and dynamic DUBs has been implied in many other catalytic domain of all DUBs contains a targeting signal. The use of a protein, rather contexts, such as apoptosis, Parkinson’s binding site for ubiquitin, and several than a small molecule, to modify a target disease and neuronal-inclusion-body DUBs bind ubiquitin at submicromolar protein confers a large interaction surface diseases, although in many cases the concentrations. Many other DUBs,

Journal of Cell Science that can be recognized by specific precise DUB involved has not been however, bind ubiquitin only very weakly receptors. In addition, the many different identified. Space limitations restrict the (Reyes-Turcu and Wilkinson, 2009). Some polymeric forms of ubiquitin allow for inclusion of these aspects in the poster. DUBs have additional binding sites with structural variation of the signal. affinity for the target protein that is Structurally different forms of DUBs are numerous and specific ubiquitylated (Ventii and Wilkinson, polyubiquitin are thought to target proteins The nearly 100 putative mammalian DUBs 2008); for example, USP7 binds to a for different cellular fates. For example, are grouped into five different families peptide sequence present in its substrates early work showed that K48-, K29-, and (Amerik and Hochstrasser, 2004; D’Andrea p53, MDM2 (murine double minute 2, an K11-linked polyubiquitin chains can target and Pellman, 1998; Wilkinson, 1997). Four oncoprotein) and the Epstein Barr nuclear proteins for degradation by the proteasome of these families are thiol proteases: antigen-1 (Hu et al., 2006). It is clear that (Chau et al., 1989; Jin et al., 2008; Koegl the ubiquitin-C-terminal hydrolases (UCHs), differently linked polyubiquitin chains et al., 1999); K63-linked polyubiquitin ubiquitin-specific proteases (USPs), have different structures, and it is thought chains participate in DNA repair and ovarian-tumor (OTU) domain DUBs and that some DUBs can distinguish between signaling kinase complexes (Deng et al., Machado-Joseph domain (MJD) DUBs. them. For example, the DUBs CYLD and 2000; Spence et al., 1995); monoubiquitin The DUBs of a fifth family contain a A20, which are involved in and K63-linked chains are involved in Jab1/MPN metalloenzyme (JAMM) downregulating the NF-κB response, only targeting cell-surface proteins for domain and act as zinc-dependent disassemble K63-linked polyubiquitin internalization and endosomal sorting metalloproteases. chains, the type that is assembled on the (Hicke and Riezman, 1996; Springael signaling components of the NF-κB et al., 1999); and monoubiquitylation of The large number of gene families, each pathway (Courtois, 2008; Heyninck and histones can influence chromatin structure with multiple members, suggests that Beyaert, 2005). Recent structures of and transcription (Levinger and selective pressure to evolve such catalysts CYLD and A20 suggest that these proteins Varshavsky, 1980). Recent mass has occurred numerous times. In addition, achieve specific cleavage of K63-linked spectrometry analysis of ubiquitylated this diversity implies that considerable polyubiquitin chains by recognizing the proteins shows that chains with multiple substrate specificity exists. This unique surfaces of ubiquitin that are linkages can be attached to a single protein assumption is supported by the finding that juxtaposed in this type of polyubiquitin. (Bish et al., 2008; Crosas et al., 2006; Kim the mutation, deletion or downregulation Similar conclusions are supported by a Journal of Cell Science 122 (14) 2327

co-crystal structure containing the JAMM substrates. Another purpose that the many mesenchymal tumors. Furthermore, domain of the DUB AMSH (associated interaction might serve is to target deletion of the gene encoding A20 in mice molecule with the SH3 domain of STAM) the DUB for degradation via the ligase- results in severe inflammation and and K63-linked diubiquitin (Sato et al., catalyzed ubiquitylation of the associated cachexia (Singhal et al., 2008). 2008). DUB. In at least some cases, the two interaction partners are indeed The potential role of DUBs in neurological DUBs associate with ubiquitin transregulated by each other. For example, disease is even less well understood. ligases, scaffold proteins and in the absence of their substrates, the Mutation of USP14 in mice or ataxin-3 in substrate adaptors ubiquitin ligases MDM2 and Ro52 humans causes ataxia (Crimmins et al., In contrast to the specificity of DUBs that (Sjogren’s syndrome associated 2006; Duenas et al., 2006), whereas the is apparent in vivo, assays carried out using autoantigen) become autoubiquitylated, S18Y allele of human UCH-L1 confers artificial substrates in vitro often indicate and this is reversed by the activity of their protection against sporadic Parkinson’s that DUBs show little specificity. This can, associated DUBs, USP7 and USP4, disease. UCH-L1 is concentrated in a in part, be attributed to the qualitative respectively (Clegg et al., 2008; variety of neuronal inclusion bodies in nature of many assays that do not measure Meulmeester et al., 2005; Wada and humans, and loss-of-function mutations the rates of substrate cleavage, although Kamitani, 2006). Conversely, USP4 can be in this protein cause axonal degeneration in this factor alone cannot fully explain the ubiquitylated by Ro52 and subsequently neurons that terminate at the Gracile apparent lack of specificity. A more likely degraded. However, another function of nucleus, a region of the brainstem that explanation is that most DUBs contain these interactions might be to enforce the receives dorsal-root fibers conveying additional protein interaction domains substrate specificity of ubiquitylation: the sensory innervation of the leg and lower (which are utilized in vivo but not in in action of the DUB might ‘proofread’ trunk (Setsuie and Wada, 2007). It is vitro) that direct the binding of DUBs to ubiquitylation and prevent the assembly of possible that interfering with DUB specific scaffolds or substrate adaptors and inappropriate ubiquitin linkages. The DUB function leads to cellular stress that is not thereby confer substrate specificity. Thus, A20, which contains both a ligase and a obvious in most tissues but has a major it is thought that in vivo specificity is DUB domain on the same polypeptide, is impact in the nervous system, as the death determined mostly by the colocalization of the most extreme example of this. Its of a small number of neurons can have the DUB and its substrates, and that apparent role is to remodel the profound functional consequences. adaptors are necessary for many DUBs to polyubiquitin chains that are generated on bind to their substrates (Marfany and RIP1 (receptor-interacting protein 1) Finally, it is notable that several bacteria Denuc, 2008; Ventii and Wilkinson, 2008). during tumor necrosis factor (TNF)- (Rytkonen and Holden, 2007) and viruses For example, USP1 is known to form a mediated stimulation of the NF-κB (Lindner, 2007) have exploited the host- complex with a non-proteolytic subunit, pathway. Removing the K63-linked cell ubiquitin pathway by encoding DUBs UAF1, and the degradation of UAF1 leads polyubiquitin downregulates signaling, that play a role in infection and to proteolysis of USP1 and consequent and assembling a K48-linked chain on pathogenesis. For example, the SARS

Journal of Cell Science defects in the DNA repair functions that RIP1 drives its degradation, further coronavirus PLpro processing protease USP1 is involved in regulating (Cohn damping signaling (Heyninck and Beyaert, acts on a broad range of ubiquitylated and and D’Andrea, 2008). Similarly, the 2005). ISG15-modified host proteins and is proteasome-associated DUBs USP14, required for viral replication (Ratia et al., UCH37 and POH must all be associated Pathological conditions related to 2008); the obligate intracellular bacterium, with the proteasome for significant DUB DUB dysfunction Burkholderia mallei, expresses and activity (Schmidt et al., 2005; Ventii and Defects in DUB functions have been secretes a DUB inside infected Wilkinson, 2008). implicated in several pathological macrophages (Shanks et al., 2009); and the conditions, most notably cancer, ChlaDub1 expressed by Chlamydia Another surprising observation is that neurological disease and microbial trachomatis suppresses NF-κB activation several DUBs have been found to associate pathogenesis (de Pril et al., 2006; (Le Negrate et al., 2008). Presumably these with ubiquitin ligases, which suggests that Rytkonen and Holden, 2007; Setsuie and microbial DUBs confer a selective DUBs have a role in regulating Wada, 2007; Shackelford and Pagano, 2005; advantage on the pathogen by ubiquitylation. The proteasome has both Singhal et al., 2008; Stuffers et al., deubiquitylating host proteins and ubiquitin ligases and DUBs that associate 2008; Yang, 2007). interfering with their normal cellular with it (Crosas et al., 2006), and several functions. DUB-ligase pairs interact directly, Based on the findings that DUBs have a including BRCC36-BRCA1, BAP1- role in regulating multiple cell-cycle and The above examples describe pathological BRCA1, USP4-Ro52, USP7-MDM2, DNA repair checkpoints, in addition to conditions that are caused by expression of USP8-GRAIL, USP20-pVHL, USP33- cytokine-signaling and apoptosis heterologous DUBs or by mutations pVHL and USP44-APC (Kee and pathways, it is likely that defects in DUB of endogenous DUBs, although many other Huibregtse, 2007; Marfany and Denuc, function could contribute to the disease states or cellular functions have 2008; Ventii and Wilkinson, 2008). One development of cancer. Notably, mutations been shown to be modulated by DUBs. explanation for these associations may be in CYLD cause cylindromatosis, and the There are only a few DUB mutations that that the associated DUBs counteract the translocation of the UBP6 coding region are currently known to cause disease, but it tendency of ubiquitin ligases to downstream of heterologous promoters is is very likely that more will be recognized autoubiquitylate in the absence of other an oncogenic event that is found in in the future. It is also probable that other 2328 Journal of Cell Science 122 (14)

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