Deubiquitination is a crucial mechanism in ubiquitin-mediated signalling networks. The importance of Dubs (deubiquitinating enzymes) as regulators of diverse cellular processes is becoming ever clearer as new roles are elucidated and new pathways are shown to be affected by this mechanism. Recent work, reviewed in the present paper, provides new perspective on the widening influence of Dubs and a new tool to focus studies of not only Dub interactions, but also potentially many more cellular systems.

Introduction

The ubiquitin–proteasome system has built a complex network of signals based on the diversity of ubiquitin linkages and their specific recognition. One of the levels of complexity of this fascinating regulatory system is the reversibility of ubiquitination, which is controlled by a variety of specialized enzymes. Factors in charge of producing ubiquitin linkages include ubiquitin activators (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-ligating factors (E3 and E4) [1]. Conjugation of ubiquitin may include the attachment of one single ubiquitin group to protein targets, known as mono-ubiquitination, or the formation of branches of ubiquitin polymers linked to targets, known as polyubiquitination, which may involve all seven lysine residues in ubiquitin (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63) [2]. Thus ubiquitin conjugation generates a sophisticated code of signals in the cell, the recognition of which is carried out by specific receptors, adaptors and enzymes.

The enzymatic reaction that opposes ubiquitin conjugation is deubiquitination. The human genome encodes 79 Dubs (deubiquitinating enzymes) predicted to be active, although most of them have not been formally characterized [3]. Catalysis of deubiquitination requires a nucleophilic attack to the carbonyl group of the isopeptide bond established between the C-terminal end of ubiquitin and the amino group of the accepting lysine residue, and, in the case of linear linkages, the attack is against a peptide bond that links two ubiquitin groups in a head-to-tail fashion. Evolution has found two solutions to this type of proteolytic reaction: cysteine and zinc active sites. Dubs with cysteine active sites are more abundant and they contain a highly conserved catalytic triad, in which an aspartic acid polarizes a histidine residue, which deprotonates the cysteine. This mechanism is found in UCHs (ubiquitin C-terminal hydrolases), USPs (ubiquitin-specific proteases), OTUs (ovarian tumour proteases) and MJDs (Machado–Josephin domains), which represent more than 80% of human Dubs. In zinc active sites of Dubs, similar to other metalloenzymes, a zinc atom is attached to two histidine residues and one aspartate residue, and one polarized water molecule co-ordinates the fourth link to the metallic atom, ensuring reactivity. This type of active site is found in JAMM (JAB1/MPN/MOV34 metalloenzyme) Dubs (reviewed in [3,4]).

Given the complexity of ubiquitination, Dubs have the difficult task of discriminating their targets among an ocean of ubiquitin–ubiquitin and protein–ubiquitin linkages in different subcellular and functional contexts. Several layers of specificity have been suggested to categorize Dubs: linkage specificity (selection of ubiquitin chain topology established by lysine usage, i.e. Lys11, Lys48, Lys63, etc.), relative position of cleavage within ubiquitin chains (distal compared with proximal cut), protein specificity (protein–ubiquitin compared with ubiquitin–ubiquitin linkage identification), mono-deubiquitination specificity (Dubs recognizing mono-ubiquitinated substrates) and chain recycling (selection of unanchored compared with anchored chains) [4]. This classification reflects the diversity of features that define Dubs and the number of factors selected in these enzymes. Research on Dubs has revealed multiple regulatory roles of deubiquitination in cellular processes; however, most Dub enzymes have not yet been investigated.

Systematic analysis of Dub interactors

A recent study from the laboratory of Wade Harper presented at the 2009 meeting of the INPROTEOLYS network and published recently [5] has addressed functional diversity of human Dubs based on their bona fide interactors. In this work, the interactome of 73 human Dubs was systematically analysed using proteomics. To do so, the authors used FLAG–HA (haemagglutinin)-tagged plasmid versions of the genes expressed in HEK (human embryonic kidney)-293 cells. They isolated Dubs by affinity purification and analysed the protein content by LC (liquid chromatography)–MS/MS (tandem MS) in duplicate. To discriminate reliable interactors from non-specifically associated proteins or simply contaminants, they developed an unbiased approach based on a software platform called CompPASS (Comparative Proteomic Analysis Software Suit). For a given Dub, CompPASS assigns scores to co-purified proteins identified within parallel proteomic analysis in a way that proteins identified more abundantly in both IPs (immunoprecipitations) and not common in multiple purifications receive a higher score. HCIPs (high-confidence candidate-interacting proteins) were defined by a novel metric devised by the authors which takes into account uniqueness, abundance and reproducibility of a protein within an immunoprecipitated complex. The accuracy of the approach was scrupulously checked using IP controls, analysing Dubs which are part of well-established protein complexes and by comparing the methodology with other established techniques, as discussed below. With this tool, they generated a list of proteins likely to be functionally linked to each Dub and uncovered multiple interactive networks.

Different interactive nature of Dubs

On the basis of the number and hierarchy of Dub interactors, they found that Dubs could be assigned to seven topological groups, each representing different types of networking, as summarized in Table 1. Group 1 is defined by Dubs with a low number of candidate interactors which have not been previously described. It contains 28 members, and includes some well-characterized Dubs. Among them, UCHL1 is involved in regulation of α2-adrenergic receptor signalling and has been implicated in Parkinson's disease [6]. UCHL1 has been found to interact with a coiled-coil domain-containing protein (CCDC14). CYLD (cylindromatosis) is an important inhibitory regulator of several factors involved in NF-κB (nuclear factor κB) signal transduction [7]. An interesting interactor of CYLD is CEP192 (centrosomal protein 192), which is involved in mitotic spindle and centrosome assembly [8]. OTUD7B is also involved in NF-κB-response inhibition [9], and interacts with HIF1AN, an inhibitor of HIF1α (hypoxia-inducible factor 1α), which is regulated by proteasome degradation [10]. USP30 and USP33 are enzymes which have recently been implicated in the scattering response, one of the steps required for the invasive growth programme of epithelial cells [11]. USP30 interacts with the MPN domain-containing protein MPND and with USP4, which associates with the spliceosome. OTUB1, which has been shown to deubiquitinate and regulate oestrogen receptor α activity [12], interacts with two E2s [UBE (ubiquitin-conjugating enzyme) 2N and UBE2D2]. STAMBPL1 {STAMBP [STAM (signal-transducing adaptor molecule)-binding protein]-like 1} {or AMSH-LP [associated molecule with the SH3 (Src homology 3) domain of STAM-like protease]}, known to function in signal transduction, has been crystallized in complex with Lys63-linked polyubiquitin, providing novel structural information about this mechanism of interaction between Dub and substrate [13]. STAMBPL1 was found to interact with USP49, OTUB1, UBE2N and BAP1 [BRCA (breast cancer early-onset) 1-associated protein 1]. ATXN3 has been widely studied as a cause of the neurodegenerative Machado–Joseph disease [14] and interacts with USP13 and OTUB2. USP16 is involved in deubiquitination of histones in a key regulatory step of mitosis [15] and has been found to associate with histone H2B1 and HERC2. USP28 has roles in DNA damage response [16] and Myc stability in proliferating tumour cells [17]. USP2 is implicated in several cancers and in p53 regulation [18] as is USP5, also known as isopeptidase T [19]. USP8 regulates endosomal sorting via the epidermal growth factor receptor pathway [20]. UCHL3 has recently been associated with metabolism and obesity control in mice [21]. USP26 appears to be linked to infertility in males [22]. USP18 has roles in viral and bacterial pathogenesis, as well as oncogenic transformation [23]. Other members of topology group 1 and their interactors are shown in Table 1.

Table 1
Human Dubs and their interactors

Abbreviations used in GO processes (GO-P): A, apoptosis; B, biogenesis; Cat, catabolic; Ch, chromatin; Dev, development; Dif, differentiation; DNA-D, DNA damage; DNA-R, DNA replication; F, folding; Met, metabolic; Mit, mitosis; Mor, morphogenesis; O, other; P, proteolysis; Ph, phosphorylation; RNA, RNA processing; ST, signal transduction; Tc, transcription; Tl, translation; Ub, ubiquitin; VT, vesicle transport. Abbreviations used in GO component (GO-C): Cp, cytoplasm; Cs, cytoskeleton; ER, endoplasmic reticulum; G, Golgi; M, mitocondrion; N, nucleus; O, other; PM, plasma membrane; V, vesicle.

Group Dub Class Interactors GO-P GO-C 
Group 1 ATXN3 MJD USP13, OTUB2, KCTD10 Ub, O 
 CYLD USP SPATA2L, SPATA2, CEP192, MGEA5, CAMK2D, MYO6 Dif, Dev N, Cp 
 DUB3 USP LOC392197, LOC402164, CBX1, SET DNA-R, Ub, Ch 
 JOSD1 MJD YOD1, CALM1, TIMM8A Dev, VT, F 
 JOSD2 MJD TYSND1, KIAA0828, AHCYL1, TRAPPC2, SMARCA2 Tc, Met ER 
 LOC402164 USP BTF3, NACAP1, DUB3, LOC392197, ADAMTS1, LGMN, USP22 Ub, P 
 OTUB1 OTU STAMBP, UBE2N, UBE2D2, MSH2, CASP14, CPNE7 Ub, A 
 OTUB2 OTU GTF2I, VCPIP1 Tc, ST 
 OTUD6B OTU OTUB1, ASCC3, MTDH, BXDC1 Ub, Tc 
 OTUD7B OTU ACAD9, SLC9A3R2, HIF1AN, NUP155 
 PARP11 OTU ZNF313, WDR23, LYZ Diff, Dev, Cat, Met 
 STAMBPL1 JAMM/MPN USP49, OTUB1, CLINT1, UBE2N, SNRPA1, HMX3, BAP1 Ub, Tc 
 UCHL1 UCH CCDC14 Dev Cs 
 UCHL3 UCH CLPB, USP20 − − 
 USP2 USP LONP1, RRP15 
 USP5 USP USP13 Ub − 
 USP8 USP USP25, USP22, LOC440587 Dev Cs 
 USP16 USP DBT, HERC2, HIST1H2BL Ub, B, Met, VT 
 USP18 USP USP41, JMJD1B, MYL6, NME1 − − 
 USP26 USP LOC392197, DUB3, LOC402164 Ub − 
 USP28 USP TP53BP1, AQR, SUMO2  
 USP29 USP USP25, CTSB A, P V, M 
 USP30 USP QKI, MPND, SS18L1, USP4, TIMM8A, CLPB, SF3A1 Dev, RNA 
 USP33 USP ZFR, IFIT5, KRR1, PRPF38B O, RNA 
 USP37 USP FBXW11, ALB Ub, A, ST, O 
 USP38 USP HSPB1, HMX3, LGALS7, RPS12, RPL7 A, Tl Cp 
 USP48 USP LOC442227 − − 
 YOD1 OTU MUTED, THOC3 RNA 
Group 2 COPS5 JAMM/MPN COP9 Signalosome, Kelch domain proteins, CUL2, LRRC14, CUL4B, WDR21A, WDR23 Ub, Mit 
 COPS6 JAMM/MPN COP9 Signalosome, Kelch domain proteins, CUL4B, CUL2, LRRC14, FBXW9, BTBD2, FBXO7, BTBD9 Ub, Mit 
 EIF3S3 JAMM EIF3 complex, BAI1, MGC14327, CSNK2A2, KIAA0515, ARPC5, CSNK2B, CSNK2A1 Tl Cp 
 EIF3S5 JAMM/MPN EIF3 complex, CSNK2A2, KIAA0515, CSNK2B, ASCC3, CSNK2A1 Tl Cp 
 PSMD7 JAMM/MPN Proteasome, KTN1 Cat Cp 
 PSMD14 JAMM/MPN Proteasome, FLJ20850, SUCLA2 Cat, P Cp 
 UCHL5 UCH Proteasome, NFRKB, TFPT, TXNL1, CCDC95, PTPN2 Ub, Cat Cp 
 USP14 USP Proteasome, TXNL1, RGPD5, SRPRB Ub, Cat Cp 
Group 3 JOSD3 MJD Transcriptional complexes, POLR1E, CENPB, RRP15, POLR1C, PPAN, LOC440587 Ts 
 USP3 USP EIF3 complex, WDTC1, RIMBP2, LRP1, NXN, USP48, GNAL, GNA13, CBR3, IFRG15, PKLR, KIAA0515 Tl Cp 
 USP4 USP Spliceosome complex, EDC3, AKAP7, PRPF3, TUT1, DCP1B, BCDIN3, ADSL, USP32, EIF4E2 RNA 
 USP15 USP Spliceosome complex, LRRC15, RNF40, MYH4, SELENBP1, FABP4, VSIG8, MYH2, TUT1, PRPF3, ADSL RNA 
 USP22 USP SAGA complex, ENY2, TADA1L, LOC254571, ATXN7L(3,2), MGC21874, KIF7, FAM48A, LOC552889, USP27X Tc 
 USP39 USP Spliceosome complex, RG9MTD1, PA2G4, ZRANB2, RY1, PRPF(4B, 3, 4), TSR2, KIAA0409, C17orf79 RNA 
 ZRANB1 OTU Phosphatase scaffolding complexes, CTTNBP2NL, HECTD1, MAP4K4, GCC2, KEAP1, PGAM5, CACYBP Ub, O Cp 
Group 4 OTUD1 OTU LOC653852, FLNC, RAD23A, RAD23B, FLNB, FLNA, KEAP1 DNA-D,ST, Dev, B N, Cs 
 STAMBP JAMM/MPN VPS24, AFP, PIK3C2A, OTUB1, CLINT1, GRB2, CLTA ST, VT V, O 
 TL132L USP USP5, LOC220594, CYLD, USP13, USP32, RNH1, SEH1L, OTUB2, TGM3 Ub Cs 
 USP10 USP CUGBP1, EIF4G1, EIF4G3, G3BP2, G3BP1, NOLA1 ST, Tl, RNA Cp 
Group 5 BAP1 UCH OGT, HCFC1, RBBP7, HAT1, FOXK1, UBE20, FOXK2, ASXL2, ANKRD17, EIF4EBP3, ASXL1, IPO4, CBX3 Tc 
 BRCC3 JAMM/MPN KIAA0157, HSPC142, BRE, CCDC98, UIMC1, EXOC8, SUPT16H, E2F5, DDOST, MCM2, CAND1, SHMT2 Tc 
 OTUD4 OTU GLA, GALK1, DSG1, PARP11, MYCBP, NMD3, MOG, GPHN, BAG5, DNAJB1, FLNC, TUBA1A Cs 
 USP1 USP PKP1, DSC1, DSG1, JUP, USP3, CALML3, TAGLN2, HSPB1, CALML5, WDR48, PHLPP, MYH9, KPNA1 Dev Cs 
 USP13 USP DLST, OGDH, SMC1A, SMC3, UBL4A, UFD1L, DIABLO, KCTD3, KCTD10, ITCH, NPLOC4, UBXD8, CACYBP Ub 
 USP21 USP RBM8A, UPF3B, MARK3, UCHL1, MARK(1,2), KIAA1553, UTRN, FUCA1, MARK4, PRKCI, USP20, USP48 Ph, Dev N, Cp 
 USP25 USP PIP, LYZ, LOC124220, WRNIP1, USP28, KCTD13, ANXA1, KLHL9, KCTD10, BTBD9, MYO6, NEDD8 − 
 USP32 USP TUBA1A, CDC2, YWHAB, USP6, VPS35, LOC51035, MRPL39, ABCD3, USP11, TRIP13, TXNDC4, SMC1A 
 USP36 USP WDR36, WDR3, UTP18, PWP2, TBL3, DHX33, NUDCD1, MPND, STK25, DDX41, GNL2, CHD4, TMEM104 ST 
 USP45 USP SF3A2, RBMX, POLR2G, MYH10, CORO1C, MRLC2, RTCD1, SRBD1, MYH9, TMOD3, PIK3CG, GLTSCR2 RNA 
 USP50 USP ABCE1, SLC1A5, DYNC2H1, LUC7L, FLJ20294, IGF2R, KEAP1, AMOT, CKAP4, AFG3L2, VCP complex M, N 
 USP52 USP VBP1, TCEB2, TCEB1, PFDN2, PAN3, USP5, PLEC1, NUP93, MRPL39, ARCN1, CBWD2, CCT7 
 USPL1 USP PGD, PKLR, ANKFY1, KIAA0947, ELL, KIF5B, IGHA2, CKB, ANXA1, PIP Cp, Cs, O 
 VCPIP1 OTU NSFL1C, UFD1L, NPLOC4, VCP, UBXD4, ABCC12, LOC137886, KFZp313A2432, UBXD8, HUWE1 Ub 
Group 6 OTUD5 OTU LONRF2, GPX4, LANCL2, CTPS2, GYS1, VARS, FLNA, CACYBP, SET, GRB2, USP11, TP53, PKLR Dev 
 TNFAIP3 OTU KIF11, NDUFS1, GLDC, FBXO3, CNKSR2, TBK1, YWHAH, RNH1, YWHAB, LRRC47, ALDH9A1, PPP2R1B − Cp 
 USP12 USP USP39, WDR20, DMWD, PHLPP, PHLPPL, UCHL5, WDR48, RAD51AP1, CYLD, PAIP1, NUP160, MMP2 RNA 
 USP42 USP PLRG1, TEX10, C14orf169, AMOT, USP32, AHCYL1, DIMT1L, USP20, FECH, MRPS14, CA2, MRPS31 − 
 USP43 USP MAGI3, WDR3, YWHAH, PDCD2, YWHAB, VPS35, FN3KRP, PSME3, YWHAE A, ST Cp 
 USP44 USP CETN2, MRPL40, SARS2, POLR2G, MRPL53, MRPL23, TCOF1, KRR1, TBL2, MRPS21 Tl, O M, N 
 USP46 USP USP12, WDR20, DMWD, PHLPP, IQGAP1, PHLPPL, EIF2AK4, PPP1R9B, WDR48, RAD51AP1, PJA2, USP52 Ub, Ph Cp 
 USP53 USP ARG1, OTUD4, CAT, CSTA, BLMH, CASP14, HCFC1, AZGP1, TGM3, RPL26, RAD50, DSG1, KEAP1 Mit Cp 
Group 7 USP7 USP MCM6, NUP98, MCM4, CRKL, MCM5, RAE1, C10orf119, USP14, BRCC3, KIAA0157, FBXO38, USP19, BRE Ub, Tc 
 USP11 USP SIPA1L1, ZNF24, USP4, MRE11A, USP7, RAD50, TP53, PKN2, TCEAL(1,4), TRIP12, MPHOSPH9, OTUD1 Tc 
 USP19 USP CSE1L, CDC2, HMOX2, TNPO3, CAMK2G, LAT, DMWD, KIAA1787, HERC2, ACADSB, PARK7, UNC45A VT 
 USP20 USP PSMD6, EIF3S6, PSMD7, EIF3S8, PSMD11, PSMD12, VAPA, PLEKHA7, RAD17, SYTL4, N4BP3, UCHL3 N, Cp 
 USP49 USP PDCD2, LRPPRC, Centrin proteins, SAPS domain proteins, USP44, F7, FKBP5, SHCBP1, PPP5C, ANKRD28 VT 
Group Dub Class Interactors GO-P GO-C 
Group 1 ATXN3 MJD USP13, OTUB2, KCTD10 Ub, O 
 CYLD USP SPATA2L, SPATA2, CEP192, MGEA5, CAMK2D, MYO6 Dif, Dev N, Cp 
 DUB3 USP LOC392197, LOC402164, CBX1, SET DNA-R, Ub, Ch 
 JOSD1 MJD YOD1, CALM1, TIMM8A Dev, VT, F 
 JOSD2 MJD TYSND1, KIAA0828, AHCYL1, TRAPPC2, SMARCA2 Tc, Met ER 
 LOC402164 USP BTF3, NACAP1, DUB3, LOC392197, ADAMTS1, LGMN, USP22 Ub, P 
 OTUB1 OTU STAMBP, UBE2N, UBE2D2, MSH2, CASP14, CPNE7 Ub, A 
 OTUB2 OTU GTF2I, VCPIP1 Tc, ST 
 OTUD6B OTU OTUB1, ASCC3, MTDH, BXDC1 Ub, Tc 
 OTUD7B OTU ACAD9, SLC9A3R2, HIF1AN, NUP155 
 PARP11 OTU ZNF313, WDR23, LYZ Diff, Dev, Cat, Met 
 STAMBPL1 JAMM/MPN USP49, OTUB1, CLINT1, UBE2N, SNRPA1, HMX3, BAP1 Ub, Tc 
 UCHL1 UCH CCDC14 Dev Cs 
 UCHL3 UCH CLPB, USP20 − − 
 USP2 USP LONP1, RRP15 
 USP5 USP USP13 Ub − 
 USP8 USP USP25, USP22, LOC440587 Dev Cs 
 USP16 USP DBT, HERC2, HIST1H2BL Ub, B, Met, VT 
 USP18 USP USP41, JMJD1B, MYL6, NME1 − − 
 USP26 USP LOC392197, DUB3, LOC402164 Ub − 
 USP28 USP TP53BP1, AQR, SUMO2  
 USP29 USP USP25, CTSB A, P V, M 
 USP30 USP QKI, MPND, SS18L1, USP4, TIMM8A, CLPB, SF3A1 Dev, RNA 
 USP33 USP ZFR, IFIT5, KRR1, PRPF38B O, RNA 
 USP37 USP FBXW11, ALB Ub, A, ST, O 
 USP38 USP HSPB1, HMX3, LGALS7, RPS12, RPL7 A, Tl Cp 
 USP48 USP LOC442227 − − 
 YOD1 OTU MUTED, THOC3 RNA 
Group 2 COPS5 JAMM/MPN COP9 Signalosome, Kelch domain proteins, CUL2, LRRC14, CUL4B, WDR21A, WDR23 Ub, Mit 
 COPS6 JAMM/MPN COP9 Signalosome, Kelch domain proteins, CUL4B, CUL2, LRRC14, FBXW9, BTBD2, FBXO7, BTBD9 Ub, Mit 
 EIF3S3 JAMM EIF3 complex, BAI1, MGC14327, CSNK2A2, KIAA0515, ARPC5, CSNK2B, CSNK2A1 Tl Cp 
 EIF3S5 JAMM/MPN EIF3 complex, CSNK2A2, KIAA0515, CSNK2B, ASCC3, CSNK2A1 Tl Cp 
 PSMD7 JAMM/MPN Proteasome, KTN1 Cat Cp 
 PSMD14 JAMM/MPN Proteasome, FLJ20850, SUCLA2 Cat, P Cp 
 UCHL5 UCH Proteasome, NFRKB, TFPT, TXNL1, CCDC95, PTPN2 Ub, Cat Cp 
 USP14 USP Proteasome, TXNL1, RGPD5, SRPRB Ub, Cat Cp 
Group 3 JOSD3 MJD Transcriptional complexes, POLR1E, CENPB, RRP15, POLR1C, PPAN, LOC440587 Ts 
 USP3 USP EIF3 complex, WDTC1, RIMBP2, LRP1, NXN, USP48, GNAL, GNA13, CBR3, IFRG15, PKLR, KIAA0515 Tl Cp 
 USP4 USP Spliceosome complex, EDC3, AKAP7, PRPF3, TUT1, DCP1B, BCDIN3, ADSL, USP32, EIF4E2 RNA 
 USP15 USP Spliceosome complex, LRRC15, RNF40, MYH4, SELENBP1, FABP4, VSIG8, MYH2, TUT1, PRPF3, ADSL RNA 
 USP22 USP SAGA complex, ENY2, TADA1L, LOC254571, ATXN7L(3,2), MGC21874, KIF7, FAM48A, LOC552889, USP27X Tc 
 USP39 USP Spliceosome complex, RG9MTD1, PA2G4, ZRANB2, RY1, PRPF(4B, 3, 4), TSR2, KIAA0409, C17orf79 RNA 
 ZRANB1 OTU Phosphatase scaffolding complexes, CTTNBP2NL, HECTD1, MAP4K4, GCC2, KEAP1, PGAM5, CACYBP Ub, O Cp 
Group 4 OTUD1 OTU LOC653852, FLNC, RAD23A, RAD23B, FLNB, FLNA, KEAP1 DNA-D,ST, Dev, B N, Cs 
 STAMBP JAMM/MPN VPS24, AFP, PIK3C2A, OTUB1, CLINT1, GRB2, CLTA ST, VT V, O 
 TL132L USP USP5, LOC220594, CYLD, USP13, USP32, RNH1, SEH1L, OTUB2, TGM3 Ub Cs 
 USP10 USP CUGBP1, EIF4G1, EIF4G3, G3BP2, G3BP1, NOLA1 ST, Tl, RNA Cp 
Group 5 BAP1 UCH OGT, HCFC1, RBBP7, HAT1, FOXK1, UBE20, FOXK2, ASXL2, ANKRD17, EIF4EBP3, ASXL1, IPO4, CBX3 Tc 
 BRCC3 JAMM/MPN KIAA0157, HSPC142, BRE, CCDC98, UIMC1, EXOC8, SUPT16H, E2F5, DDOST, MCM2, CAND1, SHMT2 Tc 
 OTUD4 OTU GLA, GALK1, DSG1, PARP11, MYCBP, NMD3, MOG, GPHN, BAG5, DNAJB1, FLNC, TUBA1A Cs 
 USP1 USP PKP1, DSC1, DSG1, JUP, USP3, CALML3, TAGLN2, HSPB1, CALML5, WDR48, PHLPP, MYH9, KPNA1 Dev Cs 
 USP13 USP DLST, OGDH, SMC1A, SMC3, UBL4A, UFD1L, DIABLO, KCTD3, KCTD10, ITCH, NPLOC4, UBXD8, CACYBP Ub 
 USP21 USP RBM8A, UPF3B, MARK3, UCHL1, MARK(1,2), KIAA1553, UTRN, FUCA1, MARK4, PRKCI, USP20, USP48 Ph, Dev N, Cp 
 USP25 USP PIP, LYZ, LOC124220, WRNIP1, USP28, KCTD13, ANXA1, KLHL9, KCTD10, BTBD9, MYO6, NEDD8 − 
 USP32 USP TUBA1A, CDC2, YWHAB, USP6, VPS35, LOC51035, MRPL39, ABCD3, USP11, TRIP13, TXNDC4, SMC1A 
 USP36 USP WDR36, WDR3, UTP18, PWP2, TBL3, DHX33, NUDCD1, MPND, STK25, DDX41, GNL2, CHD4, TMEM104 ST 
 USP45 USP SF3A2, RBMX, POLR2G, MYH10, CORO1C, MRLC2, RTCD1, SRBD1, MYH9, TMOD3, PIK3CG, GLTSCR2 RNA 
 USP50 USP ABCE1, SLC1A5, DYNC2H1, LUC7L, FLJ20294, IGF2R, KEAP1, AMOT, CKAP4, AFG3L2, VCP complex M, N 
 USP52 USP VBP1, TCEB2, TCEB1, PFDN2, PAN3, USP5, PLEC1, NUP93, MRPL39, ARCN1, CBWD2, CCT7 
 USPL1 USP PGD, PKLR, ANKFY1, KIAA0947, ELL, KIF5B, IGHA2, CKB, ANXA1, PIP Cp, Cs, O 
 VCPIP1 OTU NSFL1C, UFD1L, NPLOC4, VCP, UBXD4, ABCC12, LOC137886, KFZp313A2432, UBXD8, HUWE1 Ub 
Group 6 OTUD5 OTU LONRF2, GPX4, LANCL2, CTPS2, GYS1, VARS, FLNA, CACYBP, SET, GRB2, USP11, TP53, PKLR Dev 
 TNFAIP3 OTU KIF11, NDUFS1, GLDC, FBXO3, CNKSR2, TBK1, YWHAH, RNH1, YWHAB, LRRC47, ALDH9A1, PPP2R1B − Cp 
 USP12 USP USP39, WDR20, DMWD, PHLPP, PHLPPL, UCHL5, WDR48, RAD51AP1, CYLD, PAIP1, NUP160, MMP2 RNA 
 USP42 USP PLRG1, TEX10, C14orf169, AMOT, USP32, AHCYL1, DIMT1L, USP20, FECH, MRPS14, CA2, MRPS31 − 
 USP43 USP MAGI3, WDR3, YWHAH, PDCD2, YWHAB, VPS35, FN3KRP, PSME3, YWHAE A, ST Cp 
 USP44 USP CETN2, MRPL40, SARS2, POLR2G, MRPL53, MRPL23, TCOF1, KRR1, TBL2, MRPS21 Tl, O M, N 
 USP46 USP USP12, WDR20, DMWD, PHLPP, IQGAP1, PHLPPL, EIF2AK4, PPP1R9B, WDR48, RAD51AP1, PJA2, USP52 Ub, Ph Cp 
 USP53 USP ARG1, OTUD4, CAT, CSTA, BLMH, CASP14, HCFC1, AZGP1, TGM3, RPL26, RAD50, DSG1, KEAP1 Mit Cp 
Group 7 USP7 USP MCM6, NUP98, MCM4, CRKL, MCM5, RAE1, C10orf119, USP14, BRCC3, KIAA0157, FBXO38, USP19, BRE Ub, Tc 
 USP11 USP SIPA1L1, ZNF24, USP4, MRE11A, USP7, RAD50, TP53, PKN2, TCEAL(1,4), TRIP12, MPHOSPH9, OTUD1 Tc 
 USP19 USP CSE1L, CDC2, HMOX2, TNPO3, CAMK2G, LAT, DMWD, KIAA1787, HERC2, ACADSB, PARK7, UNC45A VT 
 USP20 USP PSMD6, EIF3S6, PSMD7, EIF3S8, PSMD11, PSMD12, VAPA, PLEKHA7, RAD17, SYTL4, N4BP3, UCHL3 N, Cp 
 USP49 USP PDCD2, LRPPRC, Centrin proteins, SAPS domain proteins, USP44, F7, FKBP5, SHCBP1, PPP5C, ANKRD28 VT 

Group 2 contains Dubs that interact with or are constituents of large macromolecular regulatory complexes. Four of them are proteasomal Dubs, which, as expected, co-precipitate with all proteasome subunits. They are PSMD (proteasome 26S subunit non-ATPase) 7 (Rpn8), PSMD14 (Rpn11), UCHL5 (UCH37) and USP14 (Ubp6). UCHL5 [24] and USP14 [25] transiently associate with the regulatory particle of the proteasome, whereas Rpn11 and Rpn8 are integral non-ATPase subunits of the lid subcomplex [2628]. COPS (COP9 constitutive photomorphogenic homologue subunit) 5 and COPS6 are subunits of the COP9 signalosome [29], which associate with the signalosome complex and with Cullin and Kelch domain proteins. eIF3 (eukaryotic initiation factor 3) S5 and eIF3S3 are components of the eukaryotic initiation factor 3 complex [30]. Interestingly, the COP9 signalosome, eIF3 and the lid of the proteasome are related complexes which show similar architecture and homologous Dubs [31].

Group 3 consists of seven members with approx. 20–30 associated proteins which are often connected to complex processes such as cell cycle and transcription. USP22 and USP3 are involved in regulation of histone ubiquitination and thus the expression of key cell-cycle components. The former has been shown to be a component of the SAGA (Spt–Ada–Gcn5–acetyltransferase) complex and to regulate the expression of cell-cycle activators such as Myc [32], whereas the latter, which is required for S-phase progression and facilitating DNA replication [33], is found to associate to the eIF3 complex. USP39 has been shown to be involved in the correct expression of Aurora B kinase and other spindle checkpoint mRNAs [34]. USP4 deubiquitinates and thus regulates Ro52, itself a regulator of the p27 cell-cycle inhibitor [35]. USP15 controls stability of the tumour suppressor APC (adenomatous polyposis coli) [36]. Sowa et al. [5] have shown that USP39, USP4 and USP15 interact with the spliceosome complex. JOSD3 (Josephin domain-containing 3) is implicated in carcinogensis and RNA polymerase I transcription of rRNA [37,38], and binds to transcriptional complexes.

The four members of Group 4 are so-called ‘distributive’ Dubs with a low number of interactors (six to eight) that may function in several distinct pathways. USP10 interacts with G3BP2 [GTPase-activating protein (Src homology 3 domain)-binding protein 2], an activator of the Ras signal transduction pathway, as well as with eukaryotic initiation factors. It is also involved in the regulation of androgen receptor function [39]. STAMBP has known regulatory relationships with the calcium-sensing receptor pathway and endosomal sorting of epidermal growth factor [40]. CompPASS analysis confirmed interaction with these pathways as well as linking STAMBP to the Ras pathway.

Group 5 Dubs have approx. 10–20 interactors, some exhibiting likely interconnectivity and others which appear to be unrelated. Specific functions of many members of this group are as yet unstudied. USP36 is involved in regulation of nucleolus structure and function, affecting the synthesis of rRNA [41]. BAP1 is a putative tumour suppressor that interacts with the BRCA1 protein and has a role in DNA-damage repair [42]. USP1 deubiquitinates mono-ubiquitinated FANCD2 (Fanconi's anaemia complementation group D2), a component of the Fanconi's anemia DNA-repair pathway [43]. BRCC6 is a BRCA1/BRCA2-containing complex subunit with a role in ERK (extracellular-signal-regulated kinase) signal transduction [44]. USP25 is a Dub with multiple splice variants whose activity has been shown to be alternatively regulated by mono-ubiquitination and SUMOylation [45]. Sowa et al. [5] suggest an interaction of USP25 with a BTB/POZ domain-containing protein NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), another ubiquitin-like signalling molecule. VCPIP1 [VCP (valosin-containing protein)/p97–p47 complex-interacting protein 1] is involved in p97–p47-mediated membrane fusion and assembly of Golgi and endoplasmic reticulum structures [46].

The Group 6 Dubs are characterized by having a large number of interactors with highly distributive functional implications. TNFAIP3 (tumour necrosis factor α-induced protein 3) plays an important role in the inhibition of NF-κB responses [47]. USP44 is part of a mechanism which controls the initiation of anaphase during mitosis [48]. OTUD5 is an inhibitor of innate immune response [49].

Group 7 contains some Dubs that are known to have roles in diverse processes. Their interaction topologies are characterized by a large number of interaction candidates, where a small subset may have apparent interconnectivity, but the majority appear to be unrelated or distributive. USP7 has roles in regulation on the p53 tumour suppressor and in the life cycles of several human herpesviruses [50,51]. USP11 also acts in the p53 pathway, as well as the BRCA2 DNA-damage repair pathway and participates in the life cycle of human papillomavirus 16 [52,53,54]. USP20 has been implicated in endocytic sorting and β2-adrenergic receptor-mediated signalling [55].

Overall, human Dubs show a wide repertoire of interactors. A challenging aspect is the high number of previously unknown interactions, a selection of which is presented in Table 1.

Dub interactome validation

The efficacy of the CompPASS system at accurately identifying reproducible protein–protein interactions was confirmed both experimentally and by comparison with published literature. IPs of 25 HA-tagged interactors that were subjected to IP–MS/MS confirmed 83% of CompPASS predicted interactions. Co-immunoprecipitation experiments using Myc-tagged interactors and Dubs validated 14 of 29 predicted interactions by immunoblotting. Endogenous co-immunoprecipitation detected three of six predicted interactions. Overall, 68% of interactions tested were confirmed independently. Study of the literature revealed 332 interactions involving 51 different Dubs. CompPASS prediction shared 71% identity with published interactions identified using endogenous co-IP or co-purification. This value was 36% when compared with interactions characterized by overexpression co-IP and just 4.6% compared with interactions characterized by yeast two-hybrid systems alone. Finally, when 11 Dubs from the set were expressed in HCT116 cells and subjected to IP–MS/MS, CompPASS analysis identified 63% of the same HCIPs from previous HEK-293T cell samples. With this kind of accuracy in the identification of interacting partners, CompPASS can help to point out potentially fruitful avenues for future research into the breadth and depth of the Dub interactome.

Following validation procedures, robust interactions can be established, such as the involvement of USP13 in ERAD (endoplasmic-reticulum-associated degradation). USP13 was found to interact with the AAA (ATPase associated with various cellular activities) VCP/p97 complex and with the VCP-associated proteins UFD1 (ubiquitin fusion degradation 1), NPL4 (nuclear protein localization 4) and UBDX8. The authors performed functional studies and showed that depletion of USP13 increases the levels of the ERAD substrate TCRα (T-cell receptor α)–GFP (green fluorescent protein), suggesting an relevant role of this Dub in the pathway.

The authors also found interesting interconnectivity of related Dubs USP1, USP12 and USP46 with two phosphatases involved in Akt dephosphorylation [PHLPP (pleckstrin homology domain and leucine-rich repeat protein phosphatase) and PHLPPL (PHLPP-like)] [56] and three WD40-containing proteins [WDR48/UAF1, WDR20 and DMWD (dystrophia myotonica, WD40 repeat-containing)]. The capacity of WDR48/UAF1 to associate with USP1, USP12 and USP46 has been reported [57,58], but WDR20 and DMWD are uncharacterized. It has been shown recently that PHLPP is polyubiquitinated and degraded in a β-TrCP (β-transducin repeat-containing protein)-dependent manner and that Akt regulates this process [59], suggesting that a cognate Dub might be involved in PHLPP stability.

Additional Dub-containing networks described in the present paper are related to RNA processing, DNA-damage response and ribosome autophagy. For instance, USP39, USP15 and USP4 were found in U5/U6-snRNP (small nuclear riboprotein) and in Lsm mRNA-binding complexes.

Another interesting finding is that USP11, a p53 regulator, was verified to interact with USP7 and USP4, which have been implicated in the p53 and p27 pathways respectively [29,44,46]. Determination of relationships could lead to greater understanding of the role of ubiquitin signalling in the wider context of global cell-cycle regulation. Furthermore, USP11 and USP7 share interactors with roles in cell-cycle regulation, including RAE1 (RNA export 1) [60] and BUB3 (budding uninhibited by benzimidazoles 3) [61].

A general feature is that multiple Dubs tend to associate with a given network or pathway, suggesting that deubiquitination may regulate several steps or levels of the pathway.

An exportable workflow

The present paper defines the workflow for a new generation of systematic proteomic studies. The first step is processing raw data from proteomic analysis. The high and ever-increasing sensitivity of protein MS requires appropriate bioinformatics implementation. In this regard, CompPASS defines a successful rationale to process raw data. The second step is to provide a cellular and functional context to new interactions. By categorizing networks according to GO (Gene Ontology) and subcellular localization, a functional landscape is plotted. The third and very important step is validating novel cellular networks. The relevance of comparative proteomics analysis is that it outlines putative interactomes which define multiple novel functions for uncharacterized proteins. These putative interactomes must be validated experimentally and this is what Sowa et al. [5] have done. By reciprocal tagging of interactor candidates, and by using other techniques not based on MS analysis, they have defined robust interactions and validated novel actors. As a sample of how powerful the approach could be, by validating interactions, they propose novel roles for Dubs in Akt regulation, RNA processing, DNA-damage response, ribophagy and ERAD. Overall, this approach appears to be a tool exportable to almost any group of proteins of interest. Currently, CompPASS is a resource available online to any researcher (http://pathology.hms.harvard.edu/labs/harper/CompPASS.html). The method cannot follow up those interactions that, although being functionally relevant, are not reproduced in IPs, but this caveat is compensated for by the fact that it rationally substantiates the study of hundreds, if not more, of novel uncharacterized proteins.

Ubiquitin–Proteasome System, Dynamics and Targeting: 4th Intracellular Proteolysis Meeting, a Biochemical Society Focused Meeting held at Institut d'Estudis Catalans, Casa de Convalescència, Barcelona, Spain, 27–29 May 2009. Organized and Edited by Bernat Crosas (Institute of Molecular Biology of Barcelona, Spain), Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (University of Barcelona, Spain), Manuel Rodríguez (CIC bioGUNE, Derio, Spain) and Timothy Thomson (Institute of Molecular Biology of Barcelona, Spain)

Abbreviations

     
  • BRCA

    breast cancer early-onset

  •  
  • BAP1

    BRCA1-associated protein 1

  •  
  • CompPASS

    Comparative Proteomic Analysis Software Suit

  •  
  • COPS

    COP9 constitutive photomorphogenic homologue subunit

  •  
  • CYLD

    cylindromatosis

  •  
  • Dub

    deubiquitinating enzyme

  •  
  • eIF3

    eukaryotic initiation factor 3

  •  
  • ERAD

    endoplasmic-reticulum-associated degradation

  •  
  • GO

    Gene Ontology

  •  
  • HA

    haemagglutinin

  •  
  • HCIP

    high-confidence candidate-interacting protein

  •  
  • HEK

    human embryonic kidney

  •  
  • IP

    immunoprecipitation

  •  
  • MS/MS

    tandem MS

  •  
  • NF-κB

    nuclear factor κB

  •  
  • OTU

    ovarian tumour protease

  •  
  • PHLPP

    pleckstrin homology domain and leucine-rich repeat protein phosphatase

  •  
  • PSMD

    proteasome 26S subunit non-ATPase

  •  
  • STAM

    signal-transducing adaptor molecule

  •  
  • STAMBP

    STAM-binding protein

  •  
  • STAMBPL1

    STAMBP-like 1

  •  
  • UBE

    ubiquitin-conjugating enzyme

  •  
  • UCH

    ubiquitin C-terminal hydrolase

  •  
  • USP

    ubiquitin-specific protease

  •  
  • VCP

    valosin-containing protein

Funding

This work was supported by the Spanish Government Ministerio de Educación y Ciencia [grant number BFU2006-02928]

References

References
1
Finley
D.
Recognition and processing of ubiquitin-protein conjugates by the proteasome
Annu. Rev. Biochem.
2009
, vol. 
78
 (pg. 
477
-
513
)
2
Xu
P.
Duong
D.M.
Seyfried
N.T.
Cheng
D.
Xie
Y.
Robert
J.
Rush
J.
Hochstrasser
M.
Finley
D.
Peng
J.
Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation
Cell
2009
, vol. 
137
 (pg. 
133
-
145
)
3
Nijman
S.M.
Luna-Vargas
M.P.
Velds
A.
Brummelkamp
T.R.
Dirac
A.M.
Sixma
T.K.
Bernards
R.
A genomic and functional inventory of deubiquitinating enzymes
Cell
2005
, vol. 
123
 (pg. 
773
-
786
)
4
Komander
D.
Clague
M.J.
Urbé
S.
Breaking the chains: structure and function of the deubiquitinases
Nat. Rev. Mol. Cell Biol.
2009
, vol. 
10
 (pg. 
550
-
563
)
5
Sowa
M.E.
Bennett
E.J.
Gygi
S.P.
Harper
J.W.
Defining the human deubiquitinating enzyme interaction landscape
Cell
2009
, vol. 
138
 (pg. 
389
-
403
)
6
Weber
B.
Schaper
C.
Wang
Y.
Scholz
J.
Bein
B.
Interaction of the ubiquitin carboxyl terminal esterase L1 with α2-adrenergic receptors inhibits agonist-mediated p44/42 MAP kinase activation
Cell. Signalling
2009
, vol. 
21
 (pg. 
1513
-
1521
)
7
Trompouki
E.
Hatzivassiliou
E.
Tsichritzis
T.
Farmer
H.
Ashworth
A.
Mosialos
G.
CYLD is a deubiquitinating enzyme that negatively regulates NF-κB activation by TNFR family members
Nature
2003
, vol. 
424
 (pg. 
793
-
796
)
8
Gomez-Ferreria
M.A.
Rath
U.
Buster
D.W.
Chanda
S.K.
Caldwell
J.S.
Rines
D.R.
Sharp
D.J.
Human Cep192 is required for mitotic centrosome and spindle assembly
Curr. Biol.
2007
, vol. 
17
 (pg. 
1960
-
1966
)
9
Enesa
K.
Zakkar
M.
Chaudhury
H.
Luong le
A.
Rawlinson
L.
Mason
J.C.
Haskard
D.O.
Dean
J.L.
Evans
P.C.
NF-κB suppression by the deubiquitinating enzyme Cezanne: a novel negative feedback loop in pro-inflammatory signaling
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
7036
-
7045
)
10
Jariel-Encontre
I.
Bossis
G.
Piechaczyk
M.
Ubiquitin-independent degradation of proteins by the proteasome
Biochem. Biophys. Acta
2008
, vol. 
1786
 (pg. 
153
-
177
)
11
Buus
R.
Faronato
M.
Hammond
D.E.
Urbé
S.
Clague
M.J.
Deubiquitinase activities required for hepatocyte growth factor-induced scattering of epithelial cells
Curr. Biol.
2009
, vol. 
19
 (pg. 
1463
-
1466
)
12
Stanisic
V.
Malovannaya
A.
Qin
J.
Lonard
D.M.
O'Malley
B.W.
OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) deubiquitinates estrogen receptor (ER) α and affects ERα transcriptional activity
J. Biol. Chem.
2009
, vol. 
284
 (pg. 
16135
-
16145
)
13
Sato
Y.
Yoshikawa
A.
Yamagata
A.
Mimura
H.
Yamashita
M.
Ookata
K.
Nureki
O.
Iwai
K.
Komada
M.
Fukai
S.
Structural basis for specific cleavage of Lys63-linked polyubiquitin chains
Nature
2008
, vol. 
455
 (pg. 
358
-
362
)
14
Boeddrich
A.
Gaumer
S.
Haacke
A.
Tzvetkov
N.
Albrecht
M.
Evert
B.O.
Müller
E.C.
Lurz
R.
Breuer
P.
Schugardt
N.
, et al. 
An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis
EMBO J.
2006
, vol. 
25
 (pg. 
1547
-
1558
)
15
Joo
H.Y.
Zhai
L.
Yang
C.
Nie
S.
Erdjument-Bromage
H.
Tempst
P.
Chang
C.
Wang
H.
Regulation of cell cycle progression and gene expression by H2A deubiquitination
Nature
2007
, vol. 
449
 (pg. 
1068
-
1072
)
16
Zhang
D.
Zaugg
K.
Mak
T.W.
Elledge
S.J.
A role for the deubiquitinating enzyme USP28 in control of the DNA-damage response
Cell
2006
, vol. 
126
 (pg. 
529
-
542
)
17
Popov
N.
Wanzel
M.
Madiredjo
M.
Zhang
D.
Beijersbergen
R.
Bernards
R.
Moll
R.
Elledge
S.J.
Eilers
M.
The ubiquitin-specific protease USP28 is required for MYC stability
Nat. Cell Biol.
2007
, vol. 
9
 (pg. 
765
-
774
)
18
Stevenson
L.F.
Sparks
A.
Allende-Vega
N.
Xirodimas
D.P.
Lane
D.P.
Saville
M.K.
The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2
EMBO J.
2007
, vol. 
26
 (pg. 
976
-
986
)
19
Dayal
S.
Sparks
A.
Jacob
J.
Allende-Vega
N.
Lane
D.P.
Saville
M.K.
Suppression of the deubiquitinating enzyme USP5 causes the accumulation of unanchored polyubiquitin and the activation of p53
J. Biol. Chem.
2009
, vol. 
284
 (pg. 
5030
-
5041
)
20
Alwan
H.A.
van Leeuwen
J.E.
UBPY-mediated epidermal growth factor receptor (EGFR) de-ubiquitination promotes EGFR degradation
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
1658
-
1669
)
21
Setsuie
R.
Suzuki
M.
Kabuta
T.
Fujita
H.
Miura
S.
Ichihara
N.
Yamada
D.
Wang
Y.L.
Ezaki
O.
Suzuki
Y.
Wada
K.
Ubiquitin C-terminal hydrolase-L3-knockout mice are resistant to diet-induced obesity and show increased activation of AMP-activated protein kinase in skeletal muscle
FASEB J.
2009
, vol. 
23
 (pg. 
4148
-
4157
)
22
Ribarski
I.
Lehavi
O.
Yogev
L.
Hauser
R.
Bar-Shira Maymon
B.
Botchan
A.
Paz
G.
Yavetz
H.
Kleiman
S.E.
USP26 gene variations in fertile and infertile men
Hum. Reprod.
2009
, vol. 
24
 (pg. 
477
-
484
)
23
Yan
M.
Luo
J.K.
Ritchie
K.J.
Sakai
I.
Takeuchi
K.
Ren
R.
Zhang
D.E.
Ubp43 regulates BCR-ABL leukemogenesis via the type 1 interferon receptor signaling
Blood
2007
, vol. 
110
 (pg. 
305
-
312
)
24
Qiu
X.B.
Ouyang
S.Y.
Li
C.J.
Miao
S.
Wang
L.
Goldberg
A.L.
hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37
EMBO J.
2006
, vol. 
25
 (pg. 
5742
-
5753
)
25
Borodovsky
A.
Kessler
B.M.
Casagrande
R.
Overkleeft
H.S.
Wilkinson
K.D.
Ploegh
H.L.
A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14
EMBO J.
2001
, vol. 
20
 (pg. 
5187
-
5196
)
26
Tsurumi
C.
DeMartino
G.N.
Slaughter
C.A.
Shimbara
N.
Tanaka
K.
cDNA cloning of p40, a regulatory subunit of the human 26S proteasome, and a homolog of the Mov-34 gene product
Biochem. Biophys. Res. Commun.
1995
, vol. 
210
 (pg. 
600
-
608
)
27
Spataro
V.
Toda
T.
Craig
R.
Seeger
M.
Dubiel
W.
Harris
A.L.
Norbury
C.
Resistance to diverse drugs and ultraviolet light conferred by overexpression of a novel human 26 S proteasome subunit
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
30470
-
30475
)
28
Sharon
M.
Taverner
T.
Ambroggio
X.I.
Deshaies
R.J.
Robinson
C.V.
Structural organization of the 19S proteasome lid: insights from MS of intact complexes
PLoS Biol.
2006
, vol. 
4
 pg. 
e267
 
29
Schwechheimer
C.
The COP9 signalosome (CSN): an evolutionary conserved proteolysis regulator in eukaryotic development
Biochem. Biophys. Acta
2004
, vol. 
1695
 (pg. 
45
-
54
)
30
Masutani
M.
Sonenberg
N.
Yokoyama
S.
Imataka
H.
Reconstitution reveals the functional core of mammalian eIF3
EMBO J.
2007
, vol. 
26
 (pg. 
3373
-
3383
)
31
Scheel
H.
Hofmann
K.
Prediction of a common structural scaffold for proteasome lid, COP9-signalosome and eIF3 complexes
BMC Bioinformatics
2005
, vol. 
6
 pg. 
71
 
32
Zhang
X.Y.
Varthi
M.
Sykes
S.M.
Phillips
C.
Warzecha
C.
Zhu
W.
Wyce
A.
Thorne
A.W.
Berger
S.L.
McMahon
S.B.
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
2008
, vol. 
29
 (pg. 
102
-
111
)
33
Nicassio
F.
Corrado
N.
Vissers
J.H.
Areces
L.B.
Bergink
S.
Marteijn
J.A.
Geverts
B.
Houtsmuller
A.B.
Vermeulen
W.
Di Fiore
P.P.
Citterio
E.
Human USP3 is a chromatin modifier required for S phase progression and genome stability
Curr. Biol.
2007
, vol. 
17
 (pg. 
1972
-
1977
)
34
van Leuken
R.J.
Luna-Vargas
M.P.
Sixma
T.K.
Wolthuis
R.M.
Medema
R.H.
Usp39 is essential for mitotic spindle checkpoint integrity and controls mRNA-levels of aurora B
Cell Cycle
2008
, vol. 
7
 (pg. 
2710
-
2719
)
35
Wada
K.
Kamitani
T.
UnpEL/Usp4 is ubiquitinated by Ro52 and deubiquitinated by itself
Biochem. Biophys. Res. Commun.
2006
, vol. 
342
 (pg. 
253
-
258
)
36
Huang
X.
Langelotz
C.
Hetfeld-Pechoc
B.K.
Schwenk
W.
Dubiel
W.
The COP9 signalosome mediates β-catenin degradation by deneddylation and blocks adenomatous polyposis coli destruction via USP15
J. Mol. Biol.
2009
, vol. 
391
 (pg. 
691
-
702
)
37
Wang
L.
Bhattacharyya
N.
Chelsea
D.M.
Escobar
P.F.
Banerjee
S.
A novel nuclear protein, MGC5306 interacts with DNA polymerase β and has a potential role in cellular phenotype
Cancer Res.
2004
, vol. 
64
 (pg. 
7673
-
7677
)
38
Gorski
J.J.
Pathak
S.
Panov
K.
Kasciukovic
T.
Panova
T.
Russell
J.
Zomerdijk
J.C.
A novel TBP-associated factor of SL1 functions in RNA polymerase I transcription
EMBO J.
2007
, vol. 
26
 (pg. 
1560
-
1568
)
39
Faus
H.
Meyer
H.A.
Huber
M.
Bahr
I.
Haendler
B.
The ubiquitin-specific protease USP10 modulates androgen receptor function
Mol. Cell. Endocrinol.
2005
, vol. 
245
 (pg. 
138
-
146
)
40
Herrera-Vigenor
F.
Hernández-García
R.
Valadez-Sánchez
M.
Vázquez-Prado
J.
Reyes-Cruz
G.
AMSH regulates calcium-sensing receptor signaling through direct interactions
Biochem. Biophys. Res. Commun.
2006
, vol. 
347
 (pg. 
924
-
930
)
41
Endo
A.
Matsumoto
M.
Inada
T.
Yamamoto
A.
Nakayama
K.I.
Kitamura
N.
Komada
M.
Nucleolar structure and function are regulated by the deubiquitylating enzyme USP36
J. Cell Sci.
2009
, vol. 
122
 (pg. 
678
-
686
)
42
Jensen
D.E.
Proctor
M.
Marquis
S.T.
Gardner
H.P.
Ha
S.I.
Chodosh
L.A.
Ishov
A.M.
Tommerup
N.
Vissing
H.
Sekido
Y.
, et al. 
BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression
Oncogene
1998
, vol. 
16
 (pg. 
1097
-
1112
)
43
Nijman
S.M.
Huang
T.T.
Dirac
A.M.
Brummelkamp
T.R.
Kerkhoven
R.M.
D'Andrea
A.D.
Bernards
R.
The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway
Mol. Cell
2005
, vol. 
17
 (pg. 
331
-
339
)
44
Boudreau
H.E.
Broustas
C.G.
Gokhale
P.C.
Kumar
D.
Mewani
R.R.
Rone
J.D.
Haddad
B.R.
Kasid
U.
Expression of BRCC3, a novel cell cycle regulated molecule, is associated with increased phospho-ERK and cell proliferation
Int. J. Mol. Med.
2007
, vol. 
19
 (pg. 
29
-
39
)
45
Denuc
A.
Bosch-Comas
A.
Gonzàlez-Duarte
R.
Marfany
G.
The UBA-UIM domains of the USP25 regulate the enzyme ubiquitination state and modulate substrate recognition
PLoS ONE
2009
, vol. 
4
 pg. 
e5571
 
46
Uchiyama
K.
Jokitalo
E.
Kano
F.
Murata
M.
Zhang
X.
Canas
B.
Newman
R.
Rabouille
C.
Pappin
D.
Freemont
P.
Kondo
H.
VCIP135, a novel essential factor for p97/p47-mediated membrane fusion, is required for Golgi and ER assembly in vivo
J. Cell Biol.
2002
, vol. 
159
 (pg. 
855
-
866
)
47
Wertz
I.E.
O'Rourke
K.M.
Zhou
H.
Eby
M.
Aravind
L.
Seshagiri
S.
Wu
P.
Wiesmann
C.
Baker
R.
Boone
D.L.
, et al. 
De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling
Nature
2004
, vol. 
430
 (pg. 
694
-
699
)
48
Stegmeier
F.
Rape
M.
Draviam
V.M.
Nalepa
G.
Sowa
M.E.
Ang
X.L.
McDonald
E.R.
3rd
Li
M.Z.
Hannon
G.J.
Sorger
P.K.
, et al. 
Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities
Nature
2007
, vol. 
446
 (pg. 
876
-
881
)
49
Kayagaki
N.
Phung
Q.
Chan
S.
Chaudhari
R.
Quan
C.
O'Rourke
K.M.
Eby
M.
Pietras
E.
Cheng
G.
Bazan
J.F.
, et al. 
DUBA: a deubiquitinase that regulates type I interferon production
Science
2007
, vol. 
318
 (pg. 
1628
-
1632
)
50
Li
M.
Chen
D.
Shiloh
A.
Luo
J.
Nikolaev
A.Y.
Qin
J.
Gu
W.
Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization
Nature
2002
, vol. 
416
 (pg. 
648
-
653
)
51
Antrobus
R.
Boutell
C.
Identification of a novel higher molecular weight isoform of USP7/HAUSP that interacts with the Herpes simplex virus type-1 immediate early protein ICP0
Virus Res.
2008
, vol. 
137
 (pg. 
64
-
71
)
52
Yamaguchi
T.
Kimura
J.
Miki
Y.
Yoshida
K.
The deubiquitinating enzyme USP11 controls an IκB kinase α (IKKα)-p53 signaling pathway in response to tumor necrosis factor α (TNFα)
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
33943
-
33948
)
53
Schoenfeld
A.R.
Apgar
S.
Dolios
G.
Wang
R.
Aaronson
S.A.
BRCA2 is ubiquitinated in vivo and interacts with USP11, a deubiquitinating enzyme that exhibits prosurvival function in the cellular response to DNA damage
Mol. Cell. Biol.
2004
, vol. 
24
 (pg. 
7444
-
7455
)
54
Lin
C.H.
Chang
H.S.
Yu
W.C.
USP11 stabilizes HPV-16E7 and further modulates the E7 biological activity
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
15681
-
15688
)
55
Berthouze
M.
Venkataramanan
V.
Li
Y.
Shenoy
S.K.
The deubiquitinases USP33 and USP20 coordinate β2 adrenergic receptor recycling and resensitization
EMBO J.
2009
, vol. 
28
 (pg. 
1684
-
1696
)
56
Gao
T.
Furnari
F.
Newton
A.C.
PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth
Mol. Cell
2005
, vol. 
18
 (pg. 
13
-
24
)
57
Cohn
M.A.
Kowal
P.
Yang
K.
Haas
W.
Huang
T.T.
Gygi
S.P.
D'Andrea
A.D.
A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway
Mol. Cell
2007
, vol. 
28
 (pg. 
786
-
797
)
58
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.
2009
, vol. 
284
 (pg. 
5343
-
5351
)
59
Li
X.
Liu
J.
Gao
T.
β-TrCP-mediated ubiquitination and degradation of PHLPP1 is negatively regulated by Akt
Mol. Cell. Biol.
2009
, vol. 
29
 (pg. 
6192
-
6205
)
60
Wong
R.W.
Blobel
G.
Coutavas
E.
Rae1 interaction with NuMA is required for bipolar spindle formation
Proc. Natl. Acad. Sci. U.S.A.
2006
, vol. 
103
 (pg. 
19783
-
19787
)
61
Yoon
Y.M.
Baek
K.H.
Jeong
S.J.
Shin
H.J.
Ha
G.H.
Jeon
A.H.
Hwang
S.G.
Chun
J.S.
Lee
C.W.
WD repeat-containing mitotic checkpoint proteins act as transcriptional repressors during interphase
FEBS Lett.
2004
, vol. 
575
 (pg. 
23
-
29
)