A new map to understand deubiquitination: Table 1

Abstract

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.

Full text

Ubiquitin–Proteasome System, Dynamics and Targeting 21
A new map to understand deubiquitination
Elijah J. Katz, Marta Isasa and Bernat Crosas1
Institut de Biologia Molecular de Barcelona, CSIC, Barcelona Scientific Park, Baldiri i Reixac 15-21, 08028 Barcelona, Spain
Abstract
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,Lys
11,Lys
27,Lys
29,Lys
33,
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:
Key words: comparative proteomics, cytokine, deubiquitinating enzyme, signalling, ubiquitin–
proteasome system.
Abbreviations used: 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.
1To whom correspondence should be addressed (email bernat.crosas@ibmb.csic.es).
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,Lys
48,Lys
63, 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
Biochem. Soc. Trans. (2010) 38, 21–28; doi:10.1042/BST0380021 C
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22 Biochemical Society Transactions (2010) Volume 38, part 1
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 αactivity
[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.
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 [26–28].
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
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Ubiquitin–Proteasome System, Dynamics and Targeting 23
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 N
CYLD USP SPATA2L, SPATA2, CEP192, MGEA5, CAMK2D, MYO6 Dif, Dev N, Cp
DUB3 USP LOC392197, LOC402164, CBX1, SET DNA-R, Ub, Ch N
JOSD1 MJD YOD1, CALM1, TIMM8A Dev, VT, F M
JOSD2 MJD TYSND1, KIAA0828, AHCYL1, TRAPPC2, SMARCA2 Tc, Met ER
LOC402164 USP BTF3, NACAP1, DUB3, LOC392197, ADAMTS1, LGMN, USP22 Ub, P V
OTUB1 OTU STAMBP, UBE2N, UBE2D2, MSH2, CASP14, CPNE7 Ub, A N
OTUB2 OTU GTF2I, VCPIP1 Tc, ST N
OTUD6B OTU OTUB1, ASCC3, MTDH, BXDC1 Ub, Tc N
OTUD7B OTU ACAD9, SLC9A3R2, HIF1AN, NUP155 O N
PARP11 OTU ZNF313, WDR23, LYZ Diff, Dev, Cat, Met O
STAMBPL1 JAMM/MPN USP49, OTUB1, CLINT1, UBE2N, SNRPA1, HMX3, BAP1 Ub, Tc N
UCHL1 UCH CCDC14 Dev Cs
UCHL3 UCH CLPB, USP20 - -
USP2 USP LONP1, RRP15 P M
USP5 USP USP13 Ub -
USP8 USP USP25, USP22, LOC440587 Dev Cs
USP16 USP DBT, HERC2, HIST1H2BL Ub, B, Met, VT M
USP18 USP USP41, JMJD1B, MYL6, NME1 - -
USP26 USP LOC392197, DUB3, LOC402164 Ub -
USP28 USP TP53BP1, AQR, SUMO2 N
USP29 USP USP25, CTSB A, P V, M
USP30 USP QKI, MPND, SS18L1, USP4, TIMM8A, CLPB, SF3A1 Dev, RNA N
USP33 USP ZFR, IFIT5, KRR1, PRPF38B O, RNA N
USP37 USP FBXW11, ALB Ub, A, ST, O O
USP38 USP HSPB1, HMX3, LGALS7, RPS12, RPL7 A, Tl Cp
USP48 USP LOC442227 - -
YOD1 OTU MUTED, THOC3 RNA N
Group 2 COPS5 JAMM/MPN COP9 Signalosome, Kelch domain proteins, CUL2, LRRC14, CUL4B,
WDR21A, WDR23
Ub, Mit N
COPS6 JAMM/MPN COP9 Signalosome, Kelch domain proteins, CUL4B, CUL2, LRRC14, FBXW9,
BTBD2, FBXO7, BTBD9
Ub, Mit N
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 N
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 N
USP15 USP Spliceosome complex, LRRC15, RNF40, MYH4, SELENBP1, FABP4, VSIG8,
MYH2, TUT1, PRPF3, ADSL
RNA N
USP22 USP SAGA complex, ENY2, TADA1L, LOC254571, ATXN7L(3,2), MGC21874, KIF7,
FAM48A, LOC552889, USP27X
Tc N
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24 Biochemical Society Transactions (2010) Volume 38, part 1
Table 1 Continued
Group Dub Class Interactors GO-P GO-C
USP39 USP Spliceosome complex, RG9MTD1, PA2G4, ZRANB2, RY1, PRPF(4B, 3, 4),
TSR2, KIAA0409, C17orf79
RNA N
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 N
BRCC3 JAMM/MPN KIAA0157, HSPC142, BRE, CCDC98, UIMC1, EXOC8, SUPT16H, E2F5, DDOST,
MCM2, CAND1, SHMT2
Tc N
OTUD4 OTU GLA, GALK1, DSG1, PARP11, MYCBP, NMD3, MOG, GPHN, BAG5, DNAJB1,
FLNC, TUBA1A
OCs
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 N
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
-N
USP32 USP TUBA1A, CDC2, YWHAB, USP6, VPS35, LOC51035, MRPL39, ABCD3, USP11,
TRIP13, TXNDC4, SMC1A
ON
USP36 USP WDR36, WDR3, UTP18, PWP2, TBL3, DHX33, NUDCD1, MPND, STK25,
DDX41, GNL2, CHD4, TMEM104
ST N
USP45 USP SF3A2, RBMX, POLR2G, MYH10, CORO1C, MRLC2, RTCD1, SRBD1, MYH9,
TMOD3, PIK3CG, GLTSCR2
RNA N
USP50 USP ABCE1, SLC1A5, DYNC2H1, LUC7L, FLJ20294, IGF2R, KEAP1, AMOT, CKAP4,
AFG3L2, VCP complex
OM,N
USP52 USP VBP1, TCEB2, TCEB1, PFDN2, PAN3, USP5, PLEC1, NUP93, MRPL39, ARCN1,
CBWD2, CCT7
FN
USPL1 USP PGD, PKLR, ANKFY1, KIAA0947, ELL, KIF5B, IGHA2, CKB, ANXA1, PIP O Cp, Cs, O
VCPIP1 OTU NSFL1C, UFD1L, NPLOC4, VCP, UBXD4, ABCC12, LOC137886,
KFZp313A2432, UBXD8, HUWE1
Ub N
Group 6 OTUD5 OTU LONRF2, GPX4, LANCL2, CTPS2, GYS1, VARS, FLNA, CACYBP, SET, GRB2,
USP11, TP53, PKLR
Dev N
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 N
USP42 USP PLRG1, TEX10, C14orf169, AMOT, USP32, AHCYL1, DIMT1L, USP20, FECH,
MRPS14, CA2, MRPS31
-N
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 N
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Table 1 Continued
Group Dub Class Interactors GO-P GO-C
USP11 USP SIPA1L1, ZNF24, USP4, MRE11A, USP7, RAD50, TP53, PKN2, TCEAL(1,4),
TRIP12, MPHOSPH9, OTUD1
Tc N
USP19 USP CSE1L, CDC2, HMOX2, TNPO3, CAMK2G, LAT, DMWD, KIAA1787, HERC2,
ACADSB, PARK7, UNC45A
VT N
USP20 USP PSMD6, EIF3S6, PSMD7, EIF3S8, PSMD11, PSMD12, VAPA, PLEKHA7,
RAD17, SYTL4, N4BP3, UCHL3
ON,Cp
USP49 USP PDCD2, LRPPRC, Centrin proteins, SAPS domain proteins, USP44, F7, FKBP5,
SHCBP1, PPP5C, ANKRD28
VT N
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 comple-
mentation 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 inter-
actors. 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 charac-
terized 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
C
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26 Biochemical Society Transactions (2010) Volume 38, part 1
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 degrada-
tion 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 me-
thod 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.
Funding
This work was supported by the Spanish Government Ministerio de
Educaci ´on y Ciencia [grant number BFU2006-02928]
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Received 23 October 2009
doi:10.1042/BST0380021
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