Biochemical characterization of USP7 reveals
post-translational modification sites and structural
requirements for substrate processing and subcellular
localization
Amaury Ferna
´
ndez-Montalva
´
n
1
, Tewis Bouwmeester
2
, Gerard Joberty
2
, Robert Mader
3
,
Marion Mahnke
4
, Benoit Pierrat
1
, Jean-Marc Schlaeppi
4
, Susanne Worpenberg
1
and Bernd Gerhartz
1
1 Expertise Platform Proteases, Novartis Institutes for Biomedical Research, Basel, Switzerland
2 Cellzome AG, Heidelberg, Germany
3 Musculoskeletal Disease Area, Novartis Institutes for Biomedical Research, Basel, Switzerland
4 Biologics Centre, Novartis Institutes for Biomedical Research, Basel, Switzerland

2007, accepted 25 June 2007)
doi:10.1111/j.1742-4658.2007.05952.x
Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinat-
ing enzymes. Among other functions, USP7 is involved in the regulation of
stress response pathways, epigenetic silencing and the progress of infections
by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core,
N- and C-terminal domains required for protein–protein interactions. In
the present study, recombinant USP7 full length, along with several vari-
ants corresponding to domain deletions, were expressed in different hosts
in order to analyze post-translational modifications, oligomerization state,
enzymatic properties and subcellular localization patterns of the enzyme.
USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in
mammalian cells. In in vitro activity assays, N- and C-terminal truncations
affected the catalytic efficiency of the enzyme different. Both the protease
core alone and in combination with the N-terminal domain are over 100-
fold less active than the full length enzyme, whereas a construct including
the C-terminal region displays a rather small decrease in catalytic effi-
ciency. Limited proteolysis experiments revealed that USP7 variants con-
taining the C-terminal domain interact more tightly with ubiquitin. Besides
playing an important role in substrate recognition and processing, this
region might be involved in enzyme dimerization. USP7 constructs lacking
the N-terminal domain failed to localize in the cell nucleus, but no nuclear
localization signal could be mapped within the enzyme’s first 70 amino
acids. Instead, the tumor necrosis factor receptor associated factor-like
region (amino acids 70–205) was sufficient to achieve the nuclear localiza-
tion of the enzyme, suggesting that interaction partners might be required
for USP7 nuclear import.
Abbreviations
CBP, calmodulin binding protein; DUB, deubiquitinating enzyme; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase;
NLS, nuclear localization signal; SUMO-1, small ubiquitin-like modifier protein 1; TAP, tandem affinity purification; TRAF, tumor necrosis

ubiquitinated and polyneddylated [16]. The sites or
regions involved in these events have not been mapped
so far. The N-terminal of USP7 part displays sequence
homology to the TNF receptor associated factors
(TRAFs) and was shown to interact with several
TRAF family proteins [17]. This domain also binds
fragments derived from p53, MDM2 and the Epstein–
Barr virus nuclear antigen 1 (EBNA1) proteins in vitro
[14,15,18–21]. Recently, elucidation of the 3D-structure
of an USP7 fragment containing amino acids 54–204
disclosed an eight-stranded beta sandwich fold typical
for the TRAF protein family [15]. Further cocrystal
structures with substrate-derived peptides, revealed
that a P ⁄ AXXS consensus sequence is recognized
mainly by residues W165 and N169 located in a shal-
low surface groove on the TRAF domain [15,19,21].
Limited proteolysis identified two digestion resistant
fragments in the C-terminal region of USP7, mapping
to amino acids 622–801 and 885–1061 [18]. The first of
these polypeptides was shown to mediate the inter-
action of USP7 with the herpes virus protein ICP0
in vitro [18]. Additionally, a yeast two hybrid screen
revealed a region including amino acids 705–1102 was
required for association with Ataxin-1 [22] (Fig. 1A).
Further structural–functional features of this domain
are currently unknown. Sequence analysis anticipated
a protease domain with conserved Cys and His
boxes delimited by the N- and C-terminal regions [3].
Ataxin binding
Ubiquitin binding

structure. The N-terminal TRAF-like domain (amino acids 50–205) is
preceded by a Q-rich region not represented here. This domain has
been reported to interact with p53, MDM2 and Epstein–Barr virus
nuclear antigen 1. The protease core (amino acids 208–560) con-
tains the catalytic triad formed by the conserved residues C223,
H464 and D481. Two protein–protein interaction sites at amino
acids 599–801 and 705–1102 were described in this region for ICP-
0 and Ataxin-1. (B) Design of USP7 variants used in this work. Con-
structs comprising USP7 full length (FL) and amino acids 1–560,
208–560 and 208–1102, were prepared for expression in different
hosts. Constructs expressed using the baculovirus system (all
except the protease core) had a C-terminal hexahistidine tag. The
catalytic domain was expressed as a GST-6XHis N-terminal fusion
protein. Variants designed for expression in mammalian cells had
an N-terminal 3XFLAG tag and a C-terminal Myc tag. USP7-FL con-
structs used for proteomics analysis contained either N- or C-termi-
nal CBP-Protein A tags separated by a TEV-protease cleavage site.
A. Ferna
´
ndez-Montalva
´
n et al. Biochemical characterization of USP7
FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4257
Matching these predictions, limited proteolysis and
X-ray crystallography disclosed amino acids 208–560
as the protease core of USP7 [20] (Fig. 1A). Two
crystal structures of this fragment alone and in
complex with ubiquitin (Ub)-aldehyde revealed a
‘Fingers’, ‘Palm’ and ‘Thumb’ three-domain archi-
tecture, apparently conserved throughout the USPs

and purification system was used for the production of
several USP7 domain deletion variants (Fig. 1B) in
Baculovirus-infected insect cells. The procedure yielded
approximately 6 mg (USP7 full length), 5 mg (1–560)
and 4 mg (208–1102) of purified recombinant protein
per litre of insect cell culture. In addition, an average
of 7 mg USP7 208-560 per litre of Escherichia coli fer-
mentation broth was obtained from the soluble cell
fraction. The recombinant proteins were purified to
homogeneity (‡ 90%) based on SDS ⁄ PAGE (Fig. 2)
and reversed phase HPLC analysis. N-terminal
sequencing showed that both USP7-FL and USP7
1-560 expressed in insect cells were N-terminally blocked
by acetylation, as confirmed by MALDI-TOF-MS.
LC-MS analysis of USP7-FL revealed two protein
masses of 130 464.0 and 130 540.0 Da, corresponding
very likely to acetylated and single phosphorylated
USP7, respectively. Again, two masses of 65 919.5 and
65 999.5 were found for USP7 1-560, corresponding
likewise to acetylated and single phosphorylated
USP7 1-560, respectively. This post-translational
modification was later confirmed in USP7 purified
from mammalian cells (see below). LC-MS analysis
of USP7 208-1102 showed that around 60% of the
protein had a three amino acid truncation at the
N-terminus. None of these modifications or hetero-
geneities was observed in the 208-560 protein produced
in E. coli.
All USP7 variants were subjected to limited proteo-
lysis by trypsin under native conditions, in order to

ously described [18,20].
Identification of post-translational modifications
in USP7 purified from mammalian cells
The observation that USP7 expressed in insect cells
was phosphorylated in its N-terminal region motivated
us to investigate post-translational modifications on
tandem affinity purification (TAP)-tagged USP7 puri-
fied from mammalian cells. LC-MS ⁄ MS analysis
revealed the presence of two phosphopeptides AGE
QQLSEPEDMEMEAGDTDDPPR, corresponding to
amino acids 12 to 35, and IIGVHQEDELLECLSP
ATSR, corresponding to amino acids 949–968. Manual
verification of the corresponding MS ⁄ MS spectra
allowed for the assignment of the phosphoacceptor res-
idues to S18 and S963, respectively (Fig. 3A). USP7
was previously described to be ubiquitinylated and
neddylated. Western analysis showed that affinity puri-
fied TAP-tagged USP7 is (mono)-ubiquitinylated in
HeLa cells (Fig. 3B). LC-MS ⁄ MS identified a single
ubiquitinylated ⁄ neddylated peptide, DLLQFFKPR
corresponding to amino acids 863–871. Manual inspec-
tion of the MS ⁄ MS spectra showed that the diglycine
remnant was conjugated to K869. The strong identifi-
cation of ubiquitin in the same gel band as USP7,
combined with the absence of Nedd8, strongly suggests
that the modified site is indeed ubiquitinylated.
Analysis of USP7 oligomerization: possible role
of the C-terminal region
USP7 was reported to exist both as dimer in cells [16],
and as a monomer in solution [18,20]. Interestingly,

USP7 as described above revealed the presence of the
nontagged USP7 N-terminal peptide (MNHQQQQQ
QQK) derived from the endogenous enzyme (not
shown). Interestingly, variants lacking the C-terminal
region ran as a single band in the native nonreducing
PAGE (supplementary Fig. S1B), suggesting a role for
the C-terminal in the oligomerization event.
Substrate specificity and enzymatic properties
of USP7
As part of the characterization of USP7 biochemical
properties, we have measured its kinetic parameters for
the hydrolysis of ubiquitin C-terminal 7-amido-4-meth-
ylcoumarin (Ub-AMC), a fluorogenic substrate which
has proven to be an useful tool with a number of
deubiquitinating enzymes [28–31]. In order to assess its
substrate specificity, USP7 activities on small ubiqu-
itin-like modifier protein 1 (SUMO-1)-AMC, Nedd8-
AMC and Z-LRGG-AMC, a synthetic peptide
substrate representing the C-terminus of ubiquitin,
were investigated. In addition, we evaluated the
hydrolysis by the enzyme of ubiquitin C-terminal-Lys-
tetramethylrhodamine (Ub-K-TAMRA) and Ub-K-
peptide-TAMRA, two substrates with the fluorophore
group attached as isoamide bond. The Ub-AMC assay
described in the experimental section was linear for at
least 1 h at enzyme concentrations up to 5 nm. Using
similar conditions with SUMO-1-AMC and Nedd8-
AMC as substrates, no USP7 activity could be
detected, indicating a high specificity for ubiquitin,
despite the well known homologies among ubiquitin-

b10 2+
567.9
600.4
b10
1134.7
750.4
b9
1021.7
b11
1247.9
b12
1376.8
MH
3
3+
-H
3
PO
4
y''6-
H
3
PO
4
LE/EL
b2
227.2
y'‘8 2+
y8
971.6

0
100
%
b2
229.1
a2
201.1
y''5
808.5
y''2
272.2
y''4
661.4
b3
342.2 y''3
514.3
y''6
936.6
y''7
1049.7
(GG)K
Fig. 3. Characterization of USP7 post-translational modifications. (A) LC-MS ⁄ MS spectrum of the USP7 tryptic peptide IIGVHQEDELLECL ⁄
(pS)PATSR containing the phosphorylated residue S963. (B) Left panel: western blot detection of TAP-tagged USP7 and ubiquitinylated proteins
throughout the two-step tandem affinity purification from mammalian cells using anti-CBP and anti-ubiquitin sera. Cells were either nontreated
or pretreated with the proteasome inhibitor MG132. Right panel: LC-MS ⁄ MS spectrum of the USP7 tryptic peptide DLLQFF ⁄ (Ub-K)PR
containing the ubiquitinated residue K869.
Biochemical characterization of USP7 A. Ferna
´
ndez-Montalva
´

and 0.1% (w ⁄ v) Chaps using 1.56 n
M of USP7 full length. Ub-AMC, SUMO-1-AMC and Nedd8-AMC were at 1 lM. Each data point repre-
sents the average of at least two independent experiments with two replicas each. (B,C) Dependence of enzyme velocity on the pH (B),
ionic strength or viscosity (C) for the USP7-catalyzed hydrolysis of Ub-AMC. Reactions were conducted at room temperature in appropriate
buffers for each pH (see experimental section) or in 25 m
M Tris ⁄ HCl, buffer, pH 7.5, 5 mM dithiothreitol and 0.1% (w ⁄ v) CHAPS at the indi-
cated concentrations of NaCl (j), NaSCN (d), Na-citrate (m) or glycerol (h). In these experiments, the nominal concentration of USP7 was
5n
M and Ub-AMC was at 1 lM.(D) Linearity range of the Ub-AMC hydrolysis reactions catalyzed by USP7-FL (j), USP7 1-560 (d), USP7
208-560 (m) and USP7 208-1102 (.). These experiments were conducted at room temperature in 50 m
M Tris ⁄ HCl buffer, pH 7.5, 1 mM
EDTA, 5 mM dithiothreitol, 100 mM NaCl and 0.1% (w ⁄ v) Chaps with 1 lM Ub-AMC and the enzyme concentrations indicated in the experi-
mental section.
A. Ferna
´
ndez-Montalva
´
n et al. Biochemical characterization of USP7
FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4261
variants at increasing enzyme concentrations revealed
that different amounts of each protein were required
to attain comparable reaction velocities (Fig. 4D). The
kinetic parameters for these reactions were determined
by measuring their rates at increasing substrate con-
centrations. To this end, enzyme concentrations that
allowed assay linearity for at least 1 h were used. As
shown in Table 1, the deletion variants recognized
both substrates with similar affinities, but remarkable
differences were observed in the turnover (k
cat

ubiquitin (Fig. 5). N-terminal sequencing of them con-
firmed that the cleavage sites corresponded to those
observed in the experiment described above (Fig. 2).
However, stabilization of some proteolysis products in
the presence of ubiquitin was observed, demonstrating
a partial protection of some trypsin cleavage
sequences. The main fragment stabilized in the full
length enzyme contained amino acids I36 to R558.
This effect was less pronounced in USP7 1-560. In
variants lacking the N-terminal domain, a fragment
corresponding to amino acids K209 to R559 was stabi-
lized by the presence of ubiquitin. Interestingly, this
behavior was more evident for USP7 208-1102. In
both digestion products, the cleavage site protected by
the presence of ubiquitin was Ser341, located in the
‘fingers’ region of the catalytic core domain involved
in the recognition of the ubiquitin core. These results
show that all USP7 variants were able to bind ubiqu-
itin through the protease core domain, suggesting that
Fig. 5. Limited proteolysis of USP7 variants in the presence and
absence of ubiquitin. SDS ⁄ PAGE (4–20% gradient gels) showing the
limited proteolysis of native USP7-FL and variants thereof by trypsin
over time with and without ubiquitin. The arrows indicate fragments
from USP7-FL and USP7 208-1102 protected from tryptic digestion by
the presence of ubiquitin. N-terminal sequences of these fragments
are shown on the right accompanied by the symbols used in Fig. 2.
Table 1. Kinetic parameters for the hydrolysis of Ub-AMC (a) and Ub-K-TAMRA (b) by USP7 domain deletion variants.
USP7 variant Substrate [Protein] (n
M) K
M

a
0.077 1.7 · 10
3
119
Ub-K-TAMRA 2000 36.8 ± 4.9
a
0.039 1.1 · 10
3
936
208–1102 Ub-AMC 5 22.8 ± 2.1 0.805 3.53 · 10
4
6
Ub-K-TAMRA 100 7.2 ± 0.8 0.33 4.58 · 10
4
23
a
K
m
values higher than the maximum substrate concentrations used for the titrations should be considered as approximate figures.
Biochemical characterization of USP7 A. Ferna
´
ndez-Montalva
´
n et al.
4262 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
the enzyme–substrate complexes were more stable in
the context of an intact C-terminal region.
Structural requirements for USP7 nuclear
localization
In order to further characterize structure–function rela-

or dimers of the enzyme were detected in vitro, whereas
in cells evidence was obtained pointing to oligomeriza-
tion events. Deletion of the N- and C-terminal
domains of USP7 affected the activity of the enzyme,
with the C-terminus having a major impact. Interest-
ingly, this region appears to be required for enzyme
oligomerization. Finally, we have observed that the
N-terminal domain of USP7, and particularly a frag-
ment including amino acids 70–205, is sufficient to
achieve nuclear localization of the enzyme.
Based on our results, USP7 can be added to the list
of deubiquitinating enzymes found to be phosphory-
lated [33–35]. In fact, phosphorylation on S18 had been
reported previously from a HeLa large scale proteomics
study [36]. This phosphorylation site is a low stringency
consensus site for casein kinase II. Noteworthy, the
casein kinase II catalytic subunits alpha1 and alpha2
and regulatory subunit beta were copurified with
tagged USP7, suggesting that CKII could indeed be the
upstream kinases responsible for the phosphorylation
at this position (data not shown). S963 phosphoryla-
tion has not been described so far and this position is
not a known consensus site for any kinase. Interest-
ingly, both sites are located near regions involved in
protein–protein interactions. By analogy with the
DUB CYLD [35] and TRAF family members such as
TANK [37,38], whose function is modulated by the
inhibitor of jB kinase, a regulatory role can be pre-
sumed for USP7 phorsphorylation. The identification
of K869 as the ubiquitination site of USP7 represents

M
¼ 0.554 lm) [25], respec-
tively, and displays a similar K
M
as USP8 (K
M
¼
10.2 lm) [39]. Differences in the ubiquitin recognition
mechanisms and in the structural rearrangements upon
substrate binding displayed by UCHL-1 [37], UCHL-3
[20,40,41] and USP5 [32,42], might account for the
variations in affinity with respect to USP7. Renatus
et al. [25] discussed recently the possible origin of the
substrate affinity divergences compared to USP7 in a
detailed analysis of the interaction of USP2 catalytic
core with ubiquitin. Despite the higher K
M
, the cata-
lytic efficiency of USP7 (k
cat
⁄ K
M
) is only weaker
A. Ferna
´
ndez-Montalva
´
n et al. Biochemical characterization of USP7
FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4263
compared to that of UCHL-3 (2.1 · 10

) [25] and USP8 (2.35 · 10
5
m
)1
Æs
)1
) [39]. This value is only higher than those
reported for USP14 (UBP6 in yeast) (1.07 ·
10
2
m
)1
Æs
)1
) [43] and the viral SARS-CoV PLpro
(2.69 · 10
2
m
)1
Æs
)1
and 1.31 · 10
4
m
)1
Æs
)1
) [28,31].
The pH and ionic strength dependencies of USP7 for
activity on Ub-AMC are similar to those described

important support for substrate processing seems to be
provided by the C-terminal domain. This is the second
known example of mutations outside the catalytic core
affecting the enzymatic properties of a DUB. In UBPt,
the testis-specific murine homologue of USP2, N-ter-
minal domain deletions mimicking splice variants of
the enzyme influenced not only its subcellular localiza-
tion [44], but also its substrate specificity [45]. In
USP7, the noncatalytic domains might be involved in
specificity determination as well. This idea is supported
by the k
cat
⁄ K
M
increase measured exclusively for the
full length enzyme when a P1¢ lysine residue was linked
to ubiquitin through an e-amino bond in order to bet-
ter mimic the a physiological substrate. Of note,
attaching of a TAMRA-labeled undecapeptide not
related to any known USP7 interaction partner rather
decreased the catalytic performance of the enzyme,
apparently due to reduced substrate affinity. Making
the assumption that the primary function of this
enzyme is to detach monoubiquitin tags from modified
proteins rather than to process of ubiquitin chains, this
observation might explain the lower catalytic efficien-
cies displayed with K48 linked diubiquitin [20], and
support the existence of substrate primed subsite speci-
ficity requirements for USP7.
We have shown that the N-terminal domain is suffi-

suggesting that nuclear localization of this enzyme
might be dependent on its interactions with one or
several of the above mentioned partners.
Although the role of USP7 is by far not fully under-
stood, evidence accumulates in favor of its potential as
therapeutic target in cancer indications. The molecular
insight provided by the crystal structure of its catalytic
domain in complex with ubiquitin will guide the design
of potent inhibitors for this enzyme. However, difficul-
ties to attain selectivity are predicted based on the
experience accumulated with other cysteine proteases.
In this context, a better understanding of the involve-
ment of noncatalyitic domains in enzyme function may
open opportunities for alternative drug discovery
approaches such as allosteric and protein–protein
interaction inhibitors.
Experimental procedures
Materials
All chemicals were purchased from Sigma (St Louis, MO,
USA) and Merck (Darmstadt, Germany) in reagent grade.
Restriction enzymes were from Roche (Manheim, Ger-
many). Pfu proofreading polymerase and other DNA modi-
fying enzymes were from Promega (Madison, WI, USA).
USP7 polyclonal antibody (BL851) was from Bethyl Labo-
ratories (Montgomery, TX, USA). Ubiquitin monoclonal
antibody (Ubi1) was from Zymed (Invitrogen, Carlsbad,
CA, USA) and calmodulin binding protein (CBP) antibody
was from Upstate (Millipore, Billerica, MA, USA). Anti-
FLAG (M2) and anti-myc (2E10) monoclonal sera were
purchased from Sigma. Rabbit anti-mouse-HRP and goat

CA, USA). The construct included an engineered PreScission
(GE Healthcare, Chalfont St Giles, UK) protease cleavage
site to remove tags. In order to create mammalian expression
vectors, DNA sequences comprising full length USP7, amino
acids 1–560, 208–560 and 208–1102 were amplified with
primers containing restriction sites for HindIII and XbaIas
5¢- and 3¢-overhangs, respectively. The PCR products
obtained were ligated into pCR2.1-TOPO and further
subcloned into p3XFLAG-myc-CMV-26
TM
(Sigma). The
resulting constructs contained a N-terminal 3XFLAG and a
C-terminal myc tag. For the generation of C-terminal EGFP
fusions, USP7 amino acids 1–205, 20–205, 50–205 and
70–205 were amplified with BamHI and AgeI5¢- and 3¢-over-
hangs, ligated into pCR2.1-TOPO and finally subcloned into
the vector pEGFPN1 (Clontech, EMD Biosciences). For
TAP experiments, four USP7 full length constructs were
prepared as described by Rigaut et al. [51]. These included
wild-type USP7 and an active site mutant (C223A) (created
with the QuickChange mutagenesis kit from Stratagene, La
Jolla, CA, USA), each of them with either N- or C-terminal
CBP-Protein A tags.
Expression and purification of USP7 variants
Three constructs, USP7 full length (USP7-FL), USP7 resi-
dues 1–560 and USP7 residues 208–1102 were prepared in
the Baculovirus expression system. Large-scale fermentation
and purification of the recombinant tagged proteins was
performed by a semiautomated process as described previ-
ously by Schlaeppi et al. [50], with a minor modification of

purification step was used to separate the tag and unpro-
cessed fusion protein from free USP7 208-560. The protein
was then concentrated and dialyzed against 10 mm
Tris ⁄ HCl buffer pH 8.0, containing 200 mm NaCl, 5%
glycerol and 5 mm dithiothreitol prior to loading on a
26 ⁄ 60 Superdex 75 size exclusion column (GE Healthcare)
equilibrated in the same buffer. The purity and integrity of
the proteins was controlled after every step by SDS ⁄ PAGE
on Novex
TM
precast 4–12% or 4–20% gradient gels and
electrophoresis chambers (Invitrogen), followed by Coo-
massie Blue staining.
Limited proteolysis
Proteins at concentrations varying from 0.1 to 0.3 gÆL
)1
were digested for 60 min with tosylphenylalanylchlorome-
thane-treated bovine trypsin (Sigma) at a molar ratio of
100 : 1 in a final volume of 150 lL. Reactions were stopped
by addition of one volume of 2 · Laemmli sample buffer,
followed by boiling at 95 °C for 5 min. The effect of ubiqu-
itin on the tryptic digestion patterns of USP7 was analyzed
using similar conditions. Each variant was incubated with
or without bovine ubiquitin (Sigma) at a final concentration
of 12.5 mm. Aliquots of 20 lL were removed 5, 15, 30 and
60 min upon addition of trypsin, mixed 1 : 1 (w ⁄ w) with
2 · Laemmli sample buffer, and boiled at 95 °C for 5 min.
In both cases, digestion fragments were separated by
SDS ⁄ PAGE (4–20% gels) and either visualized by Coomas-
sie Blue staining or transferred to Invitrogen’s Immobi-

Analytical size exclusion chromatography
coupled to light scattering analysis
Molecular weight determination of USP7 was carried out
using a custom made Sephacryl S-300 HR 10 ⁄ 600 analytical
column (GE Healthcare), with a fractionation range of
10–1500 kDa. The column was connected to an A
¨
KTA
Explorer 100 chromatography system (GE Healthcare) and
to a MiniDawn Tristar linked to an interferometric refrac-
tometer Optilab DSP (Wyatt Technology, Santa Barbara,
CA, USA). Molecular masses were calculated with astra
4.90 software (Wyatt Technology). The column was equili-
brated with NaCl ⁄ P
i
and 0.5 mL of USP7-FL protein
(1.5 mgÆmL
)1
) was injected into the column. The flow rate
was 0.5 mLÆmin
)1
. The SEC column was calibrated using
the high molecular weight protein calibration kit of GE
Healthcare.
Enzyme activity assays
Activity towards Ub-AMC (Boston Biochem, Cambridge,
MA, USA) was assayed at room temperature in 50 mm
Tris ⁄ HCl buffer at pH 7.5, with 1 mm EDTA, 5 mm dithio-
threitol, 100 mm NaCl and 0.1% (w ⁄ v) Chaps. Assays were
performed on Cliniplate black 384-well plates (Thermo

0.1% (w ⁄ v) Chaps.
Activity measurements using Ub-K-TAMRA and ubi-
quitin C-terminal-Lys- attached to the TAMRA-labeled
undecapeptide LIFAGKQLEQG (Ub-K-peptide-TAMRA)
described previously [54] as substrates were performed at
room temperature in 0.1 m Hepes pH 7.5, containing
0.5 mm EDTA, 5 mm dithioerythritol (freshly added) and
0.05% (w ⁄ v) Chaps. Assays were performed in 96-well
plates, with a total assay volume of 30 lL. USP7 variants
at the concentrations indicated in Table 1 were incubated
with substrate concentrations varying from 1 to 16 lm in
two-fold increments. Reactions were stopped at different
time points by adding 5 lLof50mm iodoacetamide. Prod-
uct formation was measured using an Agilent 1100 HPLC
instrument (Agilent Technologies, Palo Alto, CA, USA)
equipped with a Poroshell 300SB-C18 reverse phase col-
umn. Substrate and product peaks were separated with a
3.5 min linear gradient of 0–100% acetonitrile containing
0.1% (v ⁄ v) trifluoroacetic acid and visualized using excita-
tion and emission wavelengths of 543 nm and 580 nm,
respectively.
In order to calculate the kinetic parameters for the hydro-
lysis of Ub-AMC and Ub-K-TAMRA, curves obtained by
plotting the measured enzyme initial rates (v) versus the cor-
responding substrate concentrations ([S]) were subjected to
nonlinear regression fit to the Michaelis–Menten equation
V ¼ (V
max
· [S]) ⁄ ([S]+K
M

(Roskilde, Denmark) using plasmid DNA purified with an
endotoxin free Maxiprep kit from Qiagen. As DNA carrier,
FUGENE
TM
reagent (Roche) was used, following manu-
facturer’s guidelines. Twenty four hours after transfection,
cells were transferred to appropriate culture vessels (see
below).
Cell lysis, sample preparation and
immunoblotting
Extracts from nontransfected and transfected cells
were prepared in ice-cold CelLytic
TM
lysis buffer for
A. Ferna
´
ndez-Montalva
´
n et al. Biochemical characterization of USP7
FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4267
mammalian cells supplemented with a protease inhibitor
cocktail (both from Sigma) following the manufacturer’s
instructions. For immunobloting, clear lysates were mixed
1: 1 (v⁄ v) with 2 · Laemmli SDS sample buffer, boiled
5 min at 95 °C and spun down at 12 000 g in an Eppen-
dorf 5424 microcentrifuge (Eppendorf AG, Hamburg,
Germany). Proteins were separated in Novex
TM
4–12%
gradient gels and transferred onto Immobilon

Germany).
Acknowledgements
We would like to thank Ulf Eidhoff, Patrick Schwei-
gler, Peggy Brunet Lefeuvre, Yan Pouliquen, Brendan
Kerins, Magali Perret, Sonia Buri (all from Novartis,
Basel, Switzerland) and Markus Schirle, Manfred
Raida, Anne-Marie Michon and Sonja Ghidelli (Cell-
zome AG, Heidelberg, Germany) for technical sup-
port, Rita Schmitz (Novartis, Basel, Switzerland) for
providing expression vectors and USP7 cDNA as well
as Shirley Gil-Parrado, Bruno Martoglio, Martin
Renatus and Jo
¨
rg Eder (Novartis, Basel, Switzerland)
for support and helpful discussions. A. Ferna
´
ndez-
Montalva
´
n would like to dedicate this paper to the
memory of his father J. A. Ferna
´
ndez-Vidal, who died
in a failed bladder cancer surgery while it was being
written.
References
1 Amerik AY & Hochstrasser M (2004) Mechanism and
function of deubiquitinating enzymes. Biochim Biophys
Acta 1695, 189–207.
2 Nijman SM, Luna-Vargas MP, Velds A, Brummelkamp

10 van der Knaap JA, Kumar BR, Moshkin YM, Langen-
berg K, Krijgsveld J, Heck AJ, Karch F & Verrijzer CP
(2005) GMP synthetase stimulates histone H2B deubiq-
uitylation by the epigenetic silencer USP7. Mol Cell 17,
695–707.
11 van der Horst A, de Vries-Smits AM, Brenkman AB,
van Triest MH, van den Broek N, Colland F, Maurice
MM & Burgering BM (2006) FOXO4 transcriptional
activity is regulated by monoubiquitination and
USP7 ⁄ HAUSP. Nat Cell Biol 8, 1043–1045.
12 Boutell C, Canning M, Orr A & Everett RD (2005) Reci-
procal activities between herpes simplex virus type 1
regulatory protein ICP0, a ubiquitin E3 ligase, and ubiqu-
itin-specific protease USP7. J Virol 79, 12342–12354.
13 Boutell C & Everett RD (2004) Herpes simplex virus
type 1 infection induces the stabilization of p53 in a
USP7- and ATM-independent manner. J Virol 78,
8068–8077.
Biochemical characterization of USP7 A. Ferna
´
ndez-Montalva
´
n et al.
4268 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
14 Holowaty MN, Zeghouf M, Wu H, Tellam J, Athana-
sopoulos V, Greenblatt J & Frappier L (2003) Protein
profiling with Epstein–Barr nuclear antigen-1 reveals an
interaction with the herpesvirus-associated ubiquitin-
specific protease HAUSP ⁄ USP7. J Biol Chem 278,
29987–29994.

22 Hong S, Kim SJKaS, Choi I & Kang S (2002) USP7, a
ubiquitin-specific protease, interacts with ataxin-1, the
SCA1 gene product. Mol Cell Neurosci 20, 298–306.
23 Hu M, Li P, Song L, Jeffrey PD, Chenova TA,
Wilkinson KD, Cohen RE & Shi Y (2005) Structure
and mechanisms of the proteasome-associated
deubiquitinating enzyme USP14. EMBO J 24,
3747–3756.
24 Ratia K, Saikatendu KS, Santarsiero BD, Barretto N,
Baker SC, Stevens RC & Mesecar AD (2006) Severe
acute respiratory syndrome coronavirus papain-like pro-
tease: structure of a viral deubiquitinating enzyme. Proc
Natl Acad Sci USA 103, 5717–5722.
25 Renatus M, Parrado SG, D’Arcy A, Eidhoff U, Ger-
hartz B, Hassiepen U, Pierrat B, Riedl R, Vinzenz D,
Worpenberg S et al. (2006) Structural basis of ubiquitin
recognition by the deubiquitinating protease USP2.
Structure 14, 1293–1302.
26 Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia Z &
Davies PL (2002) A Ca(2+) switch aligns the active site
of calpain. Cell 108, 649–660.
27 Moldoveanu T, Hosfield CM, Lim D, Jia Z & Davies
PL (2003) Calpain silencing by a reversible intrinsic
mechanism. Nat Struct Biol 10, 371–378.
28 Barretto N, Jukneliene D, Ratia K, Chen Z, Mesecar
AD & Baker SC (2005) The papain-like protease of
severe acute respiratory syndrome coronavirus has
deubiquitinating activity. J Virol 79, 15189–15198.
29 Case A & Stein RL (2006) Mechanistic studies of ubiqu-
itin C-terminal hydrolase L1.

Chapelle JP, Bours V, Merville MP, Piette J, Beyaert R
et al. (2006) TNFalpha- and IKKbeta-mediated
TANK ⁄ I-TRAF phosphorylation: implications for
interaction with NEMO ⁄ IKKgamma and NF-kappaB
activation. Biochem J 394, 593–603.
38 Nomura F, Kawai T, Nakanishi K & Akira S (2000)
NF-kappaB activation through IKK-i-dependent
I-TRAF ⁄ TANK phosphorylation. Genes Cells 5,
191–202.
39 Avvakumov GV, Walker JR, Xue S, Finerty PJ Jr,
Mackenzie F, Newman EM & Dhe-Paganon S (2006)
Amino-terminal dimerization, NRDP1 (FLRF) ) rho-
danese interaction, and inhibited catalytic domain
A. Ferna
´
ndez-Montalva
´
n et al. Biochemical characterization of USP7
FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4269
conformation of the ubiquitin specific protease 8
(USP8 ⁄ UBPY). J Biol Chem 281, 38061–38070.
40 Johnston SC, Riddle SM, Cohen RE & Hill CP (1999)
Structural basis for the specificity of ubiquitin C-termi-
nal hydrolases. EMBO J 18, 3877–3887.
41 Misaghi S, Galardy PJ, Meester WJ, Ovaa H, Ploegh
HL & Gaudet R (2005) Structure of the ubiquitin
hydrolase UCH-L3 complexed with a suicide substrate.
J Biol Chem 280, 1512–1520.
42 Reyes-Turcu FE, Horton JR, Mullally JE, Heroux A,
Cheng X & Wilkinson KD (2006) The ubiquitin bind-

Chem 270, 25715–25721.
49 Nagai Y, Kojima T, Muro Y, Hachiya T, Nishizawa Y,
Wakabayashi T & Hagiwara M (1997) Identification of
a novel nuclear speckle-type protein, SPOP. FEBS Lett
418, 23–26.
50 Schlaeppi JM, Henke M, Mahnke M, Hartmann S,
Schmitz R, Pouliquen Y, Kerins B, Weber E, Kolbinger
F & Kocher HP (2006) A semi-automated large-scale
process for the production of recombinant tagged
proteins in the Baculovirus expression system. Protein
Expr Purif 50, 185–195.
51 Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M &
Seraphin B (1999) A generic protein purification method
for protein complex characterization and proteome
exploration. Nat Biotechnol 17, 1030–1032.
52 Bouwmeester T, Bauch A, Ruffner H, Angrand PO,
Bergamini G, Croughton K, Cruciat C, Eberhard D,
Gagneur J, Ghidelli S et al. (2004) A physical and func-
tional map of the human TNF-alpha ⁄ NF-kappa B sig-
nal transduction pathway. Nat Cell Biol 6, 97–105.
53 Gavin AC, Bosche M, Krause R, Grandi P, Marzioch
M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat
CM et al. (2002) Functional organization of the yeast
proteome by systematic analysis of protein complexes.
Nature 415, 141–147.
54 Tirat A, Schilb A, Riou V, Leder L, Gerhartz B, Zim-
mermann J, Worpenberg S, Eidhoff U, Freuler F, Stet-
tler T et al. (2005) Synthesis and characterization of
fluorescent ubiquitin derivatives as highly sensitive sub-
strates for the deubiquitinating enzymes UCH-L3 and