Báo cáo khoa học: Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization pptx

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

Deubiquitinating enzymes (DUBs) are a superfamily of
thiol- and metallo proteases specialized in the process-
ing of ubiquitin and ubiquitin-like proteins. They are
responsible for the disassembly of ubiquitin chains, and
for the cleavage of mono- and oligomers of this mole-
cule, either in precursor form or attached to small
Keywords
biochemical characterization; cysteine
protease; deubiquitinating enzyme; ubiquitin
pathway; USP7 ⁄ HAUSP
Correspondence
A. Ferna
´
ndez-Montalva
´
n, Molecular
Screening and Cellular Pharmacology,
Merck Serono S.A., 9 Chemin des Mines,
Case postale 54, CH-1211 Geneva 20,
Switzerland
Fax: +41 22 4149558
Tel: +41 22 4144977
E-mail:
B. Gerhartz, Expertise Platform Proteases,
Novartis Institutes for Biomedical Research,
CH-4002, Basel, Switzerland
Fax: +41 61696 8132
Tel: +41 61696 1204
E-mail:
(Received 20 April 2007, revised 14 June

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

factor receptor associated factor; Ub, ubiquitin; UCH, ubiquitin C-terminal hydrolase; USP, ubiquitin specific protease.
4256 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
nucleophiles and proteins [1]. Among the DUBs, the
ubiquitin specific proteases (USPs) constitute the larg-
est subfamily with 58 cysteine peptidase genes identified
so far [2]. One of the most prominent members of this
subfamily is USP7 (EC 3.1.2.15), also known as herpes
virus associated ubiquitin-specific protease (HAUSP)
due to its discovery in the promyelocytic leukemia
nuclear bodies of herpes simplex virus-infected cells [3].
Recognition and processing of ubiquitylated forms of
the tumor suppressor p53 and its negative modulator
MDM2, a RING domain E3-ligase, suggested an
important role for USP7 in cell survival pathways
[4–7]. More recently, the identification of MDMX and
DAXX (both regulatory proteins in the p53-MDM2
pathway) as USP7 substrates [8,9] has revealed a far
more complex involvement of this enzyme in cell fate
decisions than initially expected. In addition, reports
about USP7 activity on the epigenetic regulator his-
tone 2B [10] and the transcription factor FOXO4 [11]
point to further roles for this DUB in the maintenance
of cell homeostasis. Additional evidence for the crucial
role of USP7 is provided by the fact that targeting this
enzyme belongs to the strategies evolved by the herpes
simplex virus [12,13] and Epstein–Barr [14,15] viruses
for successful host infection.
USP7 is a 1102 amino acid protein with a molecular
weight of approximately 130 kDa (Fig. 1A). In cells,
the enzyme has been reported to be dimerized, poly-

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

EBNA1 / p53 /
HDM-2 binding
ICP-0 binding
1
208 560 1102
TRAF
Protease Core C-Terminal
D481
C223
A
B
H464
1
208
560
1102
USP7-FL
USP7 1-560
USP7 208-560
USP7 208-1102
EGFP
EGFP
EGFP
EGFP
USP7 1-205-EGFP
USP7 20-205-EGFP
USP7 50-205-EGFP
USP7 70-205-EGFP
Fig. 1. Structural–functional features and constructs of USP7
designed for this study. (A) Schematic representation of the USP7

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

[20,23–25]. These structures illuminated an activation
mechanism for USP7 in which a papain-like catalytic
triad (C223, H464 and D481) is assembled via con-
formational changes triggered by the interaction with
ubiquitin. A similar mechanism was described the
same year for the activation of the structural homo-
logue calpain by calcium ions [26]. Interestingly,
here the catalytic unit is significantly less active than
the full length heterodimeric enzyme [26,27]. The
individual contributions of USP7 structural domains
to the activity of the full length enzyme have not been
investigated so far.
In the present study, the biochemical properties and
structure–function relationships of USP7 were charac-
terized. We have mapped sites for phosphorylation
and ubiquitination, and studied the oligomerization
state of the enzyme in vitro and in cells. The kinetic
parameters for the hydrolysis of ubiquitin substrates
by full length USP7 and domain deletion variants have
been determined. The results suggest a role for the
C-terminus in substrate processing and oligomeriza-
tion. In addition, a fragment including amino acids
70–205 was found to be sufficient for nuclear targeting.
As this region is involved in protein–protein inter-
actions, association with nuclear proteins might be
required for USP7 subcellular localization.
Results
Heterologous expression and purification
of functional USP7 variants
In the present study, a novel semiautomated expression

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

evaluate their structural integrity and correct folding
by comparison of cleavage sites. For USP7-FL, bands
corresponding to seven main digestion fragments were
visualized by SDS ⁄ PAGE (Fig. 2). Five out of them,
with a molecular weight ‡ 25 kDa were subjected to
Fig. 2. Purity and folding of recombinant USP7 variants. SDS ⁄ PAGE
analysis (in a 4–20% gradient gel) of USP7 variants before (–) and
after (+) 1-h native limited proteolysis with tosylphenylalanylchlo-
romethane-treated trypsin as described in the experimental section.
The arrows indicate digestion products in USP7 full length sub-
jected to sequencing analysis. The N-terminal sequences of these
fragments are written on the left with special symbols used to
mark bands of similar identity derived from other USP7 variants.
These symbols were also used to represent graphically the cleav-
age sites on the schematic view of USP7 shown below.
Biochemical characterization of USP7 A. Ferna
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´
n et al.
4258 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
protein sequencing. This analysis mapped their N-ter-
mini to residues I36, K209, E557 ⁄ Q559, S341 and
I885. Identical digestion patterns were found in all
variants according to the presence or absence of the
cleavage sites in their sequences (Fig. 2), strongly indi-
cating a correct overall folding of these proteins. The
tryptic processing matched with the domain organiza-
tion proposed earlier in similar experiments (Fig. 1A),
although some cleavage sites differed from those previ-

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,

during the size exclusion chromatography step of
USP7-FL and USP7 208-1102 purification, fractions
displaying DUB activity eluted from the Superdex 200
SEC column as single peaks but at elution volumes
corresponding to significantly larger proteins. These
observations were confirmed by analysis of freshly
purified USP7-FL using analytical size exclusion chro-
matography coupled to light scattering measurement.
As shown in the supplementary Fig. S1A, USP7-FL
showed a retention time on the Sephacryl S-300
column between ferritin (440 kDa) and aldolase
(158 kDa), suggesting a molecular weight of around
250 kDa. In contrast, the light scattering measure-
ments showed an average molecular mass between
131.8 and 139.0 kDa, corresponding to the monomeric
form of USP7. Noteworthy, the light scattering results
may be indicative of a mixed population, with mostly
monomers but also a few dimers or higher aggregates.
The amount of dimers or aggregates appears to
increase, when freezing and thawing the protein (data
not shown). In native nonreducing PAGE, purified
USP7-FL migrated as two discrete bands of relative
mobilities corresponding to the monomer and putative
dimers (supplementary Fig. S1B). Accordingly, when
cell lysates containing either endogenously or ectopi-
cally expressed USP7 were subjected to native PAGE
and proteins detected by western blot again two anti-
body reactive bands were observed (supplementary
Fig. S1C). In line with this observation, LC-MS ⁄ MS
analysis of proteins copurified with the TAP-tagged

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-

like proteins (Fig. 4A). Unlike other DUBs [31,32],
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 4259
200 400 600 800 1000 1200 1400
m/
z
0
100
A
B
%
IIGVHQEDELLECL(pS)PATSR
y''5
531.4
243.2
I/L
86.1
a2
199.2
y''2
262.2
y''3
363.2
486.3
y''6
698.4

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

873.6
y'‘8-
H
3
PO
4
y9
1100.6
y7
811.5
pS
WB: CBP
WB: Ub
TAP-USP7
CBP-USP7
-+
-+
-+
MG132
Ub-USP7
97 kDa
97 kDa
Lysate
TEV-
eluate
CBP-
eluate
Tandem Affinity Purification
200 400 600 800 1000
m/z

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
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n et al.
4260 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
hydrolysis of Z-LRGG-AMC could not be measured
at maximum enzyme concentrations of 200 nm. USP7
is active on Ub-AMC in a pH range between 7.5 and
9.5 with an activity maximum at pH 8.5 (Fig. 4B).
Substrate hydrolysis was affected by increasing concen-
trations of NaCl (Fig. 4C). The effect of the chaotrop-
ic NaSCN was noticeable at lower concentrations than
with NaCl or the kosmotropes Na-citrate and glycerol
(Fig. 4C). The data shown in Table 1 and supplemen-
tary Fig. 2 demonstrate that USP7-FL recognized
Ub-AMC and Ub-K-TAMRA with slightly different
affinities. Accordingly, the catalytic efficiency of USP7-
FL for the hydrolysis of Ub-K-TAMRA was improved
by five-fold with respect to Ub-AMC. Under the
conditions chosen for the assay, saturation was not
reached with Ub-K-peptide-TAMRA.
Processing of ubiquitin synthetic substrates by
USP7-FL and domain deletion variants
Evaluation of the hydrolysis of Ub-AMC and Ub-K-
TAMRA by USP7-FL and its domain deletion
Fig. 4. Enzymatic characterization of USP7. (A) Progress curves for the USP7-catalyzed hydrolysis of Ub-AMC (j), SUMO-1-AMC (d) and
Nedd8-AMC (.). Raw fluorescence intensities (RFU) collected every 5 min with k
ex
¼ 360 nm and k
em
¼ 465 nm were plotted as a function
of the time (s). Reactions were conducted at room temperature, in 50 m
M Tris ⁄ HCl pH 7.5, 1 mM EDTA, 5 mM dithiothreitol, 100 mM NaCl

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

) and
consequently in the catalytic efficiency (k
cat
⁄ K
M
).
USP7 208-560 and USP7 1-560 were significantly less
active than the full length enzyme, whereas the enzy-
matic activity of USP7 208-1102 was rather similar to
the wild-type. These results indicate an important role
for the C-terminal domain in catalysis. The compari-
son between Ub-AMC and Ub-K-TAMRA, revealed
more pronounced differences in the catalytic efficiency
of the variants relative to USP7-FL when using the
e-amino-linked substrate.
C-terminal truncations destabilize the
ubiquitin–enzyme complex
Having realized the importance of USP7 C-terminus
for efficient substrate processing, the question was
asked whether conformational changes driven by
ubiquitin binding to the core domain, or direct inter-
actions of this region with the substrate would be
required for proper recognition and processing. In
order to address this issue USP7-FL and the domain
deletion variants were subjected to limited proteolysis
by trypsin under native conditions in the presence or
absence of a molar excess ubiquitin. Digestion was
examined over time by SDS ⁄ PAGE and Coomassie
Blue staining. Surprisingly, the fragments produced by
limited proteolysis were identical with and without

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

(lM) k
cat
(s
)1
) k
cat
⁄ K
M
(s
)1
ÆlM
)1
)
Fold decrease in
catalytic efficiency
Full length Ub-AMC 1 17.5 ± 2.0 3.56 2.03 · 10
5
1
Ub-K-TAMRA 5 6.6 ± 0.7 6.76 1.02 · 10
6
1
1–560 Ub-AMC 100 27.6 ± 3.4
a
0.045 1.6 · 10
3
127
Ub-K-TAMRA 1000 10.9 ± 1.0 0.018 1.6 · 10
3
644
208–560 Ub-AMC 100 44.2 ± 3.8

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
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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-

tionships for USP7, we studied the effect of domain
deletions in the subcellular localization patterns of the
enzyme. To this end, several mammalian cell lines were
transiently transfected with vectors encoding the USP7
variants described above (Fig. 1B). Synthesis of recom-
binant proteins was corroborated by immunoblot anal-
ysis of cell lysates with either FLAG (M2) or Myc
(9E10) specific monoclonal antibodies (not shown).
Expression levels were dependent on the construct
sequence and the cell line used. Both antibodies detected
higher quantities of USP7 1-560 and USP7 208-560
than USP7 full length and USP7 208-1102 in the
western blots (not shown). Immunofluorescent staining
revealed different subcellular localization patterns for
the constructs (Fig. 6). USP7-FL and variant 1-560
localized preferentially to the cell nucleus, whereas
USP7 208-560 and USP7 208-1102 were detected mostly
in the cytosol. A small fraction of USP7 208-560
observed in the nucleus is likely an artifact caused by
the strong over expression of this variant because USP7
208-1102 did not show this behavior. Fusion proteins
containing the N-terminal domain of USP7 (amino
acids 1–205) and variants with deletions of the first 20,
50 and 70 amino acids linked to enhanced green fluores-
cent protein (EGFP) at their C-terminus localized in the
cell nucleus (Fig. 6).
Discussion
In the present study, we have mapped S18, S963 and
K869 as phosphorylation and ubiquitination sites of
USP7. Depending on the techniques used, monomers

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

additional evidence for the interaction of the enzyme
with E3 ubiquitin ligases. Remarkably, the ubiquitina-
tion site is close to the region where it was reported to
interact with ICP-0 [18], supporting the observation
that USP7 can be ubiquitinated by this E3 ligase but
not by MDM2 [12]. Our findings indicate that USP7
could exist as a dimer in cells. The data obtained with
purified enzyme is, however, contradictory, suggesting
that further cellular components might be required to
stabilize these oligomers. Noteworthy the enzymatic
behavior of USP7 in the presence of kosmotropes cor-
responds to an enzyme that is fully active in its mono-
meric form. Further analysis is required in order to
understand the roles of the putative dimerization event.
USP7 recognizes ubiquitin with high specificity.
Moreover, its lack of activity on short peptide sub-
strates comprising the C-terminus of ubiquitin aligns
with recent data reported for USP2 [25] and USP8
[39], suggesting that recognition of both the ubiquitin
C-terminus and its core are equally important for
catalysis. The affinity of USP7 for Ub-AMC (K
M
¼
17.5 lm) was approximately 500-fold lower than in the
case of the ubiquitin C-terminal hydrolases (UCHs)
[29]. Compared to other USPs, USP7 shows slightly
lower affinities for Ub-AMC than USP5 (K
M
¼
1.4 lm) [30] and USP2 (K

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

8
m
)1
s
)1
) [29].
Otherwise the k
cat
⁄ K
M
is similar to UCHL-1 (2.9 ·
10
5
m
)1
Æs
)1
) [29], USP5 (2.4 · 10
5
m
)1
Æs
)1
) [30], USP2
(2.52 · 10
5
m
)1
Æs
)1

) [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

previously using a glutathione S-transferase (GST)-
Ubi52 as substrate [18]. These are typical for a DUB
and for cysteine proteases in general. A recent discus-
sion is provided elsewhere [29].
In USP7, the kinetic parameters for the hydrolyisis
of ubiquitin substrates appear strongly affected by the
Image-iT™
FL
208-1102
1-205-EGFP
70-205-EGFP
USP7 Merge
Fig. 6. Structural requirements for nuclear localization of USP7. Several cell lines were transiently transfected with FLAG-Myc-tagged USP7-
FL, USP7 1-560, USP7 208-560 and USP7 208-1102, as well as with USP7 1-205-EGFP, USP7 20-205-EGFP, USP7 50-205-EGFP and
USP7 70-205-EGFP. Two days later, the recombinant proteins were visualized either by immunofluorescent staining with a monoclonal anti-
FLAG (M2) serum and an Alexa 488 anti-mouse conjugate in paraformaldehyde fixed cells, or by direct detection of EGFP fluorescence (both
shown here in green). Image-iT
TM
counterstaining for the nuclei (blue) and cellular membranes (red) was applied. The results shown here
correspond to USP7-FL, USP7 208-1102, USP7 1-205-EGFP and USP7 70-205-EGFP expressed in U2OS cells.
Biochemical characterization of USP7 A. Ferna
´
ndez-Montalva
´
n et al.
4264 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
deletion of structural features outside the protease
core. Therefore, we conclude that these domains are
important for catalysis. A contribution for the TRAF-
like domain should not be neglected, but the most

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-

cient to achieve nuclear localization in USP7, an obser-
vation which is in line with previous studies [17]. Since
bioinformatics tools did not anticipate any functional
nuclear localization signal (NLS) within this domain
and most TRAF proteins localize in the cytosol [46],
we hypothesized that a novel NLS might be contained
by the first 70 N-terminal residues of USP7, a region
sharing neither sequence- nor structural similarities
with other TRAF family members. Secondary structure
prediction of this region using the GOR algorithm [47]
anticipated a coiled region between residues M1 and
E20, an alpha helix from there and up to amino acid
G28, followed by a b-sheet starting at T36 and extend-
ing to residue L49, and a larger helix including amino
acids A55 to R66. Based on these predictions, we stud-
ied the localization of deletion mutants of the first 20,
50 and 70 amino acids of USP7. Surprisingly, these
variants were found preferentially in the cell nucleus,
suggesting that the putative functional nuclear localiza-
tion sequences are located in the conserved region dis-
playing the canonical fold of TRAF proteins [15].
Among this family, only TRAF4 [46,48] and SPOP
[49] have been found exclusively in the cell nucleus so
far. In addition, TRAF1 displays both nuclear and
cytosolic localization when expressed in isolation [46].
Interestingly, TRAF4 seems to require the interaction
with a rapidly titrated endogenous factor, rather than
a NLS [46]. Remarkably, the TRAF domain of USP7
also interacts with several nuclear proteins such as p53,
mdm2 and the family members TRAF4 and TRAF1,

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

anti-rabbit-HRP secondary sera conjugates were from
Sigma and Biorad (Hercules, CA, USA), respectively. Goat
anti-rabbit-Texas red and rabbit-anti-mouse-Alexa 488 sera
conjugates for secondary detection of immunostained cells
were from Molecular Probes (Invitrogen). Mammalian cell
lines were acquired from the ATCC (Manassas, VA, USA)
and Spodoptera frugiperda (Sf9) cells from Invitrogen.
Generation of plasmids, bacmids and
baculoviruses
Full length USP7 cDNA, was amplified by PCR and inserted
into the pCR2.1-TOPO vector (Invitrogen) following the
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 4265
TOPO-TA cloning protocol provided by the manufacturer.
DNAs coding for USP7 amino acids 1–560 and 208–1102
were amplified with 5¢-BamHI and 3¢-NotI overhangs, cloned
into pCR2.1-TOPO and subcloned into a pFastBac1 vector
(Invitrogen), modified by the addition of a C-terminal His-
tag. Bacmids were prepared following thre manufacturer’s
guidelines. Generation of bacmid DNA for USP7 full length
with a C-terminal His-FLAG tag using the Gateway
TM
tech-
nology is described elsewhere [50]. USP7 208-560 was
expressed in E. coli according to Hu et al. [20] using a
pET42b(+) vector (Novagen, EMD Biosciences, San Diego,

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

the buffer used to equilibrate the size exclusion SPX200
10 ⁄ 60 column (50 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl,
5mm dithiothreitol, 5% glycerol, 100 lgÆmL
)1
phenyl-
methanesulfonyl fluoride). Characterization of the purified
proteins was done by HPLC coupled to time-of-flight mass
spectrometry (LC-MS). Bacterial expression of USP7208-
560 was performed in Rosetta (DE3) cells (Novagen).
Isopropyl thio-b-d-galactoside induction, expression and
preparation of cell lysates by sonication were accomplished
following the pET system protocols provided by Novagen.
All purification procedures were conducted at 4 °C. Chro-
matographic separations were carried out using an A
¨
kta
Purifier FPLC instrument (GE Healthcare). The cell lysate
was loaded on a Ni-nitrilotriacetic acid affinity column
(Qiagen, Hilden, Germany) and unbound proteins were
washed away with NaCl ⁄ P
i
containing 15 mm imidazole.
Following this step, NaCl ⁄ P
i
supplemented with 30 mm
EDTA was used to elute proteins attached to the affinity
matrix. The enzyme-containing fractions were pooled, and
treated with PreScission protease (GE Healthcare) follow-
ing manufacturer’s instructions in order to remove the
GST-His moiety. A second Ni-nitrilotriacetic acid affinity

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-

lon
TM
poly(vinylidene difluoride) membranes using
Novex
TM
wet transfer units (Invitrogen). Blotted proteins
were Coomassie stained and major bands were excised and
eluted in order to perform N-terminal sequencing.
Tandem affinity purification and mass
spectrometric analysis of USP7
N-terminal and C-terminal TAP-tagged USP7 fusion pro-
teins were expressed via transient transfection in HeLa cells.
USP7 isoforms were affinity purified via the tandem affinity
procedure as previously described [52,53]. Cells were treated
O ⁄ N with 5 lm MG132 prior to harvesting. Purified pro-
teins were separated by 1D SDS ⁄ PAGE, in-gel digested
with trypsin, and the resulting peptides were sequenced by
tandem mass spectrometry (LC ⁄ MS ⁄ MS). Protein identifi-
cation was performed by searching the MS data against
a curated version of the International Protein Index
Biochemical characterization of USP7 A. Ferna
´
ndez-Montalva
´
n et al.
4266 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
( using the mascot software
(Matrix Science, Boston, MA, USA). Sites of post-transla-
tional modification were verified by manual inspection of
the mass spectra.

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

Labsystems, Altrincham, UK) in a reaction volume of
30 lL. Kinetic data was collected in time intervals of 5 min
using a Genios fluorescence plate reader from Tecan (Man-
nedorf, Switzerland) at excitation and emission wavelengths
of 360 nm and 465 nm, respectively. To determine the assay
linearity range and substrate specificity serial dilutions
(from 200 nm) of each USP7 variant were used to com-
pletely hydrolyze 1 lm of Ub-AMC. Same enzyme concen-
trations were used to test USP7-FL activity on 1 lm
SUMO-1-AMC and Nedd8-AMC (Boston Biochem), as
well as on 250 lm Z-Leu-Arg-Gly-Gly-AMC (Biomol Inter-
national, Plymouth Meeting, PA, USA). For subsequent
determination of the K
M
and V
max
values for the hydrolysis
of Ub-AMC, constant enzyme concentrations (Table 1)
were used to hydrolyze substrate concentrations varying
from 0.024 to 25 lm in two-fold increments. The influence
of pH, salt and glycerol on USP7-FL activity was studied
using 5 nm enzyme and 1 lm Ub-AMC. The pH depen-
dency studies were performed in 0.2 m glycine ⁄ HCl
(pH 2.5–3.0), 0.1 m Na-acetate (pH 4.0–5.5), 0.2 m phos-
phate ⁄ citrate (pH 6.0 and 6.5), 25 mm Tris ⁄ HCl (pH 7.0–
9.0) and 0.2 m glycine ⁄ NaOH (pH 9.5–10.5). All buffers
were supplemented with fresh 5 mm dithiothreitol and
0.1% (w ⁄ v) Chaps. Salt and glycerol effects were analyzed
at the concentrations indicated in Fig. 3E,F in 25 mm
Tris ⁄ HCl buffer at pH 8.0, with 5 mm dithiothreitol and

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

) (Eqn 1), where V
max
is the
maximal velocity at saturating substrate concentrations and
K
M
the Michaelis constant. The k
cat
value was derived from
the equation k
cat
¼ V
max
⁄ [E
o
] (eqn 2) where [E
o
] is the total
enzyme concentration. Experimental data was processed
using the origin 7.5 analysis software from OriginLab,
Northampton (MA, USA).
Mammalian cell culture and transfection
HEK293, HeLa, U2OS, MCF-7, H1299 and SJSA-1 cells
were cultured in either GlutaMAX
TM
RPMI 1640 or
DMEM supplemented with 5% heat inactivated fetal
bovine serum and penicillin ⁄ streptomycin (Invitrogen) as
recommended by the ATCC for each cell line. Transfection
experiments were performed on six-well plates from Nunc

(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

TM
poly(vinylidene difluoride) membranes as described
above. Immunodetection of the proteins with specific
antibodies was performed using general protocols pro-
vided by the manufacturers. For native PAGE experi-
ments, buffers SDS- and 2-mercaptoethanol, as well as
heat denaturation of the proteins, were omitted from the
procedure.
Immunostaining and confocal fluorescence
microscopy
Nontransfected or transfected cells were seeded at densities
of 0.5–1.0 · 10
4
cells per well on Laboratory-Tek German
borosilicate glass chamber slides (Nunc) and allowed to set-
tle overnight. Cells were subsequently fixed with 2% para-
formaldehyde in the presence of 0.1% Triton X-100,
blocked with 10% newborn goat serum and incubated in
successive steps with the primary monoclonal anti-FLAG
(M2) serum (Sigma) (1 : 1000) and the secondary anti-
mouse-Alexa 488 conjugate (Molecular Probes, Invitrogen)
(1 : 5000). Nuclear and membrane counterstaining was
performed using the Image-iT
TM
kit (Molecular Probes)
following the manufacturer’s guidelines. For microscopy of
EGFP chimeras, cells were seeded in Laboratory-Tek cover
slides (Nunc), which allowed counterstaining and imaging
of living cells. Images were scanned with a Zeiss LSM 510
meta confocal microscope (Carl Zeiss, Oberkochen,

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.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Analysis of USP7 oligomerization in vitro and
in cells.
Fig. S2. Substrate titrations of USP7 variants.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Biochemical characterization of USP7 A. Ferna
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ndez-Montalva
´
n et al.
4270 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS