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-translation
Trang 1post-translational modification sites and structural
requirements for substrate processing and subcellular
localization
Amaury Ferna´ndez-Montalva´n1, Tewis Bouwmeester2, Gerard Joberty2, Robert Mader3,
Marion Mahnke4, Benoit Pierrat1, Jean-Marc Schlaeppi4, Susanne Worpenberg1
and Bernd Gerhartz1
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: amaury.fernandez@merckserono.net
B Gerhartz, Expertise Platform Proteases,
Novartis Institutes for Biomedical Research,
CH-4002, Basel, Switzerland
Fax: +41 61696 8132
Tel: +41 61696 1204
E-mail: bernd.gerhartz@novartis.com
(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 interaclocaliza-tion 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.
Trang 2nucleophiles 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
TRAF Protease Core C-Terminal
D481 C223
A
B
H464
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.
Trang 3Matching 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.
Trang 4protein 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 (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) Unubiquitin-like other DUBs [31,32],
Trang 5200 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-H3PO4
LE/EL
b2 227.2 y'‘8 2+
y8 971.6
873.6
y'‘8-H3PO4
y9 1100.6
y7 811.5
pS
TAP-USP7 CBP-USP7
MG132
Ub-USP7
97 kDa
97 kDa
Lysate TEV-eluate
CBP-eluate Tandem Affinity Purification
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.
Trang 6hydrolysis 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 notchaotrop-iceable 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 kex¼ 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 m M EDTA, 5 m M dithiothreitol, 100 m M 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 l M 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 m M 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
5 n M and Ub-AMC was at 1 l M (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 m M
EDTA, 5 m M dithiothreitol, 100 m M NaCl and 0.1% (w ⁄ v) Chaps with 1 l M Ub-AMC and the enzyme concentrations indicated in the experi-mental section.
Trang 7variants 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 (kcat) and
consequently in the catalytic efficiency (kcat⁄ KM)
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 (l M ) k cat (s)1) k cat ⁄ K M (s)1Æl M )1)
Fold decrease in catalytic efficiency
936
a Kmvalues higher than the maximum substrate concentrations used for the titrations should be considered as approximate figures.
Trang 8the 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 Deleoligomeriza-tion 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 posiphosphoryla-tion 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 (KM¼ 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 (KM¼ 1.4 lm) [30] and USP2 (KM¼ 0.554 lm) [25], respec-tively, and displays a similar KM as USP8 (KM¼ 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 KM, the cata-lytic efficiency of USP7 (kcat⁄ KM) is only weaker
Trang 9compared to that of UCHL-3 (2.1· 108m)1s)1) [29].
Otherwise the kcat⁄ KM is similar to UCHL-1 (2.9·
105m)1Æs)1) [29], USP5 (2.4· 105m)1Æs)1) [30], USP2
(2.52· 105m)1Æs)1) [25] and USP8 (2.35· 105
m)1Æs)1) [39] This value is only higher than those
reported for USP14 (UBP6 in yeast) (1.07·
102m)1Æs)1) [43] and the viral SARS-CoV PLpro
(2.69· 102m)1Æs)1 and 1.31· 104m)1Æs)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™
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-iTTMcounterstaining 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.
Trang 10deletion 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 kcat⁄ KM 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 includextend-ing 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