Biochemical characterization of USP7 reveals
`post-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
`
`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 interaction partners might be required
`for USP7 nuclear import.
`
`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
`
`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
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`

`A. Ferna´ ndez-Montalva´ n et al.
`
`Biochemical characterization of USP7
`
`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
`
`A
`
`C223
`
`H464
`
`D481
`
`TRAF
`
`Protease Core
`
`C-Terminal
`
`1
`
`208
`
`560
`
`1102
`
`Ubiquitin binding
`
`EBNA1 / p53 /
`HDM-2 binding
`
`ICP-0 binding
`
`Ataxin binding
`
`208
`
`560
`
`1102
`
`B
`
`1
`
`USP7-FL
`
`USP7 1-560
`
`USP7 208-560
`
`USP7 208-1102
`
`USP7 1-205-EGFP
`
`EGFP
`
`EGFP
`
`USP7 20-205-EGFP
`
`EGFP
`
`USP7 50-205-EGFP
`
`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.
`
`are currently unknown. Sequence analysis anticipated
`a protease domain with conserved Cys and His
`boxes delimited by the N- and C-terminal regions [3].
`
`FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
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`Biochemical characterization of USP7
`
`A. Ferna´ ndez-Montalva´ n et al.
`
`limited proteolysis and
`Matching these predictions,
`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
`
`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.
`
`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
`
`4258
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`
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`

`A. Ferna´ ndez-Montalva´ n et al.
`
`Biochemical characterization of USP7
`
`protein sequencing. This analysis mapped their N-ter-
`to residues I36, K209, E557 ⁄ Q559, S341 and
`mini
`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
`the corresponding MS ⁄ MS spectra
`verification of
`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],
`
`FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
`
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`

`

`Biochemical characterization of USP7
`
`A. Ferna´ ndez-Montalva´ n et al.
`
`A
`100
`
`y''5
`531.4
`
`LE/EL
`243.2
`
`y''6
`698.4
`
`pS
`
`IIGVHQEDELLECL(pS)PATSR
`
`y''2
`262.2
`
`y''3
`363.2
`
`
`
`MH33+ - H3PO4
`
`b10 2+
`567.9
`
`y'‘8 2+
`486.3
`
`y''6-
`H3PO4
`600.4
`
`y'‘8-
`H3PO4
`873.6
`
`750.4
`
`y7
`811.5
`
`b10
`1134.7
`
`b9
`1021.7
`y9
`1100.6
`
`y8
`971.6
`
`%
`
`b2
`227.2
`
`I/L
`86.1
`
`a2
`199.2
`
`0
`
`b11
`1247.9
`
`b12
`1376.8
`
`m/z
`
`200
`
`400
`
`600
`
`800
`
`1000
`
`1200
`
`1400
`
`Tandem Affinity Purification
`
`Lysate
`
`MG132
`
`-
`
`+
`
`TEV-
`eluate
`
`-
`
`+
`
`CBP-
`eluate
`
`-
`
`+
`
`B
`
`100
`
`b2
`229.1
`
`y''5
`808.5
`
`y''4
`661.4
`
`y''6
`936.6
`
`Ub-USP7
`
`TAP-USP7
`CBP-USP7
`
`y''7
`1049.7
`
`m/z
`
`97 kDa
`
`97 kDa
`
`WB: Ub
`
`WB: CBP
`
`(GG)K
`b3
`342.2
`
`y''3
`514.3
`
`a2
`201.1
`
`y''2
`272.2
`
`%
`
`0
`
`200
`
`400
`
`600
`
`800
`
`1000
`
`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.
`
`4260
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`FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
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`

`A. Ferna´ ndez-Montalva´ n et al.
`
`Biochemical characterization of USP7
`
`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 kex ¼ 360 nm and kem ¼ 465 nm were plotted as a function
`of the time (s). Reactions were conducted at room temperature, in 50 mM Tris ⁄ HCl pH 7.5, 1 mM EDTA, 5 mM dithiothreitol, 100 mM NaCl
`and 0.1% (w ⁄ v) Chaps using 1.56 nM 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 mM 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
`5 nM 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 mM 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.
`
`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
`
`FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
`
`4261
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`Post-Grant Review Petition for US 9,840,491
`EXHIBIT 1021
`Page 6
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`EXHIBIT 9
`DELANSORNE DECLARATION
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`214LT:20700:449507:1:ALEXANDRIA
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`

`

`Biochemical characterization of USP7
`
`A. Ferna´ ndez-Montalva´ n et al.
`
`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] (nM)
`
`Full length
`
`1–560
`
`208–560
`
`208–1102
`
`Ub-AMC
`Ub-K-TAMRA
`Ub-AMC
`Ub-K-TAMRA
`Ub-AMC
`Ub-K-TAMRA
`Ub-AMC
`Ub-K-TAMRA
`
`1
`5
`100
`1000
`100
`2000
`5
`100
`
`KM (lM)
`
`17.5 ± 2.0
`6.6 ± 0.7
`27.6 ± 3.4a
`10.9 ± 1.0
`44.2 ± 3.8a
`36.8 ± 4.9a
`22.8 ± 2.1
`7.2 ± 0.8
`
`kcat (s
`
`)1)
`
`kcat ⁄ KM (s
`
`
`
`)1ÆlM)1)
`
`Fold decrease in
`catalytic efficiency
`
`3.56
`6.76
`0.045
`0.018
`0.077
`0.039
`0.805
`0.33
`
`2.03 · 105
`1.02 · 106
`1.6 · 103
`1.6 · 103
`1.7 · 103
`1.1 · 103
`3.53 · 104
`4.58 · 104
`
`1
`1
`127
`644
`119
`936
`6
`23
`
`a Km values higher than the maximum substrate concentrations used for the titrations should be considered as approximate figures.
`
`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 (kcat) and
`efficiency (kcat ⁄ KM).
`consequently in the
`catalytic
`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
`
`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.
`
`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
`
`4262
`
`FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS
`
`Post-Grant Review Petition for US 9,840,491
`EXHIBIT 1021
`Page 7
`
`EXHIBIT 9
`DELANSORNE DECLARATION
`
`214LT:20700:449507:1:ALEXANDRIA
`
`

`

`A. Ferna´ ndez-Montalva´ n et al.
`
`Biochemical characterization of USP7
`
`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 (KM 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
`for Ub-AMC than USP5 (KM ¼
`lower affinities
`1.4 lm) [30] and USP2 (KM ¼ 0.554 lm) [25], respec-
`tively, and displays a similar KM as USP8 (KM 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