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 Fernandez-Montalvan1, Tewis Bouwmeester2, Gerard Joberty2, Robert Mader3, Marion Mahnke4, Benoit Pierrat1, Jean-Marc Schlaeppi4, Susanne Worpenberg1 and Bernd Gerhartz1 Expertise Platform Proteases, Novartis Institutes for Biomedical Research, Basel, Switzerland Cellzome AG, Heidelberg, Germany Musculoskeletal Disease Area, Novartis Institutes for Biomedical Research, Basel, Switzerland Biologics Centre, Novartis Institutes for Biomedical Research, Basel, Switzerland Keywords biochemical characterization; cysteine protease; deubiquitinating enzyme; ubiquitin pathway; USP7 ⁄ HAUSP Correspondence ´ndez-Montalvan, Molecular ´ A Ferna Screening and Cellular Pharmacology, Merck Serono S.A., 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 (USP7) belongs to the family of deubiquitinating 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 variants 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 100fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency Limited proteolysis experiments revealed that USP7 variants containing 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 localization 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 processing 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 molecule, 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 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS ´ ´ A Fernandez-Montalvan et al nucleophiles and proteins [1] Among the DUBs, the ubiquitin specific proteases (USPs) constitute the largest 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 histone 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, polyubiquitinated 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 (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 shallow 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 interaction 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 Biochemical characterization of USP7 A C223 Protease Core TRAF H464 D481 208 C-Terminal 560 1102 Ubiquitin binding ICP-0 binding EBNA1 / p53 / HDM-2 binding Ataxin binding B 208 560 1102 USP7-FL USP7 1-560 USP7 208-560 USP7 208-1102 EGFP USP7 1-205-EGFP EGFP USP7 20-205-EGFP EGFP USP7 50-205-EGFP EGFP USP7 70-205-EGFP Fig 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 The protease core (amino acids 208–560) contains 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 ICP0 and Ataxin-1 (B) Design of USP7 variants used in this work Constructs 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 constructs used for proteomics analysis contained either N- or C-terminal 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 4257 Biochemical characterization of USP7 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 architecture, 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 conformational changes triggered by the interaction with ubiquitin A similar mechanism was described the same year for the activation of the structural homologue 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 characterized 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 oligomerization 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 interactions, 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 mg (USP7 full length), mg (1–560) and mg (208–1102) of purified recombinant protein per litre of insect cell culture In addition, an average of mg USP7 208-560 per litre of Escherichia coli fermentation 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 4258 ´ ´ A Fernandez-Montalvan et al Fig 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 tosylphenylalanylchloromethane-treated trypsin as described in the experimental section The arrows indicate digestion products in USP7 full length subjected 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 cleavage 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 heterogeneities was observed in the 208-560 protein produced in E coli All USP7 variants were subjected to limited proteolysis 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 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS ´ ´ A Fernandez-Montalvan et al protein sequencing This analysis mapped their N-termini 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 indicating a correct overall folding of these proteins The tryptic processing matched with the domain organization proposed earlier in similar experiments (Fig 1A), although some cleavage sites differed from those previously 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 purified 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 residues to S18 and S963, respectively (Fig 3A) USP7 was previously described to be ubiquitinylated and neddylated Western analysis showed that affinity purified 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 inspection of the MS ⁄ MS spectra showed that the diglycine remnant was conjugated to K869 The strong identification 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 chromatography coupled to light scattering measurement As shown in the supplementary Fig S1A, USP7-FL showed a retention time on the Sephacryl S-300 Biochemical characterization of USP7 column between ferritin (440 kDa) and aldolase (158 kDa), suggesting a molecular weight of around 250 kDa In contrast, the light scattering measurements 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 ectopically expressed USP7 were subjected to native PAGE and proteins detected by western blot again two antibody 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-methylcoumarin (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 ubiquitin-like modifier protein (SUMO-1)-AMC, Nedd8AMC 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-Lystetramethylrhodamine (Ub-K-TAMRA) and Ub-Kpeptide-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 h at enzyme concentrations up to nm Using similar conditions with SUMO-1-AMC and Nedd8AMC as substrates, no USP7 activity could be detected, indicating a high specificity for ubiquitin, despite the well known homologies among ubiquitinlike 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 4259 ´ ´ A Fernandez-Montalvan et al Biochemical characterization of USP7 y''5 531.4 A 100 y''6 698.4 LE/EL 243.2 pS IIGVHQEDELLECL(pS)PATSR y''2 262.2 % MH33+ - H3PO4 b2 227.2 y''3 363.2 y'‘8 2+ 486.3 b10 2+ 567.9 y'‘8H3PO4 873.6 y''6750.4 H3PO4 y7 600.4 811.5 I/L 86.1 b10 1134.7 b9 1021.7 y9 y8 1100.6 971.6 a2 199.2 b11 1247.9 b12 1376.8 m/z 200 400 600 800 1000 1200 1400 Tandem Affinity Purification Lysate MG132 b2 229.1 WB: Ub 100 a2 201.1 - + - + 97 kDa TAP-USP7 CBP-USP7 y''5 808.5 y''4 661.4 (GG)K b3 342.2 + Ub-USP7 y''2 272.2 % - CBPeluate 97 kDa WB: CBP B TEVeluate y''6 936.6 y''3 514.3 y''7 1049.7 m/z 200 400 600 800 1000 Fig 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 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS ´ ´ A Fernandez-Montalvan et al Biochemical characterization of USP7 Fig 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 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, mM EDTA, 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 lM Each data point represents 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, mM dithiothreitol and 0.1% (w ⁄ v) CHAPS at the indicated concentrations of NaCl (j), NaSCN (d), Na-citrate (m) or glycerol (h) In these experiments, the nominal concentration of USP7 was nM and Ub-AMC was at 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, mM EDTA, mM dithiothreitol, 100 mM NaCl and 0.1% (w ⁄ v) Chaps with lM Ub-AMC and the enzyme concentrations indicated in the experimental 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 concentrations of NaCl (Fig 4C) The effect of the chaotropic NaSCN was noticeable at lower concentrations than with NaCl or the kosmotropes Na-citrate and glycerol (Fig 4C) The data shown in Table and supplementary Fig demonstrate that USP7-FL recognized Ub-AMC and Ub-K-TAMRA with slightly different affinities Accordingly, the catalytic efficiency of USP7FL 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-KTAMRA 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 ´ ´ A Fernandez-Montalvan et al Biochemical characterization of USP7 Table Kinetic parameters for the hydrolysis of Ub-AMC (a) and Ub-K-TAMRA (b) by USP7 domain deletion variants USP7 variant Substrate [Protein] (nM) KM (lM) kcat (s)1) Full length Ub-AMC Ub-K-TAMRA Ub-AMC Ub-K-TAMRA Ub-AMC Ub-K-TAMRA Ub-AMC Ub-K-TAMRA 100 1000 100 2000 100 17.5 6.6 27.6 10.9 44.2 36.8 22.8 7.2 3.56 6.76 0.045 0.018 0.077 0.039 0.805 0.33 1–560 208–560 208–1102 a ± ± ± ± ± ± ± ± 2.0 0.7 3.4a 1.0 3.8a 4.9a 2.1 0.8 kcat ⁄ KM (s)1ỈlM)1) 2.03 1.02 1.6 1.6 1.7 1.1 3.53 4.58 · · · · · · · · 105 106 103 103 103 103 104 104 Fold decrease in catalytic efficiency 1 127 644 119 936 23 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 concentrations To this end, enzyme concentrations that allowed assay linearity for at least 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 enzymatic 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 comparison 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 interactions 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 confirmed that the cleavage sites corresponded to those 4262 Fig 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 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 stabilized 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 ubiquitin through the protease core domain, suggesting that FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS ´ ´ A Fernandez-Montalvan et al 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 relationships 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 recombinant proteins was corroborated by immunoblot analysis 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 fluorescent 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 oligomerization 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 Interestingly, this region appears to be required for enzyme oligomerization Finally, we have observed that the N-terminal domain of USP7, and particularly a fragment 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 phosphorylated [33–35] In fact, phosphorylation on S18 had been reported previously from a HeLa large scale proteomics Biochemical characterization of USP7 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 phosphorylation has not been described so far and this position is not a known consensus site for any kinase Interestingly, 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 presumed 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 ubiquitination 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 corresponds to an enzyme that is fully active in its monomeric 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 substrates 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 lower affinities for Ub-AMC than USP5 (KM ¼ 1.4 lm) [30] and USP2 (KM ¼ 0.554 lm) [25], respectively, 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 in a detailed analysis of the interaction of USP2 catalytic core with ubiquitin Despite the higher KM, the catalytic efficiency of USP7 (kcat ⁄ KM) is only weaker FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS 4263 ´ ´ A Fernandez-Montalvan et al Biochemical characterization of USP7 Image-iT™ Merge 70-205-EGFP 1-205-EGFP 208-1102 FL USP7 Fig Structural requirements for nuclear localization of USP7 Several cell lines were transiently transfected with FLAG-Myc-tagged USP7FL, 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 antiFLAG (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-iTTM 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 compared to that of UCHL-3 (2.1 · 108 m)1s)1) [29] Otherwise the kcat ⁄ KM is similar to UCHL-1 (2.9 · 105 m)1Ỉs)1) [29], USP5 (2.4 · 105 m)1Ỉs)1) [30], USP2 (2.52 · 105 m)1Ỉs)1) [25] and USP8 (2.35 · 105 m)1Ỉs)1) [39] This value is only higher than those reported for USP14 (UBP6 in yeast) (1.07 · 102 m)1Ỉs)1) [43] and the viral SARS-CoV PLpro (2.69 · 102 m)1Ỉs)1 and 1.31 · 104 m)1Ỉs)1) [28,31] 4264 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 discussion is provided elsewhere [29] In USP7, the kinetic parameters for the hydrolyisis of ubiquitin substrates appear strongly affected by the FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS ´ ´ A Fernandez-Montalvan et al deletion of structural features outside the protease core Therefore, we conclude that these domains are important for catalysis A contribution for the TRAFlike 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-terminal domain deletions mimicking splice variants of the enzyme influenced not only its subcellular localization [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 better 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 efficiencies displayed with K48 linked diubiquitin [20], and support the existence of substrate primed subsite specificity requirements for USP7 We have shown that the N-terminal domain is sufficient to achieve nuclear localization in USP7, an observation 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 extending to residue L49, and a larger helix including amino acids A55 to R66 Based on these predictions, we studied 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 localization sequences are located in the conserved region displaying 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 Biochemical characterization of USP7 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 understood, 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, difficulties to attain selectivity are predicted based on the experience accumulated with other cysteine proteases In this context, a better understanding of the involvement 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, Germany) Pfu proofreading polymerase and other DNA modifying enzymes were from Promega (Madison, WI, USA) USP7 polyclonal antibody (BL851) was from Bethyl Laboratories (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) AntiFLAG (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 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS 4265 ´ ´ A Fernandez-Montalvan et al Biochemical characterization of USP7 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 Histag 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 GatewayTM technology 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 XbaI as 5¢- and 3¢-overhangs, respectively The PCR products obtained were ligated into pCR2.1-TOPO and further subcloned into p3XFLAG-myc-CMV-26TM (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 AgeI 5¢- and 3¢-overhangs, 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 residues 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 previously 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, mm dithiothreitol, 5% glycerol, 100 lgỈmL)1 phenylmethanesulfonyl fluoride) Characterization of the purified proteins was done by HPLC coupled to time-of-flight mass spectrometry (LC-MS) Bacterial expression of USP7208560 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 C Chroă matographic separations were carried out using an Akta Purifier FPLC instrument (GE Healthcare) The cell lysate was loaded on a Ni-nitrilotriacetic acid affinity column 4266 (Qiagen, Hilden, Germany) and unbound proteins were washed away with NaCl ⁄ Pi containing 15 mm imidazole Following this step, NaCl ⁄ Pi 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) following 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 unprocessed 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 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 NovexTM precast 4–12% or 4–20% gradient gels and electrophoresis chambers (Invitrogen), followed by Coomassie Blue staining Limited proteolysis Proteins at concentrations varying from 0.1 to 0.3 gỈL)1 were digested for 60 with tosylphenylalanylchloromethane-treated bovine trypsin (Sigma) at a molar ratio of 100 : in a final volume of 150 lL Reactions were stopped by addition of one volume of · Laemmli sample buffer, followed by boiling at 95 °C for The effect of ubiquitin 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 upon addition of trypsin, mixed : (w ⁄ w) with · Laemmli sample buffer, and boiled at 95 °C for In both cases, digestion fragments were separated by SDS ⁄ PAGE (4–20% gels) and either visualized by Coomassie Blue staining or transferred to Invitrogen’s ImmobilonTM poly(vinylidene difluoride) membranes using NovexTM 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 proteins 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 lm MG132 prior to harvesting Purified proteins 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 identification was performed by searching the MS data against a curated version of the International Protein Index FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS ´ ´ A Fernandez-Montalvan et al (http://www.ebi.ac.uk/IPI/) using the mascot software (Matrix Science, Boston, MA, USA) Sites of post-translational 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 ă 101500 kDa The column was connected to an AKTA Explorer 100 chromatography system (GE Healthcare) and to a MiniDawn Tristar linked to an interferometric refractometer Optilab DSP (Wyatt Technology, Santa Barbara, CA, USA) Molecular masses were calculated with astra 4.90 software (Wyatt Technology) The column was equilibrated with NaCl ⁄ Pi and 0.5 mL of USP7-FL protein (1.5 mgỈmL)1) was injected into the column The flow rate was 0.5 mLỈmin)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 mm EDTA, mm dithiothreitol, 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 using a Genios fluorescence plate reader from Tecan (Mannedorf, 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 completely hydrolyze lm of Ub-AMC Same enzyme concentrations were used to test USP7-FL activity on lm SUMO-1-AMC and Nedd8-AMC (Boston Biochem), as well as on 250 lm Z-Leu-Arg-Gly-Gly-AMC (Biomol International, Plymouth Meeting, PA, USA) For subsequent determination of the KM and Vmax 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 nm enzyme and lm Ub-AMC The pH dependency 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 phosphate ⁄ 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 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 Biochemical characterization of USP7 Tris ⁄ HCl buffer at pH 8.0, with mm dithiothreitol and 0.1% (w ⁄ v) Chaps Activity measurements using Ub-K-TAMRA and ubiquitin 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, 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 were incubated with substrate concentrations varying from to 16 lm in two-fold increments Reactions were stopped at different time points by adding lL of 50 mm iodoacetamide Product formation was measured using an Agilent 1100 HPLC instrument (Agilent Technologies, Palo Alto, CA, USA) equipped with a Poroshell 300SB-C18 reverse phase column Substrate and product peaks were separated with a 3.5 linear gradient of 0–100% acetonitrile containing 0.1% (v ⁄ v) trifluoroacetic acid and visualized using excitation and emission wavelengths of 543 nm and 580 nm, respectively In order to calculate the kinetic parameters for the hydrolysis of Ub-AMC and Ub-K-TAMRA, curves obtained by plotting the measured enzyme initial rates (v) versus the corresponding substrate concentrations ([S]) were subjected to nonlinear regression fit to the Michaelis–Menten equation V ¼ (Vmax · [S]) ⁄ ([S] + KM) (Eqn 1), where Vmax is the maximal velocity at saturating substrate concentrations and KM the Michaelis constant The kcat value was derived from the equation kcat ¼ Vmax ⁄ [Eo] (eqn 2) where [Eo] 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 GlutaMAXTM 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, FUGENETM reagent (Roche) was used, following manufacturer’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 CelLyticTM lysis buffer for FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS 4267 ´ ´ A Fernandez-Montalvan et al Biochemical characterization of USP7 mammalian cells supplemented with a protease inhibitor cocktail (both from Sigma) following the manufacturer’s instructions For immunobloting, clear lysates were mixed : (v ⁄ v) with · Laemmli SDS sample buffer, boiled at 95 °C and spun down at 12 000 g in an Eppendorf 5424 microcentrifuge (Eppendorf AG, Hamburg, Germany) Proteins were separated in NovexTM 4–12% gradient gels and transferred onto ImmobilonTM poly(vinylidene difluoride) membranes as described above Immunodetection of the proteins with specific antibodies was performed using general protocols provided by the manufacturers For native PAGE experiments, 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 · 104 cells per well on Laboratory-Tek German borosilicate glass chamber slides (Nunc) and allowed to settle overnight Cells were subsequently fixed with 2% paraformaldehyde 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 antimouse-Alexa 488 conjugate (Molecular Probes, Invitrogen) (1 : 5000) Nuclear and membrane counterstaining was performed using the Image-iTTM 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 Schweigler, 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 (Cellzome AG, Heidelberg, Germany) for technical support, Rita Schmitz (Novartis, Basel, Switzerland) for providing expression vectors and USP7 cDNA as well as Shirley Gil-Parrado, Bruno Martoglio, Martin Renatus and Jorg Eder (Novartis, Basel, Switzerland) ă for support and helpful discussions A Fernandez´ Montalvan would like to dedicate this paper to the ´ memory of his father J A Fernandez-Vidal, who died in a failed bladder cancer surgery while it was being written 4268 References Amerik AY & Hochstrasser M (2004) Mechanism and function of deubiquitinating enzymes Biochim Biophys Acta 1695, 189–207 Nijman SM, Luna-Vargas MP, Velds A, Brummelkamp TR, Dirac AM, Sixma TK & 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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 http://www.blackwell-synergy.com 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 corresponding author for the article FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR) Journal compilation ª 2007 FEBS ... 1102 USP7- FL USP7 1-560 USP7 208-560 USP7 208-1102 EGFP USP7 1-205-EGFP EGFP USP7 20-205-EGFP EGFP USP7 50-205-EGFP EGFP USP7 70-205-EGFP Fig Structural? ??functional features and constructs of USP7. .. Fernandez-Montalvan et al Biochemical characterization of USP7 Fig Enzymatic characterization of USP7 (A) Progress curves for the USP7- catalyzed hydrolysis of Ub-AMC (j), SUMO-1-AMC (d) and Nedd8-AMC... 208-1102 FL USP7 Fig Structural requirements for nuclear localization of USP7 Several cell lines were transiently transfected with FLAG-Myc-tagged USP7FL, USP7 1-560, USP7 208-560 and USP7 208-1102,