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

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Biochemical characterization of USP7 revealspost-translational modification sites and structuralrequirements for substrate processing and subcellularlocalizationAmaury Ferna´ndez-Montalva´n1, Tewis Bouwmeester2, Gerard Joberty2, Robert Mader3,Marion Mahnke4, Benoit Pierrat1, Jean-Marc Schlaeppi4, Susanne Worpenberg1and Bernd Gerhartz11 Expertise Platform Proteases, Novartis Institutes for Biomedical Research, Basel, Switzerland2 Cellzome AG, Heidelberg, Germany3 Musculoskeletal Disease Area, Novartis Institutes for Biomedical Research, Basel, Switzerland4 Biologics Centre, Novartis Institutes for Biomedical Research, Basel, SwitzerlandDeubiquitinating enzymes (DUBs) are a superfamily ofthiol- and metallo proteases specialized in the process-ing of ubiquitin and ubiquitin-like proteins. They areresponsible for the disassembly of ubiquitin chains, andfor the cleavage of mono- and oligomers of this mole-cule, either in precursor form or attached to smallKeywordsbiochemical characterization; cysteineprotease; deubiquitinating enzyme; ubiquitinpathway; USP7 ⁄ HAUSPCorrespondenceA. Ferna´ndez-Montalva´n, MolecularScreening and Cellular Pharmacology,Merck Serono S.A., 9 Chemin des Mines,Case postale 54, CH-1211 Geneva 20,SwitzerlandFax: +41 22 4149558Tel: +41 22 4144977E-mail: amaury.fernandez@merckserono.netB. Gerhartz, Expertise Platform Proteases,Novartis Institutes for Biomedical Research,CH-4002, Basel, SwitzerlandFax: +41 61696 8132Tel: +41 61696 1204E-mail: bernd.gerhartz@novartis.com(Received 20 April 2007, revised 14 June2007, accepted 25 June 2007)doi:10.1111/j.1742-4658.2007.05952.xUbiquitin specific protease 7 (USP7) belongs to the family of deubiquitinat-ing enzymes. Among other functions, USP7 is involved in the regulation ofstress response pathways, epigenetic silencing and the progress of infectionsby DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core,N- and C-terminal domains required for protein–protein interactions. Inthe present study, recombinant USP7 full length, along with several vari-ants corresponding to domain deletions, were expressed in different hostsin 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 inmammalian cells. In in vitro activity assays, N- and C-terminal truncationsaffected the catalytic efficiency of the enzyme different. Both the proteasecore alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct includingthe 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. Besidesplaying an important role in substrate recognition and processing, thisregion might be involved in enzyme dimerization. USP7 constructs lackingthe N-terminal domain failed to localize in the cell nucleus, but no nuclearlocalization signal could be mapped within the enzyme’s first 70 aminoacids. Instead, the tumor necrosis factor receptor associated factor-likeregion (amino acids 70–205) was sufficient to achieve the nuclear localiza-tion of the enzyme, suggesting that interaction partners might be requiredfor USP7 nuclear import.AbbreviationsCBP, 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 necrosisfactor 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 FEBSnucleophiles and proteins [1]. Among the DUBs, theubiquitin specific proteases (USPs) constitute the larg-est subfamily with 58 cysteine peptidase genes identifiedso far [2]. One of the most prominent members of thissubfamily is USP7 (EC 3.1.2.15), also known as herpesvirus associated ubiquitin-specific protease (HAUSP)due to its discovery in the promyelocytic leukemianuclear bodies of herpes simplex virus-infected cells [3].Recognition and processing of ubiquitylated forms ofthe tumor suppressor p53 and its negative modulatorMDM2, a RING domain E3-ligase, suggested animportant role for USP7 in cell survival pathways[4–7]. More recently, the identification of MDMX andDAXX (both regulatory proteins in the p53-MDM2pathway) as USP7 substrates [8,9] has revealed a farmore complex involvement of this enzyme in cell fatedecisions than initially expected. In addition, reportsabout 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 maintenanceof cell homeostasis. Additional evidence for the crucialrole of USP7 is provided by the fact that targeting thisenzyme belongs to the strategies evolved by the herpessimplex virus [12,13] and Epstein–Barr [14,15] virusesfor successful host infection.USP7 is a 1102 amino acid protein with a molecularweight of approximately 130 kDa (Fig. 1A). In cells,the enzyme has been reported to be dimerized, poly-ubiquitinated and polyneddylated [16]. The sites orregions involved in these events have not been mappedso far. The N-terminal of USP7 part displays sequencehomology to the TNF receptor associated factors(TRAFs) and was shown to interact with severalTRAF family proteins [17]. This domain also bindsfragments 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-structureof an USP7 fragment containing amino acids 54–204disclosed an eight-stranded beta sandwich fold typicalfor the TRAF protein family [15]. Further cocrystalstructures with substrate-derived peptides, revealedthat a P ⁄ AXXS consensus sequence is recognizedmainly by residues W165 and N169 located in a shal-low surface groove on the TRAF domain [15,19,21].Limited proteolysis identified two digestion resistantfragments in the C-terminal region of USP7, mappingto amino acids 622–801 and 885–1061 [18]. The first ofthese polypeptides was shown to mediate the inter-action of USP7 with the herpes virus protein ICP0in vitro [18]. Additionally, a yeast two hybrid screenrevealed a region including amino acids 705–1102 wasrequired for association with Ataxin-1 [22] (Fig. 1A).Further structural–functional features of this domainare currently unknown. Sequence analysis anticipateda protease domain with conserved Cys and Hisboxes delimited by the N- and C-terminal regions [3].Ataxin bindingUbiquitin bindingEBNA1 / p53 / HDM-2 bindingICP-0 binding 1208 560 1102TRAFProtease Core C-TerminalD481C223ABH46412085601102USP7-FLUSP7 1-560USP7 208-560USP7 208-1102EGFPEGFPEGFPEGFPUSP7 1-205-EGFPUSP7 20-205-EGFPUSP7 50-205-EGFPUSP7 70-205-EGFPFig. 1. Structural–functional features and constructs of USP7designed for this study. (A) Schematic representation of the USP7structure. The N-terminal TRAF-like domain (amino acids 50–205) ispreceded by a Q-rich region not represented here. This domain hasbeen reported to interact with p53, MDM2 and Epstein–Barr virusnuclear 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 aminoacids 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 differenthosts. Constructs expressed using the baculovirus system (allexcept the protease core) had a C-terminal hexahistidine tag. Thecatalytic domain was expressed as a GST-6XHis N-terminal fusionprotein. Variants designed for expression in mammalian cells hadan N-terminal 3XFLAG tag and a C-terminal Myc tag. USP7-FL con-structs used for proteomics analysis contained either N- or C-termi-nal CBP-Protein A tags separated by a TEV-protease cleavage site.A. Ferna´ndez-Montalva´n et al. Biochemical characterization of USP7FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4257Matching these predictions, limited proteolysis andX-ray crystallography disclosed amino acids 208–560as the protease core of USP7 [20] (Fig. 1A). Twocrystal structures of this fragment alone and incomplex 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 activationmechanism for USP7 in which a papain-like catalytictriad (C223, H464 and D481) is assembled via con-formational changes triggered by the interaction withubiquitin. A similar mechanism was described thesame year for the activation of the structural homo-logue calpain by calcium ions [26]. Interestingly,here the catalytic unit is significantly less active thanthe full length heterodimeric enzyme [26,27]. Theindividual contributions of USP7 structural domainsto the activity of the full length enzyme have not beeninvestigated so far.In the present study, the biochemical properties andstructure–function relationships of USP7 were charac-terized. We have mapped sites for phosphorylationand ubiquitination, and studied the oligomerizationstate of the enzyme in vitro and in cells. The kineticparameters for the hydrolysis of ubiquitin substratesby full length USP7 and domain deletion variants havebeen determined. The results suggest a role for theC-terminus in substrate processing and oligomeriza-tion. In addition, a fragment including amino acids70–205 was found to be sufficient for nuclear targeting.As this region is involved in protein–protein inter-actions, association with nuclear proteins might berequired for USP7 subcellular localization.ResultsHeterologous expression and purificationof functional USP7 variantsIn the present study, a novel semiautomated expressionand purification system was used for the production ofseveral USP7 domain deletion variants (Fig. 1B) inBaculovirus-infected insect cells. The procedure yieldedapproximately 6 mg (USP7 full length), 5 mg (1–560)and 4 mg (208–1102) of purified recombinant proteinper litre of insect cell culture. In addition, an averageof 7 mg USP7 208-560 per litre of Escherichia coli fer-mentation broth was obtained from the soluble cellfraction. The recombinant proteins were purified tohomogeneity (‡ 90%) based on SDS ⁄ PAGE (Fig. 2)and reversed phase HPLC analysis. N-terminalsequencing showed that both USP7-FL and USP71-560 expressed in insect cells were N-terminally blockedby acetylation, as confirmed by MALDI-TOF-MS.LC-MS analysis of USP7-FL revealed two proteinmasses of 130 464.0 and 130 540.0 Da, correspondingvery likely to acetylated and single phosphorylatedUSP7, respectively. Again, two masses of 65 919.5 and65 999.5 were found for USP7 1-560, correspondinglikewise to acetylated and single phosphorylatedUSP7 1-560, respectively. This post-translationalmodification was later confirmed in USP7 purifiedfrom mammalian cells (see below). LC-MS analysisof USP7 208-1102 showed that around 60% of theprotein had a three amino acid truncation at theN-terminus. None of these modifications or hetero-geneities was observed in the 208-560 protein producedin E. coli.All USP7 variants were subjected to limited proteo-lysis by trypsin under native conditions, in order toevaluate their structural integrity and correct foldingby comparison of cleavage sites. For USP7-FL, bandscorresponding to seven main digestion fragments werevisualized by SDS ⁄ PAGE (Fig. 2). Five out of them,with a molecular weight ‡ 25 kDa were subjected toFig. 2. Purity and folding of recombinant USP7 variants. SDS ⁄ PAGEanalysis (in a 4–20% gradient gel) of USP7 variants before (–) andafter (+) 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 thesefragments are written on the left with special symbols used tomark bands of similar identity derived from other USP7 variants.These symbols were also used to represent graphically the cleav-age sites on the schematic view of USP7 shown below.Biochemical characterization of USP7 A. Ferna´ndez-Montalva´n et al.4258 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBSprotein sequencing. This analysis mapped their N-ter-mini to residues I36, K209, E557 ⁄ Q559, S341 andI885. Identical digestion patterns were found in allvariants according to the presence or absence of thecleavage sites in their sequences (Fig. 2), strongly indi-cating a correct overall folding of these proteins. Thetryptic 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 modificationsin USP7 purified from mammalian cellsThe observation that USP7 expressed in insect cellswas phosphorylated in its N-terminal region motivatedus to investigate post-translational modifications ontandem affinity purification (TAP)-tagged USP7 puri-fied from mammalian cells. LC-MS ⁄ MS analysisrevealed the presence of two phosphopeptides AGEQQLSEPEDMEMEAGDTDDPPR, corresponding toamino acids 12 to 35, and IIGVHQEDELLECLSPATSR, corresponding to amino acids 949–968. Manualverification of the corresponding MS ⁄ MS spectraallowed for the assignment of the phosphoacceptor res-idues to S18 and S963, respectively (Fig. 3A). USP7was previously described to be ubiquitinylated andneddylated. Western analysis showed that affinity puri-fied TAP-tagged USP7 is (mono)-ubiquitinylated inHeLa cells (Fig. 3B). LC-MS ⁄ MS identified a singleubiquitinylated ⁄ neddylated peptide, DLLQFFKPRcorresponding to amino acids 863–871. Manual inspec-tion of the MS ⁄ MS spectra showed that the diglycineremnant was conjugated to K869. The strong identifi-cation of ubiquitin in the same gel band as USP7,combined with the absence of Nedd8, strongly suggeststhat the modified site is indeed ubiquitinylated.Analysis of USP7 oligomerization: possible roleof the C-terminal regionUSP7 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 ofUSP7-FL and USP7 208-1102 purification, fractionsdisplaying DUB activity eluted from the Superdex 200SEC column as single peaks but at elution volumescorresponding to significantly larger proteins. Theseobservations were confirmed by analysis of freshlypurified USP7-FL using analytical size exclusion chro-matography coupled to light scattering measurement.As shown in the supplementary Fig. S1A, USP7-FLshowed a retention time on the Sephacryl S-300column between ferritin (440 kDa) and aldolase(158 kDa), suggesting a molecular weight of around250 kDa. In contrast, the light scattering measure-ments showed an average molecular mass between131.8 and 139.0 kDa, corresponding to the monomericform of USP7. Noteworthy, the light scattering resultsmay be indicative of a mixed population, with mostlymonomers but also a few dimers or higher aggregates.The amount of dimers or aggregates appears toincrease, when freezing and thawing the protein (datanot shown). In native nonreducing PAGE, purifiedUSP7-FL migrated as two discrete bands of relativemobilities corresponding to the monomer and putativedimers (supplementary Fig. S1B). Accordingly, whencell lysates containing either endogenously or ectopi-cally expressed USP7 were subjected to native PAGEand proteins detected by western blot again two anti-body reactive bands were observed (supplementaryFig. S1C). In line with this observation, LC-MS ⁄ MSanalysis of proteins copurified with the TAP-taggedUSP7 as described above revealed the presence of thenontagged USP7 N-terminal peptide (MNHQQQQQQQK) derived from the endogenous enzyme (notshown). Interestingly, variants lacking the C-terminalregion ran as a single band in the native nonreducingPAGE (supplementary Fig. S1B), suggesting a role forthe C-terminal in the oligomerization event.Substrate specificity and enzymatic propertiesof USP7As part of the characterization of USP7 biochemicalproperties, we have measured its kinetic parameters forthe hydrolysis of ubiquitin C-terminal 7-amido-4-meth-ylcoumarin (Ub-AMC), a fluorogenic substrate whichhas proven to be an useful tool with a number ofdeubiquitinating enzymes [28–31]. In order to assess itssubstrate specificity, USP7 activities on small ubiqu-itin-like modifier protein 1 (SUMO-1)-AMC, Nedd8-AMC and Z-LRGG-AMC, a synthetic peptidesubstrate representing the C-terminus of ubiquitin,were investigated. In addition, we evaluated thehydrolysis by the enzyme of ubiquitin C-terminal-Lys-tetramethylrhodamine (Ub-K-TAMRA) and Ub-K-peptide-TAMRA, two substrates with the fluorophoregroup attached as isoamide bond. The Ub-AMC assaydescribed in the experimental section was linear for atleast 1 h at enzyme concentrations up to 5 nm. Usingsimilar conditions with SUMO-1-AMC and Nedd8-AMC as substrates, no USP7 activity could bedetected, indicating a high specificity for ubiquitin,despite the well known homologies among ubiquitin-like proteins (Fig. 4A). Unlike other DUBs [31,32],A. Ferna´ndez-Montalva´n et al. Biochemical characterization of USP7FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4259200 400 600 800 1000 1200 1400m/z0100AB%IIGVHQEDELLECL(pS)PATSRy''5531.4243.2I/L86.1a2199.2y''2262.2y''3363.2486.3y''6698.4b10 2+567.9600.4b101134.7750.4b91021.7b111247.9b121376.8MH33+ -H3PO4y''6-H3PO4LE/ELb2227.2y'‘8 2+y8971.6873.6y'‘8-H3PO4y91100.6y7811.5pSWB: CBPWB: UbTAP-USP7CBP-USP7-+-+-+MG132Ub-USP797 kDa97 kDaLysate TEV-eluateCBP-eluateTandem Affinity Purification200 400 600 800 1000m/z0100%b2229.1a2201.1y''5808.5y''2272.2y''4661.4b3342.2 y''3514.3y''6936.6y''71049.7(GG)KFig. 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 proteinsthroughout the two-step tandem affinity purification from mammalian cells using anti-CBP and anti-ubiquitin sera. Cells were either nontreatedor pretreated with the proteasome inhibitor MG132. Right panel: LC-MS ⁄ MS spectrum of the USP7 tryptic peptide DLLQFF ⁄ (Ub-K)PRcontaining the ubiquitinated residue K869.Biochemical characterization of USP7 A. Ferna´ndez-Montalva´n et al.4260 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBShydrolysis of Z-LRGG-AMC could not be measuredat maximum enzyme concentrations of 200 nm. USP7is active on Ub-AMC in a pH range between 7.5 and9.5 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 thanwith 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 recognizedUb-AMC and Ub-K-TAMRA with slightly differentaffinities. Accordingly, the catalytic efficiency of USP7-FL for the hydrolysis of Ub-K-TAMRA was improvedby five-fold with respect to Ub-AMC. Under theconditions chosen for the assay, saturation was notreached with Ub-K-peptide-TAMRA.Processing of ubiquitin synthetic substrates byUSP7-FL and domain deletion variantsEvaluation of the hydrolysis of Ub-AMC and Ub-K-TAMRA by USP7-FL and its domain deletionFig. 4. Enzymatic characterization of USP7. (A) Progress curves for the USP7-catalyzed hydrolysis of Ub-AMC (j), SUMO-1-AMC (d) andNedd8-AMC (.). Raw fluorescence intensities (RFU) collected every 5 min with kex¼ 360 nm and kem¼ 465 nm were plotted as a functionof 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 NaCland 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 appropriatebuffers 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 was5nM 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), USP7208-560 (m) and USP7 208-1102 (.). These experiments were conducted at room temperature in 50 mM Tris ⁄ HCl buffer, pH 7.5, 1 mMEDTA, 5 mM dithiothreitol, 100 mM NaCl and 0.1% (w ⁄ v) Chaps with 1 lM Ub-AMC and the enzyme concentrations indicated in the experi-mental section.A. Ferna´ndez-Montalva´n et al. Biochemical characterization of USP7FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4261variants at increasing enzyme concentrations revealedthat different amounts of each protein were requiredto attain comparable reaction velocities (Fig. 4D). Thekinetic parameters for these reactions were determinedby measuring their rates at increasing substrate con-centrations. To this end, enzyme concentrations thatallowed assay linearity for at least 1 h were used. Asshown in Table 1, the deletion variants recognizedboth substrates with similar affinities, but remarkabledifferences were observed in the turnover (kcat) andconsequently in the catalytic efficiency (kcat⁄ KM).USP7 208-560 and USP7 1-560 were significantly lessactive than the full length enzyme, whereas the enzy-matic activity of USP7 208-1102 was rather similar tothe wild-type. These results indicate an important rolefor the C-terminal domain in catalysis. The compari-son between Ub-AMC and Ub-K-TAMRA, revealedmore pronounced differences in the catalytic efficiencyof the variants relative to USP7-FL when using thee-amino-linked substrate.C-terminal truncations destabilize theubiquitin–enzyme complexHaving realized the importance of USP7 C-terminusfor efficient substrate processing, the question wasasked whether conformational changes driven byubiquitin binding to the core domain, or direct inter-actions of this region with the substrate would berequired for proper recognition and processing. Inorder to address this issue USP7-FL and the domaindeletion variants were subjected to limited proteolysisby trypsin under native conditions in the presence orabsence of a molar excess ubiquitin. Digestion wasexamined over time by SDS ⁄ PAGE and CoomassieBlue staining. Surprisingly, the fragments produced bylimited proteolysis were identical with and withoutubiquitin (Fig. 5). N-terminal sequencing of them con-firmed that the cleavage sites corresponded to thoseobserved in the experiment described above (Fig. 2).However, stabilization of some proteolysis products inthe presence of ubiquitin was observed, demonstratinga partial protection of some trypsin cleavagesequences. The main fragment stabilized in the fulllength enzyme contained amino acids I36 to R558.This effect was less pronounced in USP7 1-560. Invariants lacking the N-terminal domain, a fragmentcorresponding to amino acids K209 to R559 was stabi-lized by the presence of ubiquitin. Interestingly, thisbehavior was more evident for USP7 208-1102. Inboth digestion products, the cleavage site protected bythe presence of ubiquitin was Ser341, located in the‘fingers’ region of the catalytic core domain involvedin the recognition of the ubiquitin core. These resultsshow that all USP7 variants were able to bind ubiqu-itin through the protease core domain, suggesting thatFig. 5. Limited proteolysis of USP7 variants in the presence andabsence of ubiquitin. SDS ⁄ PAGE (4–20% gradient gels) showing thelimited proteolysis of native USP7-FL and variants thereof by trypsinover time with and without ubiquitin. The arrows indicate fragmentsfrom USP7-FL and USP7 208-1102 protected from tryptic digestion bythe presence of ubiquitin. N-terminal sequences of these fragmentsare 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] (nM) KM(lM) kcat(s)1) kcat⁄ KM(s)1ÆlM)1)Fold decrease incatalytic efficiencyFull length Ub-AMC 1 17.5 ± 2.0 3.56 2.03 · 1051Ub-K-TAMRA 5 6.6 ± 0.7 6.76 1.02 · 10611–560 Ub-AMC 100 27.6 ± 3.4a0.045 1.6 · 103127Ub-K-TAMRA 1000 10.9 ± 1.0 0.018 1.6 · 103644208–560 Ub-AMC 100 44.2 ± 3.8a0.077 1.7 · 103119Ub-K-TAMRA 2000 36.8 ± 4.9a0.039 1.1 · 103936208–1102 Ub-AMC 5 22.8 ± 2.1 0.805 3.53 · 1046Ub-K-TAMRA 100 7.2 ± 0.8 0.33 4.58 · 10423aKmvalues higher than the maximum substrate concentrations used for the titrations should be considered as approximate figures.Biochemical characterization of USP7 A. Ferna´ndez-Montalva´n et al.4262 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBSthe enzyme–substrate complexes were more stable inthe context of an intact C-terminal region.Structural requirements for USP7 nuclearlocalizationIn order to further characterize structure–function rela-tionships for USP7, we studied the effect of domaindeletions in the subcellular localization patterns of theenzyme. To this end, several mammalian cell lines weretransiently transfected with vectors encoding the USP7variants 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 constructsequence and the cell line used. Both antibodies detectedhigher quantities of USP7 1-560 and USP7 208-560than USP7 full length and USP7 208-1102 in thewestern blots (not shown). Immunofluorescent stainingrevealed different subcellular localization patterns forthe constructs (Fig. 6). USP7-FL and variant 1-560localized preferentially to the cell nucleus, whereasUSP7 208-560 and USP7 208-1102 were detected mostlyin the cytosol. A small fraction of USP7 208-560observed in the nucleus is likely an artifact caused bythe strong over expression of this variant because USP7208-1102 did not show this behavior. Fusion proteinscontaining the N-terminal domain of USP7 (aminoacids 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 thecell nucleus (Fig. 6).DiscussionIn the present study, we have mapped S18, S963 andK869 as phosphorylation and ubiquitination sites ofUSP7. Depending on the techniques used, monomersor dimers of the enzyme were detected in vitro, whereasin cells evidence was obtained pointing to oligomeriza-tion events. Deletion of the N- and C-terminaldomains 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 enzymeoligomerization. Finally, we have observed that theN-terminal domain of USP7, and particularly a frag-ment including amino acids 70–205, is sufficient toachieve nuclear localization of the enzyme.Based on our results, USP7 can be added to the listof deubiquitinating enzymes found to be phosphory-lated [33–35]. In fact, phosphorylation on S18 had beenreported previously from a HeLa large scale proteomicsstudy [36]. This phosphorylation site is a low stringencyconsensus site for casein kinase II. Noteworthy, thecasein kinase II catalytic subunits alpha1 and alpha2and regulatory subunit beta were copurified withtagged USP7, suggesting that CKII could indeed be theupstream kinases responsible for the phosphorylationat this position (data not shown). S963 phosphoryla-tion has not been described so far and this position isnot a known consensus site for any kinase. Interest-ingly, both sites are located near regions involved inprotein–protein interactions. By analogy with theDUB CYLD [35] and TRAF family members such asTANK [37,38], whose function is modulated by theinhibitor of jB kinase, a regulatory role can be pre-sumed for USP7 phorsphorylation. The identificationof K869 as the ubiquitination site of USP7 representsadditional evidence for the interaction of the enzymewith E3 ubiquitin ligases. Remarkably, the ubiquitina-tion site is close to the region where it was reported tointeract with ICP-0 [18], supporting the observationthat USP7 can be ubiquitinated by this E3 ligase butnot by MDM2 [12]. Our findings indicate that USP7could exist as a dimer in cells. The data obtained withpurified enzyme is, however, contradictory, suggestingthat further cellular components might be required tostabilize these oligomers. Noteworthy the enzymaticbehavior 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 tounderstand 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 alignswith recent data reported for USP2 [25] and USP8[39], suggesting that recognition of both the ubiquitinC-terminus and its core are equally important forcatalysis. The affinity of USP7 for Ub-AMC (KM¼17.5 lm) was approximately 500-fold lower than in thecase of the ubiquitin C-terminal hydrolases (UCHs)[29]. Compared to other USPs, USP7 shows slightlylower affinities for Ub-AMC than USP5 (KM¼1.4 lm) [30] and USP2 (KM¼ 0.554 lm) [25], respec-tively, and displays a similar KMas USP8 (KM¼10.2 lm) [39]. Differences in the ubiquitin recognitionmechanisms and in the structural rearrangements uponsubstrate binding displayed by UCHL-1 [37], UCHL-3[20,40,41] and USP5 [32,42], might account for thevariations in affinity with respect to USP7. Renatuset al. [25] discussed recently the possible origin of thesubstrate affinity divergences compared to USP7 in adetailed analysis of the interaction of USP2 catalyticcore with ubiquitin. Despite the higher KM, the cata-lytic efficiency of USP7 (kcat⁄ KM) is only weakerA. Ferna´ndez-Montalva´n et al. Biochemical characterization of USP7FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4263compared to that of UCHL-3 (2.1 · 108m)1s)1) [29].Otherwise the kcat⁄ KMis 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 · 105m)1Æs)1) [39]. This value is only higher than thosereported for USP14 (UBP6 in yeast) (1.07 ·102m)1Æs)1) [43] and the viral SARS-CoV PLpro(2.69 · 102m)1Æs)1and 1.31 · 104m)1Æs)1) [28,31].The pH and ionic strength dependencies of USP7 foractivity on Ub-AMC are similar to those describedpreviously using a glutathione S-transferase (GST)-Ubi52 as substrate [18]. These are typical for a DUBand for cysteine proteases in general. A recent discus-sion is provided elsewhere [29].In USP7, the kinetic parameters for the hydrolyisisof ubiquitin substrates appear strongly affected by theImage-iT™FL208-11021-205-EGFP70-205-EGFPUSP7 MergeFig. 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 andUSP7 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 (bothshown here in green). Image-iTTMcounterstaining for the nuclei (blue) and cellular membranes (red) was applied. The results shown herecorrespond to USP7-FL, USP7 208-1102, USP7 1-205-EGFP and USP7 70-205-EGFP expressed in U2OS cells.Biochemical characterization of USP7 A. Ferna´ndez-Montalva´n et al.4264 FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBSdeletion of structural features outside the proteasecore. Therefore, we conclude that these domains areimportant for catalysis. A contribution for the TRAF-like domain should not be neglected, but the mostimportant support for substrate processing seems to beprovided by the C-terminal domain. This is the secondknown example of mutations outside the catalytic coreaffecting the enzymatic properties of a DUB. In UBPt,the testis-specific murine homologue of USP2, N-ter-minal domain deletions mimicking splice variants ofthe enzyme influenced not only its subcellular localiza-tion [44], but also its substrate specificity [45]. InUSP7, the noncatalytic domains might be involved inspecificity determination as well. This idea is supportedby the kcat⁄ KMincrease measured exclusively for thefull length enzyme when a P1¢ lysine residue was linkedto ubiquitin through an e-amino bond in order to bet-ter mimic the a physiological substrate. Of note,attaching of a TAMRA-labeled undecapeptide notrelated to any known USP7 interaction partner ratherdecreased the catalytic performance of the enzyme,apparently due to reduced substrate affinity. Makingthe assumption that the primary function of thisenzyme is to detach monoubiquitin tags from modifiedproteins rather than to process of ubiquitin chains, thisobservation might explain the lower catalytic efficien-cies displayed with K48 linked diubiquitin [20], andsupport 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]. Sincebioinformatics tools did not anticipate any functionalnuclear localization signal (NLS) within this domainand most TRAF proteins localize in the cytosol [46],we hypothesized that a novel NLS might be containedby the first 70 N-terminal residues of USP7, a regionsharing neither sequence- nor structural similaritieswith other TRAF family members. Secondary structureprediction of this region using the GOR algorithm [47]anticipated a coiled region between residues M1 andE20, an alpha helix from there and up to amino acidG28, followed by a b-sheet starting at T36 and extend-ing to residue L49, and a larger helix including aminoacids 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, thesevariants 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 sofar. In addition, TRAF1 displays both nuclear andcytosolic localization when expressed in isolation [46].Interestingly, TRAF4 seems to require the interactionwith a rapidly titrated endogenous factor, rather thana NLS [46]. Remarkably, the TRAF domain of USP7also interacts with several nuclear proteins such as p53,mdm2 and the family members TRAF4 and TRAF1,suggesting that nuclear localization of this enzymemight be dependent on its interactions with one orseveral of the above mentioned partners.Although the role of USP7 is by far not fully under-stood, evidence accumulates in favor of its potential astherapeutic target in cancer indications. The molecularinsight provided by the crystal structure of its catalyticdomain in complex with ubiquitin will guide the designof potent inhibitors for this enzyme. However, difficul-ties to attain selectivity are predicted based on theexperience accumulated with other cysteine proteases.In this context, a better understanding of the involve-ment of noncatalyitic domains in enzyme function mayopen opportunities for alternative drug discoveryapproaches such as allosteric and protein–proteininteraction inhibitors.Experimental proceduresMaterialsAll 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 monoclonalantibody (Ubi1) was from Zymed (Invitrogen, Carlsbad,CA, USA) and calmodulin binding protein (CBP) antibodywas from Upstate (Millipore, Billerica, MA, USA). Anti-FLAG (M2) and anti-myc (2E10) monoclonal sera werepurchased from Sigma. Rabbit anti-mouse-HRP and goatanti-rabbit-HRP secondary sera conjugates were fromSigma and Biorad (Hercules, CA, USA), respectively. Goatanti-rabbit-Texas red and rabbit-anti-mouse-Alexa 488 seraconjugates for secondary detection of immunostained cellswere from Molecular Probes (Invitrogen). Mammalian celllines were acquired from the ATCC (Manassas, VA, USA)and Spodoptera frugiperda (Sf9) cells from Invitrogen.Generation of plasmids, bacmids andbaculovirusesFull length USP7 cDNA, was amplified by PCR and insertedinto the pCR2.1-TOPO vector (Invitrogen) following theA. Ferna´ndez-Montalva´n et al. Biochemical characterization of USP7FEBS Journal 274 (2007) 4256–4270 ª 2007 Novartis Institutes for Biomedical Research (NIBR). Journal compilation ª 2007 FEBS 4265[...]... 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) ¨ ´ for support and helpful discussions A Fernandez´ Montalvan... 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... 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... 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. .. in time intervals of 5 min 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 1 lm of Ub-AMC Same enzyme concentrations were used to test USP7- FL activity...´ ´ 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... 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... with a Poroshell 300SB-C18 reverse phase column Substrate and product peaks were separated with a 3.5 min linear gradient of 0–100% acetonitrile containing 0.1% (v ⁄ v) trifluoroacetic acid and visualized using 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... final volume of 150 lL Reactions were stopped by addition of one volume of 2 · Laemmli sample buffer, followed by boiling at 95 °C for 5 min The effect of 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 min upon... (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 . Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization Amaury. C-TerminalD481C223ABH46412085601102 USP7- FL USP7 1-560 USP7 208-560 USP7 208-1102EGFPEGFPEGFPEGFP USP7 1-205-EGFP USP7 20-205-EGFP USP7 50-205-EGFP USP7 70-205-EGFPFig. 1. Structural functional
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