USP7, a Ubiquitin-Specific Protease, Interacts with Ataxin-1, the SCA1 Gene Product

MCN

Molecular and Cellular Neuroscience 20, 298 –306 (2002) doi:10.1006/mcne.2002.1103

USP7, a Ubiquitin-Specific Protease, Interacts with Ataxin-1, the SCA1 Gene Product Sunghoi Hong, Sung-Jo Kim, Sojeong Ka, Inho Choi, and Seongman Kang 1 Graduate School of Biotechnology, Korea University, 1,5-ka Anam-dong, Sungbuk-ku, Seoul 136-701, Korea

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration. SCA1 has been known to associate with elongated polyglutamine tract in ataxin-1, the SCA1 gene product. Using the yeast two-hybrid system, we have found that USP7, a ubiquitin-specific protease, binds to ataxin-1. Further experiments with deletion mutants indicated that the C-terminal region of ataxin-1 was essential for the interaction. Liquid ␤-galactosidase assay and coimmunoprecipitation experiments revealed that the strength of the interaction between USP7 and ataxin-1 is influenced by the length of the polyglutamine tract in the ataxin-1; weaker interaction was observed in mutant ataxin-1 with longer polyglutamine tract and USP7 was not recruited to the mutant ataxin-1 aggregates in the Purkinje cells of SCA1 transgenic mice. Our results suggest that altered function of the ubiquitin system can be involved in the pathogenesis of spinocerebellar ataxia type 1.

INTRODUCTION Expansion of CAG trinucleotide repeats encoding the polyglutamine tract has been identified as a common pathogenic mutation in a number of neurodegenerative disorders, which include spinocerebellar ataxia type 1 (SCA1) (Orr et al., 1993), spinal and bulbar muscular atrophy (SBMA) (La Spada et al., 1991), Huntington disease (HD) (The Huntington’s Disease Collaborative Research Group, 1993), dentatorubralpallidoluysian atrophy (DRPLA) (Koida et al., 1994; Nagafuchi et al., 1994), MachadoJoseph disease (MJD) (Kawaguchi et al., 1994), SCA2 (Sanpei et al., 1996; Pulst et al., 1996; Imbert et al., 1996), SCA6 (Zhuchenko et al., 1997), and SCA7 (David et al., 1997), and

1 To whom correspondence and reprint requests should be addressed. Fax: 82-2-927-9028. E-mail: [email protected].

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the number of disorders caused by the same mechanism is expected to increase further. SCA1 is an autosomal dominant disorder characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells (Zoghbi, et al., 1995). The protein ataxin-1, an SCA1 gene product, is found predominantly in the nucleus and cytoplasm of neurons and peripheral tissues, respectively (Servadio et al., 1995). A selective degeneration of cerebellar Purkinje cells and brainstem neurons occurs in SCA1 (Zoghbi et al., 1995), despite the broad expression patterns of ataxin-1 in the central nervous system as well as in the non-neuronal tissues. Thus, cell-specific proteins may mediate the pathogenesis of SCA1 and other polyglutamine neurodegenerative disorders. Proteins found to associate with ataxin-1 include the leucine-rich acidic nuclear protein (LANP) (Matilla et al., 1997) and the widely expressed glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Burke et al., 1996). GAPDH interacts also with the HD, DRPLA, and SBMA gene products (Burke et al., 1996; Koshy et al., 1996), and has been suggested to interact directly with polyglutamine in the proteins. Taking into consideration the selective degeneration of cerebellar Purkinje cell in SCA1, LANP, which is Purkinje cell-enriched, is intriguing as a possible pathogenic cofactor. In particular, the interaction between LANP and ataxin-1 is significantly stronger when the number of glutamines is increased. However, it remains to be elucidated how ataxin-1 contributes to the cellular and molecular mechanisms involved in the pathogenesis of SCA1. To explore the function of ataxin-1, we performed a yeast two-hybrid screen and identified an ataxin-1-interacting protein that has complete amino acid identity to USP7, which was found to strongly interact with the wildtype ataxin-1 but weakly with the mutant ataxin-1. Our results suggest that the altered function of the ubiquitin system may contribute to the SCA1 pathogenesis. 1044-7431/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

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FIG. 1. Ataxin-1 interacts with USP7. (A) A schematic representation of proteins used (ataxin-1) in and obtained (USP7) through the yeast two-hybrid screening. The numbers indicate the positions of amino acids in ataxin-1 and USP7. ␣-Helix, hatched boxes; polyglutamine tract, black box; self-association region (S.A.R.), dotted box. (B) Determination of protein regions essential for the interaction between ataxin-1 and USP7 using liquid ␤-galactosidase assay. Fragments of human ataxin-1 shown in the left panel were cloned into the yeast expression vector pLexA-BD. ␤-Galactosidase activity was quantified after the yeast was co-transformed with the indicated construct and pB42AD-USP7 705–1102 (a.a. 705–1102). As the negative control, atrophin-1, an unrelated protein gene, was subcloned into pLex-BD.

RESULTS Identification of USP7 as an Ataxin-1-Interacting Protein Protein–protein interaction is a common mechanism in various cellular processes. To identify proteins that interact with the wild-type ataxin-1, we performed a yeast two-hybrid screen using a C-terminal portion of ataxin-1 as a bait (Fig. 1A). Yeast expressing LexA-ataxin-1 was transformed with a human brain cDNA library, and six positive clones were identified by screening approximately 2.5 ⫻ 10 6 Trp ⫹ , Leu ⫹ auxotrophic transformants. In database searches performed at NCBI using the BLASTX program, one plasmid revealed a perfect match to the sequence of a ubiquitin-specific protease, USP7. Through sequence analyses, the USP7 cDNA fragment was revealed to contain a 1188-bp C-terminal

region of the entire open reading frame (ORF) of USP7. The entire USP7 ORF encodes a 1102-aminoacid polypeptide (Z72499) with a molecular mass of ⬃130 kDa (Fig. 1A). The reading frame was further confirmed by expressing the cloned USP7 cDNA in COS-7 mammalian cells. To determine whether the activation of the reporter genes lacZ and LEU2 reflects a specific interaction between USP7 and ataxin-1, pB42AD-USP7 was retransformed into yeast expressing LexA-ataxin-1 or LexABD, and the Trp ⫹, Leu ⫹ yeast transformants were then selected. LexA-ataxin-1 and pB42AD-USP7-expressing yeast cells, grown on a selective medium lacking Trp and Leu, showed a strong ␤-galactosidase activity (Fig. 1B), whereas no ␤-galactosidase activity was detected in cells containing pB42AD-USP7 and LexA-BD (data not shown), indicating that a specific interaction exists between USP7 and ataxin-1.

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Binding Determinants in Ataxin-1 To define the protein region responsible for the interaction between ataxin-1 and USP7, deleted versions of ataxin-1 were expressed as LexA-BD fusion proteins in yeast. LacZ reporter genes were activated both in cells coexpressing the LexA-ataxin-1 C-terminus (a.a. 539 – 816) and pB42AD-USP7 C-terminus (a.a. 705–1102) fusion proteins and the LexA-full length ataxin-1 and pB42AD-USP7 C-terminus, even though a weaker activation was observed in the latter case (Fig. 1B). However, no detectable ␤-galactosidase activity was observed in cells expressing the protein pairs LexAataxin-1 N-terminal stretch (a.a. 1–594) and pB42ADUSP7 C-terminus. These results suggest that the C-terminal region of ataxin-1 including the self-association region (SAR) is essential for the observed protein– protein interaction. Further experiments revealed that LexA-ataxin-1 278 – 645 or LexA-ataxin-1 589 – 816 did not activate the lacZ gene (Fig. 1B). In control experiments performed to verify our results, coexpression of pB42AD-USP7 C-terminus and LexABD-atrophin-1 (DRPLA gene product) C-terminus showed no ␤-galactosidase activity, while an activation did occur with LexA-p53 and pB42AD-T Ag, which was performed as the positive control (data not shown). Taken together, these results indicate that the C-terminal region of ataxin-1 serves as the interaction motif.

In Vivo Interaction of Ataxin-1 and USP7 To investigate whether ataxin-1 and USP7 can physically interact in mammalian cells, in vivo binding assays using GST-fusion proteins were performed. COS-7 cells were harvested 48 h after co-transfection either with pcDNA3.1/HisA-USP7 and pcDNA3/GSTataxin-1 or pcDNA3.1/HisA-USP7 705–1102 and pcDNA3/ GST-ataxin-1 539 – 816. The transfected cell extracts were mixed with glutathione-Sepharose beads, and the proteins bound to the beads were then probed with an anti-Xpress antibody (Fig. 2A) or an anti-USP7 serum r201 (Fig. 2B) to detect the precipitated USP7 proteins. USP7 705–1102 and full-length USP7 proteins were bound to the GST-ataxin-1 539 – 816 fusion protein (lane 3 in Fig. 2A) and GST–full-length ataxin-1 fusion protein (lane 3 in Fig. 2B), respectively, but no significant interaction was observed with USP7 and the GST proteins (lane 2 in Figs. 2A and 2B). These results suggest that USP7 binds to ataxin-1 under in vivo conditions, and, furthermore, confirm the in vivo interaction detected through the yeast two-hybrid assays. To further verify whether USP7 interacts with

FIG. 2. Determination of interaction between ataxin-1 and USP7 through in vivo assays. (A) COS-7 cells were cotransfected with the pcDNA3-GST and pcDNA3.1-USP7 705–1102 or pcDNA3-GST-fusion ataxin-1 539 – 816 and pcDNA3.1-USP7 705–1102 constructs. The transfected cell extracts were mixed with glutathione-Sepharose beads. The beads were then washed with PBST, and the proteins bound to the beads were Western-blotted and probed with an anti-Xpress antibody to detect the precipitated USP7 705–1102 proteins. Lane 2 indicates that USP7 705–1102 proteins were not coprecipitated with GST only, and lane 3 indicates that USP7 705–1102 proteins were coprecipitated with GSTfusion ataxin-1 539 – 816 proteins. Lanes 1 and 4 contain lysates from cotransfected cells as positive controls. (B) pcDNA3-GST and pcDNA3.1-USP7 1–1102, or pcDNA3-GST-fusion ataxin-1 (30Q) 1– 816 and pcDNA3.1-USP7 1–1102 were expressed in COS-7 cells. The proteins bound to the beads were blotted and probed with an anti-USP7 serum r201. Lane 2 indicates that USP7 1–1102 proteins were not coprecipitated with GST only, and lane 3 indicates that USP7 1–1102 proteins were coprecipitated with GST-fusion ataxin-1 (30Q) proteins. Lanes 1 and 4 contain lysates from cotransfected cells as positive controls.

ataxin-1 in mammalian cells, we performed coimmunoprecipitation assays. Constructs containing the wildtype full-length ataxin-1 with 30 glutamines, or mutant ataxin-1 with 82 glutamines and USP7, were transfected into COS-7 cells. After 48 h posttransfection, total cell extracts were prepared and immunoprecipitated with M2 anti-FLAG or anti-USP7 serum r201. Figure 3A shows that the wild-type ataxin-1 (lane 4) was coimmunoprecipitated with USP7, but not with pcDNA3.1/ HisA empty vector (lane 3), an indication that a strong

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FIG. 3. Influence of the polyglutamine length on the strength of USP7-ataxin-1 interaction. (A) COS-7 cells were cotransfected with the empty vector, wild-type ataxin-1 (30Q) 1– 816 or mutant ataxin-1 (82Q) 1– 868, and the USP7 constructs as indicated on the top. The cell lysates were immunoprecipitated with an anti-USP7 rabbit serum r201. For immunodetection, monoclonal anti-FLAG was used for detecting the FLAGtagged ataxin-1 protein. Lane 3 indicates that wild-type ataxin-1 proteins were not coimmunoprecipitated with the empty vector. Lanes 4 and 5 indicate that the USP7 proteins were coimmunoprecipitated with wild-type ataxin-1 and with mutant ataxin-1 proteins, respectively. Lanes 1 and 2 indicate cell lysates as positive controls. The blot was stripped and reprobed with the anti-USP7 to detect the precipitated USP7 protein (bottom). (B) Plasmids encoding LexA fusion proteins, which include a full-length wild-type ataxin-1 (30Q), mutant ataxin-1 (82Q), or atrophin-1 917–1184 (negative control), were transformed into the yeast expressing the protein pB42AD-USP7 1–1102. The transformants were used for liquid ␤-galactosidase assays. LexA-BD domain, light gray box.

interaction exists in vivo between the wild-type ataxin-1 and USP7. Influences of Polyglutamine Tract Length on USP7 Interaction A strong interaction was detected between the wildtype ataxin-1 and USP7 (Fig. 3A, lane 4), whereas a weak one was detected between the mutant ataxin-1 and USP7 (lane 5), indications that the length of polyglutamine tract affects the interaction strength, which was further inves-

tigated through the yeast two-hybrid system. The interaction between a full-length USP7 and wild-type or mutant ataxin-1 was quantified through a liquid ␤-galactosidase assay. The level of ␤-galactosidase activity in the presence of wild-type ataxin-1 (30Q) was found to be sevenfold higher than that observed in the presence of mutant ataxin-1 (82Q) (Fig. 3B). In contrast, no interaction was observed between the full-length USP7 and atrophin-1 C-terminus (DRPLA gene product), a control protein. The above results thus confirm those of the in vivo coimmunoprecipitation experiments.

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FIG. 4. USP7 is colocalized with ataxin-1. COS-7 cells were transfected with the indicated constructs and immunolabeled with polyclonal A539 anti-ataxin-1 coupled to Texas red-conjugated antibody (red) and anti-USP7 coupled to FITC-conjugated antibody (green). Superimposition of both proteins under a fluorescent microscope is indicated in yellow (right).

Colocalization of Ataxin-1 and USP7 We next looked into the cellular localization of USP7 and ataxin-1 to further investigate the in vivo interaction. Localization of the proteins was determined through the immunofluorescence microscopy, which revealed that endogeneous USP7 proteins were distributed in the nuclei of the COS-7 cells not transfected with ataxin-1 (data not shown). However, in COS-7 cells transfected with the wild-type ataxin-1, endogeneous USP7 proteins formed aggregates with the ataxin-1 proteins (Figs. 4A– 4C), indicating that USP7 and the wildtype ataxin-1 interact and colocalize in the nucleus. Interestingly, colocalizations could also be detected between USP7 and the mutant ataxin-1 (82Q) (Figs. 4D– 4F). We believe that the phenomenon was caused due to the overexpression of mutant ataxin-1, since the mutant ataxin-1 was transfected into the COS-7 cells and observed to interact with USP7 in the experiments of the yeast two-hybrid assay although the strength of the interaction was much weaker. Whereas wild-type

ataxin-1 aggregates show homogenous appearance, mutant ataxin-1 shows a ring-like appearance, suggesting that the penetration of the antibody into the inclusion may be variable.

USP7 Is Not Recruited Mutant Ataxin-1 Aggregates in the Purkinje Cells of Transgenic Mice We next examined the interaction between USP7 and ataxin-1 in the Purkinje cells of nontransgenic mice or transgenic mice expressing a mutant allele (B05 line containing 82Q) through the immunohistochemistry. In B05 transgenic mice, a single large nuclear inclusion was observed in the Purkinje cells (Fig. 5A). All nuclear inclusions were ubiquitin-positive (Fig. 5A) as well as ataxin-1 positive (data not shown), but USP7 was not colocalized to the nuclear inclusion (Fig. 5B), indicating that USP7 does not interact with mutant ataxin-1. In the Purkinje cells of nontransgenic mice, no ataxin-1 nuclear inclusion was observed (Fig. 5C) and USP7 was

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FIG. 5. USP7 is not recruited to mutant ataxin-1 aggregates in transgenic Purkinje cells. (A) Purkinje cells of SCA1 transgenic mouse were stained with anti-ubiquitin antibody. Most of Purkinje cells show ubiquitin-positive ataxin-1 nuclear inclusions. (B) Purkinje cells of SCA1 transgenic mouse were stained with anti-USP7 antibody. Arrows indicate Purkinje cells. (C) Purkinje cells of nontransgenic mouse were stained with anti-ubiquitin antibody. Ubiquitin staining is diffusely localized both in the nucleus and cytoplasm. (D) Purkinje cells of nontransgenic mouse were stained with anti-USP7 antibody. USP7 is detected in the nucleus and cytoplasm.

distributed both in the nucleus and cytoplasm although the staining was considerablely faint (Fig. 5D). We could not confirm the USP7-ataxin-1 interaction because there was no ataxin-1 aggregates in the Purkinje cells of nontransgenic mice. Interestingly, the USP7 staining in the B05 nucleus was fainter than that seen in the nontransgenic control, implying that the distribution of USP7 in the nucleus might be influenced by the formation of mutant ataxin-1 aggregates.

DISCUSSION In this study, USP7, a new ataxin-1-interacting protein that preferentially binds to the wild-type ataxin-1, was identified. USP7 proteins, though predominantly expressed in the nucleus, are also present in a minimum

of the nuclear bodies (NBs) (Everett et al., 1997), which are punctate structures associated with the nuclear matrix. Although the exact biological function of NBs is still unclear, a striking feature is that NB-associated proteins (PML, Sp100, CBP, and pRB) are involved in a number of pathological situations. Recently, the mutant ataxin-1 containing 82Q has been reported to colocalize with PML, thus altering the normal nuclear distribution of PML (Skinner et al., 1997). In addition to PML, the mutant ataxin-1 expression redistributes other matrixassociated proteins and alters the subcellular localization of the HDJ-2/HSDJ chaperon and 20 S proteasome in the nuclei of SCA1 patient neurons and transgenic mouse Purkinje cells (Cummings et al., 1998). Furthermore, a number of cell culture and transgenic mouse model studies have implicated nucleus as a site of pathogenesis. The importance of ataxin-1 localization in

304 the nucleus involving SCA1 disorder was clearly revealed through the ataxin-1 K772T transgenic mice (Klement et al., 1998). Even in the absence of nuclear aggregates, the toxic gain of function of mutant ataxin-1 seems to be targeted toward the Purkinje cell nucleus. Our liquid ␤-galactosidase assay and coimmunoprecipitation experiments (Fig. 3) revealed a weak interaction between the mutant ataxin-1 and USP7, which is a component of PML nuclear bodies, suggesting that the mutant ataxin-1 may alter the normal protein–protein interactions in the nucleus; a cell-specific protein or process and nuclear structures can be disrupted by the elongated polyglutamine tract. The decreased affinity of USP7 for mutant ataxin-1 is, therefore, due to a change in the protein conformation of the wild-type ataxin-1, which is induced by the elongated polyglutamine tract. USP7 may accelerate the degradation of ataxin-1 through a proper trimming of polyubiquitin attached to ataxin-1. Therefore, the decreased affinity of USP7 for mutant ataxin-1 in the subcellular structure of the nucleus may be directly involved in the SCA1 pathogenesis. Our next work involves the generation of mice that lack USP7 in the Purkinje cell and crossing these animals with the SCA1 transgenic mice. Such animal models should provide further insights into the role of ubiquitin-specific protease in SCA1 pathology.

EXPERIMENTAL METHODS

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by PCR from the pT7-HAUSP. The cDNAs were then subcloned into pcDNA3.1/HisA (Invitrogen) and pB42AD. To confirm the in-frame insertion, all PCR products and junctions of the constructs were sequenced using the ABI 310 Genetic Analyzer (PerkinElmer, U.S.A.). Yeast Two-Hybrid Screening for Ataxin-1-Interacting Proteins pLexA-ataxin-1 was transformed into the yeast strain EGY48, host strain transformed with the reporter plasmid p8op-lacZ. For the library selection, a single colony of EGY48 cells transformed with pLexA-ataxin-1 was grown at 30°C overnight in a minimal medium lacking uracil and histidine and was then transformed with a human fetal brain cDNA library constructed into pB42AD (Matchmaker, Clontech). A total of 2.5 ⫻ 10 6 independent transformants were plated on a minimal medium lacking uracil, histidine, tryptophan, and leucine, but containing X-gal (5-Bromo-4-chloro-3-indolyl-␤-d-galactoside). After incubation at 30°C for 4 to 6 days, positive colonies were picked. The inserts of selected positive clones were sequenced and aligned in NCBI BLAST database. To verify the interaction, the library plasmids were transformed into KC8 harboring pLexA-BD alone, pLexA-ataxin-1 or pLexA-atrophin-1. The resulting transformants were subsequently plated on the above-mentioned minimal medium for the activation of lacZ and LEU2 reporter genes.

Plasmid Constructs Wild-type ataxin-1 was amplified from the human fetal brain cDNA library (Clontech) using polymerase chain reaction (PCR) with Pfu polymerase (Stratagene). The amplification product was cloned into pBluscript KS(⫹) (Stratagene) and pGEX-5X-1 (Pharmacia). Both ataxin-1 and GST-fusion ataxin-1 were then subcloned into pcDNA3.1/HisC, pcDNA3 (Invitrogen), pLexA-BD, and pB42AD (Clontech). For the constructions of pLexA-ataxin-1 539 – 816, and pcDNA3/GST-ataxin1 539 – 816, the truncated ataxin-1 539 – 816, ataxin-1 589 – 816 and ataxin-1 278 – 645 were amplified using PCR. The cDNAs were cloned into pLexA-BD and pcDNA3. The plasmid pcDNA/amp FlagSCA1[30] and [82] were kind gifts from H. T. Orr (Institute of Human Genetics, University of Minnesota, Minnesota), and R. D. Everett (Institute of Virology, University of Glasgow, Scotland) kindly provided the plasmid pT7-HAUSP. For the constructions of pcDNA3.1/HisA-USP71–1102, pcDNA3.1/HisA-USP7705–1102, pB42AD-USP7 1–1102, and pB42AD-USP7 705–1102, BamHI– XhoI USP7 1–1102, and USP7 705–1102 cDNA were amplified

Antibodies To generate polyclonal antibodies against ataxin-1, the GST fusion protein, ataxin-1 C-terminus (leucine 539 to lysine 816), was expressed in Escherichia coli, purified through gel electrophoresis, and used for the immunization of rats. Anti-A539, the resulting immune serum, could detect both the wild-type and the mutant ataxin-1 through Western blots and immunofluorescence of the transfected COS-7 cells. The identity of the protein as ataxin-1 was confirmed using FLAG- or Xpress-tagged antibodies. The anti-USP7 rabbit serum r201 was kindly provided by R. D. Everett. M2 antiFLAG, and mouse monoclonal anti-Xpress were, respectively, purchased from Eastman Kodak, Invitrogen, and Santa Cruz. Interaction of GST-Ataxin-1 with USP7 in Vivo COS7 cells were grown in Dulbecco’s modified Eagle medium (GIBCO-BRL) supplemented with 10% fetal

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bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 ␮g/ml). Transient transfections were performed GST-ataxin-1 and USP7 constructs using Lipofectamine (GIBCO-BRL). Cells were harvested 48 h after transfection and lysed in a 0.5% NP-40 lysis buffer (25 mM Hepes, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1 mM DTT, and 0.5% NP-40) supplemented with protease inhibitors (2 mM PMSF, 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin, and 1 ␮g/ml aprotinin). Total proteins were mixed with glutathione–Sepharose beads and incubated for 2 h at 4°C with end-over-end mixing. The beads were pelleted for 5 min, and washed three times with 1X PBS supplemented with 0.5% NP-40 and protease inhibitors. Resin-bound protein complexes were eluted from the beads by boiling in 25 ␮l elution buffer [20 mM glutathione, 50 mM Tris–HCl (pH 8.0)] and 25 ␮l SDS–PAGE sample buffer. The proteins were applied to 12% SDS–PAGE gel and transferred onto PVDF membranes (Millipore). The membrane was blocked with 5% non-fat milk and reacted with an anti-Xpress antibody (1:5000, Invitrogen) for 2 h at room temperature. Detections were performed using the Enhanced Chemiluminescence (ECL) western blotting system (Amersham).

Coimmunoprecipitation Experiments Cells were harvested 48 h after transfection with ataxin-1 and USP7 constructs. They were then washed with cold PBS, scraped, pelleted via centrifugation, and lysed on ice for 30 min applying end-over-end mixing in a 0.5% NP-40 lysis buffer supplemented with protease inhibitors. Cell extracts were centrifuged at 13,000g for 15 min at 4°C, and the supernatants (total soluble extracts) were used for immunoprecipitations. For each immunoprecipitation experiment, 250 –300 ␮g protein in 0.5 ml NP-40 lysis buffer was used, to which a polyclonal antibody, either r201 anti-USP7 (1:200) or M2 anti-FLAG (1:500; Eastman Kodak, New Haven, CT), was added. After incubation overnight at 4°C with rotation, 30 ␮l of protein A–Sepharose (Sigma) was added, and the reaction mixture was incubated for 2 h at 4°C with rotation. The beads were pulled down through centrifugation and washed three times with 1 ml NP-40 lysis buffer containing protease inhibitors. Bound proteins were eluted from the beads with 1X SDS-sample buffer, boiled for 5 min, and analyzed by Western blotting. Detections were performed using r201 anti-USP7 (1:2000), anti-A539, and M2 anti-FLAG (1:3000) antibodies.

Immunofluorescence Forty-eight hours after transfection, COS-7 cells were prepared for immunofluorescence and confocal microscopy. Cells were washed three times in PBS, fixed in 3.7% formaldehyde for 15 min, rinsed three times in PBS, and quenched for 10 min in PBS containing 50 mM NH 4Cl. Subsequently, the cells were again rinsed three times with PBS and permeabilized for 10 min in PBS containing 0.1% Triton X-100. Coverslips with the cells were then incubated in a block buffer (2% bovine serum albumin in PBS) for 1 h at 4°C. The cells were reincubated for 1 h at room temperature with the following primary antibodies diluted in a block buffer: r201 antiUSP7 (1:500), M2 anti-FLAG (1:500), or anti-A539. The coverslips were rinsed three times with PBS and incubated for 1 h with goat anti-rabbit FITC (Jackson Laboratories, West Grove, PA) and/or goat anti-mouse Texas-red (Jackson Laboratories), each at 1:800 in a block buffer. They were again rinsed three times with PBS and mounted on glass slides using FluoroGuard Antifade Reagent (Bio-Rad Laboratories, Hercules, CA). Confocal images were obtained from a Leica TCS-NT laser confocal microscope (Heidelberg, Germany), and images were processed at the Adobe Photoshop.

Immunohistochemistry SCA1 transgenic mice (line B05) and wild-type nontransgenic mice were generously provided by Dr H. T. Orr. Formalin-fixed paraffin sections of mouse brain were immunohistochemically stained with antibodies to USP7, ubiquitin, or ataxin-1. The procedures used have been described elsewhere (Vig et al., 1998). Briefly, 8- to 10-␮m-thick tissue sections were deparaffinized and were then incubated with 5% blocking goat, rabbit, or mouse serum for 10 min with through intervening washes with PBS. The sections were incubated for 48 h at 4°C with antibodies. After washing in PBS, the sections were stained with hematoxylin and photomicrographed using a Carl Zeiss microscope.

ACKNOWLEDGMENTS This work was supported in part by a grant of the Korea Health 21 R&D project, Ministry of Health & Welfare, Republic of Korea (HMP00-B-21300-00202), and by a grant of the Korea research Foundation (00-B-21300-0072).

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