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The Deubiquitinating Enzyme Ataxin-3, a Polyglutamine Disease Protein, Edits Lys63 Linkages in Mixed Linkage Ubiquitin Chains

2008; Elsevier BV; Volume: 283; Issue: 39 Linguagem: Inglês

10.1074/jbc.m803692200

ISSN

1083-351X

Autores

Brett J Winborn, Sue M. Travis, Sokol V. Todi, K. Matthew Scaglione, Ping Xu, Aislinn Williams, Robert E. Cohen, Junmin Peng, Henry L. Paulson,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

Ubiquitin chain complexity in cells is likely regulated by a diverse set of deubiquitinating enzymes (DUBs) with distinct ubiquitin chain preferences. Here we show that the polyglutamine disease protein, ataxin-3, binds and cleaves ubiquitin chains in a manner suggesting that it functions as a mixed linkage, chain-editing enzyme. Ataxin-3 cleaves ubiquitin chains through its amino-terminal Josephin domain and binds ubiquitin chains through a carboxyl-terminal cluster of ubiquitin interaction motifs neighboring the pathogenic polyglutamine tract. Ataxin-3 binds both Lys48- or Lys63-linked chains yet preferentially cleaves Lys63 linkages. Ataxin-3 shows even greater activity toward mixed linkage polyubiquitin, cleaving Lys63 linkages in chains that contain both Lys48 and Lys63 linkages. The ubiquitin interaction motifs regulate the specificity of this activity by restricting what can be cleaved by the protease domain, demonstrating that linkage specificity can be determined by elements outside the catalytic domain of a DUB. These findings establish ataxin-3 as a novel DUB that edits topologically complex chains. Ubiquitin chain complexity in cells is likely regulated by a diverse set of deubiquitinating enzymes (DUBs) with distinct ubiquitin chain preferences. Here we show that the polyglutamine disease protein, ataxin-3, binds and cleaves ubiquitin chains in a manner suggesting that it functions as a mixed linkage, chain-editing enzyme. Ataxin-3 cleaves ubiquitin chains through its amino-terminal Josephin domain and binds ubiquitin chains through a carboxyl-terminal cluster of ubiquitin interaction motifs neighboring the pathogenic polyglutamine tract. Ataxin-3 binds both Lys48- or Lys63-linked chains yet preferentially cleaves Lys63 linkages. Ataxin-3 shows even greater activity toward mixed linkage polyubiquitin, cleaving Lys63 linkages in chains that contain both Lys48 and Lys63 linkages. The ubiquitin interaction motifs regulate the specificity of this activity by restricting what can be cleaved by the protease domain, demonstrating that linkage specificity can be determined by elements outside the catalytic domain of a DUB. These findings establish ataxin-3 as a novel DUB that edits topologically complex chains. The conjugation of ubiquitin to proteins regulates diverse cellular processes ranging from protein degradation to DNA repair (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar, 2Johnson E.S. Ma P.C.M. Ota I.M. Varshavsky A. J. Biol. Chem. 1995; 270: 17442-17456Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar, 3Pickart C. FASEB J. 1997; 11: 1055-1066Crossref PubMed Scopus (308) Google Scholar, 4Pickart C.M. Fushman D. Curr. Opin. Chem. 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Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1319) Google Scholar, 11Tenno T. Fujiwara K. Tochio H. Iwai K. Morita E.H. Hayashi H. Murata S. Hiroaki H. Sato M. Tanaka K. Shirakawa M. Genes Cells. 2004; 9: 865-875Crossref PubMed Scopus (131) Google Scholar, 12Varadan R. Walker O. Pickart C. Fushman D. J. Mol. Biol. 2002; 324: 637-647Crossref PubMed Scopus (229) Google Scholar, 13Varadan R. Assfalg M. Haririnia A. Raasi S. Pickart C. Fushman D. J. Biol. Chem. 2004; 279: 7055-7063Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Lys48-linked chains, the best studied type of chain, function in the ubiquitin-proteasome pathway of protein degradation (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar, 2Johnson E.S. Ma P.C.M. Ota I.M. Varshavsky A. J. Biol. Chem. 1995; 270: 17442-17456Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar, 3Pickart C. FASEB J. 1997; 11: 1055-1066Crossref PubMed Scopus (308) Google Scholar, 6Varshavsky A. Trends Biochem. Sci. 2005; 30: 283-286Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). In contrast, less is known about the functions of chains linked through other lysines, including Lys63. Chain complexity has recently emerged as critically important to the regulation of various cellular pathways (5Sun L. Chen Z.J. Curr. Opin. Cell Biol. 2004; 16: 119-126Crossref PubMed Scopus (375) Google Scholar). Mixed linkage and multiply branched chains, for which evidence is now emerging, likely add to this complexity (10Peng J.S.D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1319) Google Scholar, 14Ben-Saadon R. Zaaroor D. Ziv T. Ciechanover A. Mol. Cell. 2006; 24: 701-711Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 15Kim H.T. Kim K.P. Lledias F. Kisselev A.F. Scaglione K.M. Skowyra D. Gygi S.P. Goldberg A.L. J. Biol. Chem. 2007; 282: 17375-17386Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Mixed linkage chains, for example, may impede some cellular processes such as the efficient handling of substrates by the proteasome (15Kim H.T. Kim K.P. Lledias F. Kisselev A.F. Scaglione K.M. Skowyra D. Gygi S.P. Goldberg A.L. J. Biol. Chem. 2007; 282: 17375-17386Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). However, little is known about how chain complexity is regulated, especially concerning how deubiquitinating enzymes (DUBs) 2The abbreviations used are: polyQpolyglutamineUIMubiquitin-interacting motifDUBdeubiquitinating enzymeCHIPcarboxyl terminus of HSC70-interacting proteinUbubiquitinHMWhigh molecular weightHAhemagglutininGAPDHglyceraldehyde-3-phosphate dehydrogenaseGSTglutathione S-transferaseRNAiRNA interferenceFRT,FLPrecombinase target. act on specific types of chains. polyglutamine ubiquitin-interacting motif deubiquitinating enzyme carboxyl terminus of HSC70-interacting protein ubiquitin high molecular weight hemagglutinin glyceraldehyde-3-phosphate dehydrogenase glutathione S-transferase RNA interference recombinase target. Ubiquitin signaling is regulated by dozens of DUBs (16Amerik A.Y. Hochstrasser M. Biochim. Biophys. Acta. 2004; 1695: 189-207Crossref PubMed Scopus (757) Google Scholar, 17Nijman S.M.B. Luna-Vargas M.P.A. Velds A. Brummelkamp T.R. Dirac A.M.G. Sixma T.K. Bernards R. Cell. 2005; 123: 773-786Abstract Full Text Full Text PDF PubMed Scopus (1430) Google Scholar). By removing ubiquitin from substrates, DUBs can facilitate substrate entry into the proteasome as well as terminate ubiquitin signals underlying various ubiquitin-dependent pathways. One such DUB is ataxin-3, the disease protein in the polyglutamine (polyQ) neurodegenerative disorder spinocerebellar ataxia type 3, also known as Machado-Joseph disease (18Kawaguchi Y. Okamoto T. Taniwaki M. Aizawa M. Inoue M. Katayama S. Kawakami H. Nakamura S. Nishimura M. Akiguchi I. Kimura J. Narumiya S. Kakizuka A. Nat. Genet. 1994; 8: 221-228Crossref PubMed Scopus (1565) Google Scholar). Ataxin-3 possesses a catalytic amino-terminal Josephin domain and a carboxyl-terminal ubiquitin-binding domain that contains three ubiquitin interacting motifs (UIMs) (see Fig. 1A) (19Hofmann K. Falquet L. Trends Biochem. Sci. 2001; 26: 347-350Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 20Scheel H. Tomiuk S. Hofmann K. Hum. Mol. Genet. 2003; 12: 2845-2852Crossref PubMed Scopus (139) Google Scholar). Mutating the catalytic cysteine residue (Cys14) in ataxin-3 abolishes protease activity, whereas mutating conserved residues in the UIMs diminishes ubiquitin chain binding (21Berke S.J.S. Chai Y. Marrs G.L. Wen H. Paulson H.L. J. Biol. Chem. 2005; 280: 32026-32034Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 22Burnett B. Li F. Pittman R.N. Hum. Mol. Genet. 2003; 12: 3195-3205Crossref PubMed Scopus (314) Google Scholar, 23Chai Y. Berke S.S. Cohen R.E. Paulson H.L. J. Biol. Chem. 2004; 279: 3605-3611Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 24Mao Y. Senic-Matuglia F. Di Fiore P.P. Polo S. Hodsdon M.E. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12700-12705Crossref PubMed Scopus (135) Google Scholar). The polyQ tract, which is expanded in persons afflicted with spinocerebellar ataxia type 3, resides between the second and third UIMs. The normal role of this polyQ tract and the consequences of its expansion on ataxin-3 activity are poorly understood. At least nine neurodegenerative disorders are caused by polyQ expansions in otherwise unrelated proteins (25Orr H.T. Zoghbi H.Y. Annu. Rev. Neurosci. 2007; 30: 575-621Crossref PubMed Scopus (1112) Google Scholar, 26Perutz M.F. Trends Biochem. Sci. 1999; 24: 58-63Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). PolyQ diseases are marked by perturbations in protein homeostasis, including disease protein and ubiquitin chain accumulation in the brain (27Bennett E.J. Shaler T.A. Woodman B. Ryu K.Y. Zaitseva T.S. Becker C.H. Bates G.P. Schulman H. Kopito R.R. Nature. 2007; 448: 704-708Crossref PubMed Scopus (429) Google Scholar, 28Michalik A. Van Broeckhoven C. Hum. Mol. Genet. 2003; 12: R173-R186Crossref PubMed Scopus (201) Google Scholar, 29Paulson H.L. Perez M.K. Trottier Y. Trojanowski J.Q. Subramony S.H. Das S.S. Vig P. Mandel J.-L. Fischbeck K.H. Pittman R.N. Neuron. 1997; 19: 333-344Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar, 30Taylor J.P. Hardy J. Fischbeck K.H. Science. 2002; 296: 1991-1995Crossref PubMed Scopus (1023) Google Scholar). Although protein misfolding contributes to disease pathogenesis, the events leading to neuronal dysfunction and cell death have not been fully elucidated for any polyQ disorder. It is nevertheless clear that the protein context in which expansion occurs plays a key role (31La Spada A.R. Taylor J.P. Neuron. 2003; 38: 681-684Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Growing evidence implicates ataxin-3 in protein quality control pathways. For example, ataxin-3 interacts with ubiquitin-proteasome pathway components (32Doss-Pepe E.W. Stenroos E.S. Johnson W.G. Madura K. Mol. Cell Biol. 2003; 23: 6469-6483Crossref PubMed Scopus (187) Google Scholar, 33Todi S.V. Laco M.N. Winborn B.J. Travis S.M. Wen H.M. Paulson H.L. J. Biol. Chem. 2007; 282: 29348-29358Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 34Wang Q. Li L. Ye Y. J. Cell Biol. 2006; 174: 963-971Crossref PubMed Scopus (154) Google Scholar), localizes to protein inclusions in various disease states (35Takahashi J. Tanaka J. Arai K. Funata N. Hattori T. Fukuda T. Fujigasaki H. Uchihara T. J. Neuropathol. Exp. Neurol. 2001; 60: 36-376Google Scholar, 36Uchihara T.F.H. Koyano S. Nakamura A. Yagishita S. Iwabuchi K. Acta Neuropathol. 2001; 102: 149-152Crossref PubMed Scopus (70) Google Scholar), regulates the formation of aggresomes (37Burnett B.G. Pittman R.N. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4330-4335Crossref PubMed Scopus (141) Google Scholar), and assists in degrading substrates retrotranslocated from the endoplasmic reticulum (34Wang Q. Li L. Ye Y. J. Cell Biol. 2006; 174: 963-971Crossref PubMed Scopus (154) Google Scholar, 38Zhong X. Pittman R.N. Hum. Mol. Genet. 2006; 15: 2409-2420Crossref PubMed Scopus (162) Google Scholar). In Drosophila, wild type ataxin-3 can suppress polyQ-mediated neurodegeneration through its ubiquitin-binding and cleavage activities, and expanded ataxin-3 retains this suppressor activity despite its pathogenic mutation (39Warrick J.M. Morabito L.M. Bilen J. Gordesky-Gold B. Faust L.Z. Paulson H.L. Bonini N.M. Mol. Cell. 2005; 18: 37-48Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). These unusual properties of ataxin-3 underscore both the importance of quality control pathways in polyQ diseases and the need to define the function of ataxin-3 in such pathways. Here we present evidence that ataxin-3 possesses novel ubiquitin chain binding and cleavage properties that suggest it functions as the first identified, mixed linkage ubiquitin chain-editing enzyme. Plasmids—The ataxin-3 variant used in all assays is isoform 1, variant 1 (NM_004993), which contains all 11 exons and encodes the full protein with all three UIMs. Ataxin-3 plasmids pGEX6P1-ATX3-WT, -C14A, and -SA were generated by digesting the corresponding pcDNA3-Myc-ATX3 (23Chai Y. Berke S.S. Cohen R.E. Paulson H.L. J. Biol. Chem. 2004; 279: 3605-3611Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) plasmids with BamHI and NotI and inserting the released fragments into pGEX-6P1 (GE Healthcare) vector. For the truncation mutant containing only the Josephin domain, the wild type construct was mutated with forward primer 5′-GAC CAA CTC CTG CAG ATG ATT CGC TAA ATA AGA ATG CGG CCG CAT CG and reverse primer 5′-CG ATG CGG CCG CAT TCT TAT TTA GCG AAT CAT CTG CAG GAG TTG GTC to introduce a stop codon after residue 182. SDS-PAGE and Immunoblotting—SDS-PAGE was performed with constant voltage on 10% (see Figs. 1, B and C, and 2A and supplemental Fig. S1), 15% (see Figs. 2, C, D, and G, 3B, 4B, and 5A and supplemental Fig. S2), 10–20% (see Fig. 2, B and F, 4A, and 5, B and C) or 4–15% (see Fig. 5, D–G, and supplemental Fig. S3) gels. Immunoblotting was performed as described previously (33Todi S.V. Laco M.N. Winborn B.J. Travis S.M. Wen H.M. Paulson H.L. J. Biol. Chem. 2007; 282: 29348-29358Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The following antibodies were used: anti-ataxin-3 polyclonal (Machado-Joseph disease, 1:20,000 (29Paulson H.L. Perez M.K. Trottier Y. Trojanowski J.Q. Subramony S.H. Das S.S. Vig P. Mandel J.-L. Fischbeck K.H. Pittman R.N. Neuron. 1997; 19: 333-344Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar)), anti-ataxin-3 monoclonal (1H9, kindly provided by Y. Trottier, 1:2,000), anti-HA polyclonal (Y11, Santa Cruz, 1:500), anti-ubiquitin monoclonal (P4D1, Santa Cruz, 1:10,000), anti-ubiquitin polyclonal (Dako, 1:1,000), anti-tubulin monoclonal (Sigma, 1:50,000), anti-FLAG polyclonal (Sigma, 1:1,000), anti-GAPDH monoclonal (Chemicon, 1:1,000), goat anti-mouse peroxidase-conjugated secondary (Jackson Laboratories, 1:15,000), and goat anti-rabbit peroxidase-conjugated secondary antibodies (Jackson Laboratories, 1:15,000).FIGURE 3Ataxin-3 cleaves mixed linkage ubiquitin chains. A, diagram of mixed linkage Ub4 chain with Lys48- and Lys63-linkages used in this study. B, comparison of ataxin-3 (Gln22) cleavage of Ub4 chains with pure or mixed linkages. GST-ataxin-3 was incubated with the indicated Ub4 chains for 1 h at 37 °C before analysis by SDS-PAGE and Sypro Ruby staining. The arrow indicates Lys48-linked Ub2 reaction product.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4UIMs restrict Lys48-linked ubiquitin chain cleavage by ataxin-3. A, effect of UIM mutations on Lys48- and Lys63-linked ubiquitin proteolysis. Performed as in Fig. 2B, except that UIM-mutated (UIM*) ataxin-3 (Gln22) was incubated with Lys48- or Lys63-linked Ub6 for the indicated times. B, similar Ub6 cleavage by UIM-mutated ataxin-3 (Gln22) and Josephin domain alone. As in A, except Josephin domain-only (Jos) or UIM-mutated (UIM*) ataxin-3 were incubated with Lys48- or Lys63-linked Ub6.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Effect of polyQ expansion on ataxin-3 activity. A, absence of significant effect of polyQ expansion on Lys63-linked ubiquitin chain proteolysis in vitro. GST-ataxin-3 (250 nm) with normal (Gln22) or expanded (Gln80) glutamine repeat was incubated with Lys63-linked Ub6 (250 nm) at 37 °C for the indicated times and then analyzed by anti-ubiquitin immunoblotting. B, absence of effect of polyQ expansion on Lys63 linkage proteolysis by ataxin-3 immunopurified from mammalian FLP-In 293 cells. As in A, except immunopurified FLAG-ataxin-3 (Gln22 or Gln80) was incubated with Lys63-linked Ub6. C, absence of effect of polyQ expansion on ataxin-3 chain cleavage preference. As in A, except immunopurified FLAG-ataxin-3 (Gln22 or Gln80) was incubated with Lys48- or Lys63-linked Ub6. D, ataxin-3 levels in stably transfected HEK293 cell lines. Lysates from vector control line expressing no exogenous ataxin-3 (FRT) and cell lines expressing normal (WT-Q22) or expanded (WT-Q80) FLAG-ataxin-3 were immunoblotted with the indicated antibodies. Anti-ATX3 recognizes all ataxin-3, whereas anti-FLAG recognizes only exogenous ataxin-3. E, effects of normal or expanded ataxin-3 expression on cellular protein ubiquitination. HEK293 cells stably expressing normal or expanded FLAG-ataxin-3 were transfected with HA-ubiquitin, lysed, and analyzed by immunoblotting. Densitometric quantification of ubiquitin smears (relative to tubulin and normalized to FRT control) shows significantly reduced ubiquitination in ataxin-3-Gln22-expressing cells compared with FRT control cells or ataxin-3-Gln80-expressing cells (p < 0.05; n = 3; mean ± S.D.). F, effect of ataxin-3 knockdown on endogenous cellular protein ubiquitination. The cells transfected with RNAi duplexes were lysed and immunoblotted with anti-ubiquitin and anti-GAPDH antibodies. Ataxin-3 knockdown resulted in increased ubiquitin levels relative to GAPDH when compared with control (p < 0.01; n = 6 for control duplexes and n = 12 for ataxin-3-targeting duplexes; mean ± S.D.). G, levels of ataxin-3 after RNAi knockdown. 293 cells were transfected with the indicated RNAi duplexes at 0 and 48 h, lysed at 72 h, and immunoblotted with ataxin-3 and GAPDH antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Recombinant Protein Purification—GST fusion proteins were purified as described previously (33Todi S.V. Laco M.N. Winborn B.J. Travis S.M. Wen H.M. Paulson H.L. J. Biol. Chem. 2007; 282: 29348-29358Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), except that columns were washed two additional times with Buffer A (50 mm HEPES, 0.5 mm EDTA, 1 mm dithiothreitol, and 0.1 mg/ml bovine albumin at pH 7.5) or Buffer B (50 mm HEPES, 0.5 mm EDTA, 1 mm dithiothreitol, and 0.1 mg/ml ovalbumin at pH 7.5). Binding Assays—GST fusion proteins (250 nm) were incubated with 250 nm (assuming average size of 40 kDa) mixed length (Ub3–7) ubiquitin chains (Boston Biochem) for 30 min. Incubations were performed at 4 °C to minimize proteolysis. Unbound supernatant fractions were removed and added to loading buffer. The beads were washed four times with Buffer A (see Fig. 1, B and C) or Buffer A with 0.1% Nonidet P-40 (supplemental Fig. S1). Protease Assays—Ubiquitin conjugation reactions were performed as described previously (15Kim H.T. Kim K.P. Lledias F. Kisselev A.F. Scaglione K.M. Skowyra D. Gygi S.P. Goldberg A.L. J. Biol. Chem. 2007; 282: 17375-17386Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar) and were stopped by adding 10 mm EDTA. GST fusion proteins were incubated with ubiquitinated proteins or 250 nm ubiquitin chains (Boston Biochem) at 37 °C in conjugation buffer with EDTA (see Fig. 2A), Buffer A (see Figs. 2, B–F, 3B, and 4A), or Buffer B (see Figs. 4B and supplemental Fig. S2). Protein concentrations were determined by UV absorbance with a spectrophotometer (Nano-Drop, ND-1000) for Figs. 2 (C and D) and 3. FLAG-tagged proteins immunopurified from FLP-In 293 cells were incubated with 100 or 250 nm ubiquitin chains (Boston Biochem) at 37 °C in Buffer D (Buffer A with 1% Sigma Protease Inhibitor mixture P8340). Because equal numbers of cells from the different stable cell lines did not always yield the same amount of enzyme (see Fig. 5A), we standardized FLAG-ataxin-3 concentrations by comparison with known amounts of purified ataxin-3 (data not shown) for other experiments (Fig. 5C and data not shown). The reactions were stopped with loading buffer at room temperature. Quantification of immunoblots was performed with QuantityOne (Bio-Rad), using the rolling disk method of background subtraction. For Fig. 2H, significances were determined by single factor analysis of variance and Tukey's test. Immunoaffinity Purification—FLP-In 293 cells (33Todi S.V. Laco M.N. Winborn B.J. Travis S.M. Wen H.M. Paulson H.L. J. Biol. Chem. 2007; 282: 29348-29358Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) were lysed in ice-cold Buffer C (1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, 50 mm Tris-HCl, 150 mm NaCl, and 10% Sigma Protease Inhibitor mixture P8340 at pH7.4). The lysates were incubated with 25 μl of anti-FLAG M2 affinity beads (Sigma) for 2 h at 4 °C. The beads were washed four times with Buffer C and twice with Buffer A. The proteins were eluted with 3× FLAG peptide in 50 μl of Buffer A. Immunopurified proteins were frozen in liquid nitrogen and stored at –80 °C. Ubiquitin Accumulation Experiments—FLP-In cell lines were transiently transfected with HA-tagged ubiquitin, using Lipofectamine-PLUS per the manufacturer's instructions (Invitrogen). The cells were harvested with SDS sample buffer 48 h later. After immunoblotting with anti-HA antibody, the intensities of the HA-ubiquitin smears were quantified by densitometry (ImageJ) (40Abramoff M. Magelhaes P. Ram S. Biophotonics Int. 2004; 11: 36-42Google Scholar). Student's t test was used to evaluate significance (n = 5, p < 0.05). RNAi Knockdown—Ataxin-3 knockdown was performed with IDT TriFECTA dicer substrate (DsiRNA) duplexes (HSC.RNAI.N001024631.3), including three DsiRNAs targeting ataxin-3 mRNA (accession number NM_001024631) and a scrambled negative control DsiRNA. 293 cells were transfected with 5–45 nm DsiRNA duplexes using Lipofectamine 2000 (Invitrogen) per manufacturer's instructions at 0 and 48 h. The cells were then lysed in Laemmli buffer at 72 h, and the lysates were analyzed by immunoblotting and quantification by densitometry with ImageJ (40Abramoff M. Magelhaes P. Ram S. Biophotonics Int. 2004; 11: 36-42Google Scholar). Two-tailed t tests were used to evaluate significance (n = 7, p < 0.01). Ataxin-3 Binds Lys48- and Lys63-linked Ubiquitin Chains—We sought to determine the preferred ubiquitin chain substrates for ataxin-3, which is shown schematically in Fig. 1A. Unless otherwise noted, ataxin-3 with a nonexpanded polyglutamine length of 22 was used for these studies. We first performed pull-down binding assays of GST-ataxin-3 with ladders of Lys48- or Lys63-linked polyubiquitin employing extensive washes. As shown in Fig. 1 (B and C), wild type or catalytically inactive (C14A) ataxin-3 bound both Lys48- and Lys63-linked chains, provided the chain contained at least four ubiquitin moieties. Ataxin-3, in which critical conserved serine residues in each UIM had been mutated to alanine (UIM*) displayed negligible binding in this assay. Normal and expanded ataxin-3 bound similarly to both Lys48- and Lys63-linked chains (supplemental Fig. S1). Ataxin-3 bound nearly all Lys48- and Lys63-linked chains with five or more ubiquitins. In contrast, Ub4 was partially bound and Ub3 poorly bound, revealing a length dependence to binding. Therefore, ataxin-3 preferentially binds longer ubiquitin chains, and binding is not restricted to Lys48 linkages. Ataxin-3 Preferentially Cleaves Longer Chains and Lys63 Linkages—Consistent with its binding of longer chains, ataxin-3 has been shown to cleave long ubiquitin chains (22Burnett B. Li F. Pittman R.N. Hum. Mol. Genet. 2003; 12: 3195-3205Crossref PubMed Scopus (314) Google Scholar, 37Burnett B.G. Pittman R.N. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4330-4335Crossref PubMed Scopus (141) Google Scholar). To verify that ataxin-3 cleaves chains attached to a substrate, we incubated it with auto-ubiquitinated CHIP (an E3 ligase) generated in vitro under conditions where all ubiquitin chain linkage types can be formed (15Kim H.T. Kim K.P. Lledias F. Kisselev A.F. Scaglione K.M. Skowyra D. Gygi S.P. Goldberg A.L. J. Biol. Chem. 2007; 282: 17375-17386Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Ataxin-3 rapidly and efficiently cleaved highly ubiquitinated CHIP but failed to deubiquitinate CHIP conjugated to shorter chains (Fig. 2A). This selective cleavage only of heavily ubiquitinated substrate suggests that ataxin-3 is inefficient at, or restricted from, processing most chains containing fewer than approximately six ubiquitins. The ability of ataxin-3 to bind both Lys48- and Lys63-linked chains suggested that it might also cleave both. Alternatively, given the distinct topologies of Lys48- and Lys63-linked chains (9Eddins M.J. Varadan R. Fushman D. Pickart C.M. Wolberger C. J. Mol. Biol. 2007; 367: 204-211Crossref PubMed Scopus (134) Google Scholar, 11Tenno T. Fujiwara K. Tochio H. Iwai K. Morita E.H. Hayashi H. Murata S. Hiroaki H. Sato M. Tanaka K. Shirakawa M. Genes Cells. 2004; 9: 865-875Crossref PubMed Scopus (131) Google Scholar, 13Varadan R. Assfalg M. Haririnia A. Raasi S. Pickart C. Fushman D. J. Biol. Chem. 2004; 279: 7055-7063Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), ataxin-3 could possess divergent catalytic activity toward the two chain types. To address this question, we compared the DUB activity of ataxin-3 in a controlled system containing chains of defined linkage and length (Lys48-Ub6 or Lys63-Ub6). These studies revealed that ataxin-3 preferentially cleaves Lys63-linked polyubiquitin (Fig. 2, B–E). Cleavage of Lys63-linked Ub6 led to the accumulation of smaller reaction products (Ub1 through Ub5), whereas little cleavage was observed with Lys48-linked Ub6. We could not determine a precise kcat for ataxin-3 because the total number of linkages cleaved by ataxin-3, in both reactant ubiquitin chains and lower molecular weight reaction products, is unclear. Mutating the catalytic cysteine residue (Cys14) abolished protease activity toward either chain type (Figs. 2F and 5B and data not shown). Ataxin-3 immunopurified from mammalian cells also cleaved Lys63-linked Ub6 more efficiently than Lys48-linked Ub6 (shown later in Fig. 5C), indicating that preferential Lys63 chain cleavage is an intrinsic property of ataxin-3, whether expressed in bacterial or mammalian cells. We noticed that ataxin-3 possessed robust activity toward a subset of Ub6 chains that electrophorese on denaturing gels as much higher molecular weight (HMW) chains, whether derived from Lys48- or Lys63-linked chains (Fig. 2B). Mutating the catalytic cysteine abolished activity toward these HMW chains (Fig. 2F), indicating that ataxin-3 acts on HMW chains through its DUB activity rather than by simply dissociating HMW chain complexes. By quantitative mass spectrometry, HMW chains were nearly uniformly Lys48- or Lys63-linked (supplemental Table S1). These HMW chains are likely covalently linked complexes of Ub6 because they are resistant to various denaturants including heat, SDS, urea, and ethylene glycol (data not shown) and are present whether or not samples are boiled. The pattern of Ub reaction products generated by ataxin-3 sheds light on the nature of chain cleavage. With Ub6 chains, ataxin-3 does not primarily produce Ub1 and thus is not exclusively a processive enzyme. In addition, Ub4 and Ub2 are more prevalent reaction products than Ub3, implying nonrandom cleavage (Fig. 2, B–D). These results suggest that ataxin-3 recognizes a four-ubiquitin patch and cleaves adjacent Lys63 linkages internally within the chain. To define the chain length dependence of ataxin-3 activity, we explored cleavage of Lys48- and Lys63-linked chains containing three to six ubiquitin moieties (Fig. 2, G and H). Proteolysis of Lys63-linked chains did not occur with Ub3 but increased incrementally as chains were lengthened from four to six. In contrast, ataxin-3 possessed very little activity toward any tested length of Lys48-linked ubiquitin chains (data not shown). Ataxin-3 Cleaves Mixed Linkage Ubiquitin Chains—The discrepancy between which linkages ataxin-3 can bind and which it can cleave led us to analyze its activity against ubiquitin chains of greater topological complexity. Ataxin-3 activity was tested against mixed linkage Ub4 chains with a central Lys63 linkage flanked by Lys48 linkages (Fig. 3A). Analysis by Sypro Ruby staining of parallel reactions with Lys48, Lys63, or mixed linkage Ub4 incubated with varying ataxin-3 concentrations revealed enhanced catalytic activity toward mixed linkage chains (Fig. 3B). Observable reaction products appeared at 10-fold lower ataxin-3 concentration from mixed linkage chains than from Lys63-linked chains (10 and 100 nm, respectively). Cleavage of heterotypic Ub4 containing a central

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