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• W1995989097 abstract "Polyglutamine proteins that cause neurodegenerative disease are known to form proteinaceous aggregates, such as nuclear inclusions, in the neurons of affected patients. Although polyglutamine proteins have been shown to form fibrillar aggregates in a variety of contexts, the mechanisms underlying the aberrant conformational changes and aggregation are still not well understood. In this study, we have investigated the hypothesis that polyglutamine expansion in the protein ataxin-3 destabilizes the native protein, leading to the accumulation of a partially unfolded, aggregation-prone intermediate. To examine the relationship between polyglutamine length and native state stability, we produced and analyzed three ataxin-3 variants containing 15, 28, and 50 residues in their respective glutamine tracts. At pH 7.4 and 37 °C, Atax3(Q50), which lies within the pathological range, formed fibrils significantly faster than the other proteins. Somewhat surprisingly, we observed no difference in the acid-induced equilibrium and kinetic un/folding transitions of all three proteins, which indicates that the stability of the native conformation was not affected by polyglutamine tract extension. This has led us to reconsider the mechanisms and factors involved in ataxin-3 misfolding, and we have developed a new model for the aggregation process in which the pathways of un/folding and misfolding are distinct and separate. Furthermore, given that native state stability is unaffected by polyglutamine length, we consider the possible role and influence of other factors in the fibrillization of ataxin-3. Polyglutamine proteins that cause neurodegenerative disease are known to form proteinaceous aggregates, such as nuclear inclusions, in the neurons of affected patients. Although polyglutamine proteins have been shown to form fibrillar aggregates in a variety of contexts, the mechanisms underlying the aberrant conformational changes and aggregation are still not well understood. In this study, we have investigated the hypothesis that polyglutamine expansion in the protein ataxin-3 destabilizes the native protein, leading to the accumulation of a partially unfolded, aggregation-prone intermediate. To examine the relationship between polyglutamine length and native state stability, we produced and analyzed three ataxin-3 variants containing 15, 28, and 50 residues in their respective glutamine tracts. At pH 7.4 and 37 °C, Atax3(Q50), which lies within the pathological range, formed fibrils significantly faster than the other proteins. Somewhat surprisingly, we observed no difference in the acid-induced equilibrium and kinetic un/folding transitions of all three proteins, which indicates that the stability of the native conformation was not affected by polyglutamine tract extension. This has led us to reconsider the mechanisms and factors involved in ataxin-3 misfolding, and we have developed a new model for the aggregation process in which the pathways of un/folding and misfolding are distinct and separate. Furthermore, given that native state stability is unaffected by polyglutamine length, we consider the possible role and influence of other factors in the fibrillization of ataxin-3. The polyglutamine diseases are now well established as a group of genetic neurodegenerative disorders arising from the expansion of a repeated glutamine tract in specific proteins (1Margolis R.L. Ross C.A. Trends Mol. Med. 2001; 7: 479-482Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 2Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1097) Google Scholar). Each of the nine currently identified polyglutamine diseases is associated with a particular protein, with the manifestation of disease generally observed when the polyglutamine tract of the relevant protein is elongated to over a threshold of ∼40 residues. The protein ataxin-3, which has recently been associated with ubiquitin binding and deubiquitinating functions (3Chai 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, 4Donaldson K.M. Li W. Ching K.A. Batalov S. Tsai C.C. Joazeiro C.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8892-8897Crossref PubMed Scopus (183) Google Scholar, 5Doss-Pepe E.W. Stenroos E.S. Johnson W.G. Madura K. Mol. Cell. Biol. 2003; 23: 6469-6483Crossref PubMed Scopus (185) Google Scholar, 6Burnett B. Li F. Pittman R.N. Hum. Mol. Genet. 2003; 12: 3195-3205Crossref PubMed Scopus (311) Google Scholar), is responsible for the condition spinocerebellar ataxia type 3, also known as Machado-Joseph Disease (7Kawaguchi Y. Okamoto T. Taniwaki M. Aizawa M. Inoue M. Katayama S. Kawakami H. Nakamura S. Nishimura M. Akiguchi I. Kimmura J. Narumiya S. Kakizuka A. Nat. Genet. 1994; 8: 221-228Crossref PubMed Scopus (1553) Google Scholar), in which the polyglutamine tract of ataxin-3 is expanded to over 45 residues. 1A. Srivastava, personal communication.1A. Srivastava, personal communication. The different polyglutamine proteins share little or no structural or functional homology outside of the glutamine tract, yet fundamental similarities are consistently observed in the various disease states, with progressive dysfunction and death of neurons resulting in neurodegeneration and eventual death. The expanded polyglutamine tract is generally accepted to be the key causal element of the disease process, especially because increasingly longer polyglutamine proteins are associated with earlier onset and more severe manifestation of the disease state (2Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1097) Google Scholar). Another striking feature of polyglutamine diseases is the formation of aggregates such as nuclear inclusions (NIs), 2The abbreviations used are: NI, nuclear inclusion; Atax3, ataxin-3; bis-ANS, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid; PBS, phosphate-buffered saline; ThioT, thioflavin T.2The abbreviations used are: NI, nuclear inclusion; Atax3, ataxin-3; bis-ANS, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid; PBS, phosphate-buffered saline; ThioT, thioflavin T. which contain the expanded form of disease-associated protein, within the neurons of affected patients (8DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2312) Google Scholar, 9Holmberg M. Duyckaerts C. Durr A. Cancel G. Gourfinkel-An I. Damier P. Faucheux B. Trottier Y. Hirsch E.C. Agid Y. Brice A. Hum. Mol. Genet. 1998; 7: 913-918Crossref PubMed Scopus (300) Google Scholar, 10Paulson 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 (725) Google Scholar). The exact role and significance of NIs is as yet uncertain; however, they are inseparably associated with the manifestation of polyglutamine disease. The mechanisms of polyglutamine toxicity are still largely unelucidated; however, an increasing number of reports indicate that the polyglutamine disorders belong to a wider range of diseases that associated with protein misfolding, including Alzheimer's disease, Parkinson's disease, and a range of amyloidoses (11Stefani M. Dobson C.M. J. Mol. Med. 2003; 81: 678-699Crossref PubMed Scopus (1339) Google Scholar, 12Chow M.K. Lomas D.A. Bottomley S.P. Curr. Med. Chem. 2004; 11: 491-499Crossref PubMed Scopus (34) Google Scholar, 13Ellisdon A.M. Bottomley S.P. IUBMB Life. 2004; 56: 119-123Crossref PubMed Scopus (22) Google Scholar, 14Zerovnik E. Eur. J. Biochem. 2002; 269: 3362-3371Crossref PubMed Scopus (198) Google Scholar). All of these disorders involve the formation and deposition of protein aggregates within diseased tissues and/or cells. One of the most commonly observed forms of such aggregates is that of amyloid plaques, or fibrils, which have a characteristic cross-β-sheet structure (15Dobson C.M. Sem. Cell Dev. Biol. 2004; 15: 3-16Crossref PubMed Scopus (720) Google Scholar). In the case of polyglutamine diseases, the formation of NIs and the presence of proteasomal proteins in aggregates suggest that similar protein misfolding processes take place. In vivo, NIs display a range of morphologies, including granular, fibrillar, and amorphous forms (8DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2312) Google Scholar, 10Paulson 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 (725) Google Scholar, 16Zander C. Takahashi J. El Hachimi K.H. Fujigasaki H. Albanese V. Lebre A.S. Stevanin G. Duyckaerts C. Brice A. Hum. Mol. Genet. 2001; 10: 2569-2579Crossref PubMed Scopus (85) Google Scholar, 17Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar, 18McGowan D.P. van Roon-Mom W. Holloway H. Bates G.P. Mangiarini L. Cooper G.J. Faull R.L. Snell R.G. Neuroscience. 2000; 100: 677-680Crossref PubMed Scopus (79) Google Scholar). In vitro, expanded forms of polyglutamine-containing proteins and peptides have been shown to form fibrils very readily (17Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar, 19Bevivino A.E. Loll P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11955-11960Crossref PubMed Scopus (151) Google Scholar, 20Chen S. Berthelier V. Hamilton J.B. O'Nuallain B. Wetzel R. Biochemistry. 2002; 41: 7391-7399Crossref PubMed Scopus (281) Google Scholar, 21Poirier M.A. Li H. Macosko J. Cai S. Amzel M. Ross C.A. J. Biol. Chem. 2002; 277: 41032-41037Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 22Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It has also been shown that nonpathological length proteins can also form fibrils, albeit generally at slower rates or under specific solution conditions (23Chen S. Berthelier V. Yang W. Wetzel R. J. Mol. Biol. 2001; 311: 173-182Crossref PubMed Scopus (280) Google Scholar, 24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar, 25Shehi E. Fusi P. Secundo F. Pozzuolo S. Bairati A. Tortora P. Biochemistry. 2003; 42: 14626-14632Crossref PubMed Scopus (36) Google Scholar, 26Scherzinger E. Sittler A. Schweiger K. Heiser V. Lurz R. Hasenbank R. Bates G.P. Lehrach H. Wanker E.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4604-4609Crossref PubMed Scopus (577) Google Scholar). Recent work from our laboratory and others have shown that destabilization of the native state of nonpathological variants of ataxin-3 by various stresses such as chemical denaturation and heat can result in the formation of fibrillar aggregates (24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar, 25Shehi E. Fusi P. Secundo F. Pozzuolo S. Bairati A. Tortora P. Biochemistry. 2003; 42: 14626-14632Crossref PubMed Scopus (36) Google Scholar, 27Marchal S. Shehi E. Harricane M.C. Fusi P. Heitz F. Tortora P. Lange R. J. Biol. Chem. 2003; 278: 31554-31563Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Based on various results, we and others have proposed that in the pathological state, the elongation of the polyglutamine tract disrupts the stability of the native conformation of the ataxin-3 (19Bevivino A.E. Loll P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11955-11960Crossref PubMed Scopus (151) Google Scholar, 24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar). We have hypothesized that the loss of native state stability leads to the formation and accumulation of a partially unfolded, aggregation-prone species, resulting in fibrillization. This is supported by an earlier study showing that expansion of a polyglutamine tract inserted into myoglobin results in a progressive loss of conformational stability of the protein (22Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and is also concordant with the generalized model of protein misfolding associated with a wider variety of conformational diseases (11Stefani M. Dobson C.M. J. Mol. Med. 2003; 81: 678-699Crossref PubMed Scopus (1339) Google Scholar, 12Chow M.K. Lomas D.A. Bottomley S.P. Curr. Med. Chem. 2004; 11: 491-499Crossref PubMed Scopus (34) Google Scholar, 28Kelly J.W. Curr. Opin. Struct. Biol. 1998; 8: 101-106Crossref PubMed Scopus (951) Google Scholar, 29Horwich A. J. Clin. Invest. 2002; 110: 1221-1232Crossref PubMed Scopus (202) Google Scholar). Although expanded polyglutamine proteins have been shown to form fibrillar aggregates much more rapidly than shorter length proteins (22Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 23Chen S. Berthelier V. Yang W. Wetzel R. J. Mol. Biol. 2001; 311: 173-182Crossref PubMed Scopus (280) Google Scholar, 26Scherzinger E. Sittler A. Schweiger K. Heiser V. Lurz R. Hasenbank R. Bates G.P. Lehrach H. Wanker E.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4604-4609Crossref PubMed Scopus (577) Google Scholar), to date, no study has examined the relative stabilities of variants ranging into the pathological length of ataxin-3 or any other polyglutamine protein. We have successfully produced milligram quantities of a pathological length variant, Atax3(Q50), which contains 50 glutamine repeats. This has allowed us to perform a comparative study with two other nonpathological variants, containing 15 and 28 residues in their polyglutamine tract (Atax3(Q15) and Atax3(Q28)) (see Fig. 1A). Building on previous results (24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar), we have performed a comprehensive investigation into the effects of polyglutamine length on the conformational changes involved in folding, unfolding, and native state stability of ataxin-3. Interestingly, our results show that upon polyglutamine expansion, the unfolding and folding transitions of ataxin-3 are essentially unchanged, indicating that the hypothesized mechanism of native state destabilization upon polyglutamine expansion in fact does not apply to ataxin-3. This has led us to consider other alternative pathways, mechanisms, and factors that may be significant in the pathogenic processes of aggregation and fibrillization. Phenylmethylsulfonyl fluoride and thioflavin T (ThioT) were obtained from Sigma. The serine protease inhibitor Pefabloc SC (4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride) was purchased from Roche Applied Science. 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) was purchased from Molecular Probes. Atax3(Q15)—A construct encoding Atax3(Q15) was engineered by modification of the pQE30 Atax3(Q28) vector that had been created previously (24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar) from a vector encoding the MJD1a gene variant (7Kawaguchi Y. Okamoto T. Taniwaki M. Aizawa M. Inoue M. Katayama S. Kawakami H. Nakamura S. Nishimura M. Akiguchi I. Kimmura J. Narumiya S. Kakizuka A. Nat. Genet. 1994; 8: 221-228Crossref PubMed Scopus (1553) Google Scholar, 30Goto J. Watanabe M. Ichikawa Y. Yee S.B. Ihara N. Endo K. Igarashi S. Takiyama Y. Gaspar C. Maciel P. Tsuji S. Rouleau G.A. Kanazawa I. Neurosci. Res. 1997; 28: 373-377Crossref PubMed Scopus (66) Google Scholar). EcoN1 sites flanking the polyglutamine-encoding region were introduced using the QuikChange technique sites coupled with a cassette mutagenesis approach allowing for the manipulation of the length of the polyglutamine tract without introducing or substituting amino acids within the protein. The following oligonucleotides were used: N-terminal end of the polyglutamine-coding region, sense, 5′-gaagcctactttaggaaacagcagcag-3′, and antisense, 3′-cttcggatgaaatcctttgtcgtcgtc-5′; and C-terminal of the polyglutamine-coding region, sense, 5′-cagcagcagcagcacctgcagcagggggacctatca-3′, and antisense, 3′-gtcgtcgtcgtcgtggacgtcgtccccctggatagt-5′. A Q15 cassette was then created with EcoNI restriction sites at either end using the following complementary oligonucleotides: 5′-tttgaaaaacagcagcaaaagcagcagcagcagcagcagcagcagcagc-3′ and 3′-aactttttgtcgtcgttttcgtcgtcgtcgtcgtcgtcgtcgtcgtcgt-5′. The oligonucleotides were allowed to anneal slowly prior to their ligation into the EcoN1 digested vector. The nature of the sites allowed for directional cloning of the cassette, with the disappearance of the EcoNI sites as proof of successful ligation. DNA sequencing was used to verify the integrity of the construct. Atax3(Q50)—The cDNA encoding human Atax3(Q50) was a gift from Henry Paulson. A HindIII digestion site was engineered into the 3′ end of the gene. By exploiting this HindIII site and the 5′ BamHI site, the cDNA was then subcloned into the pQE30 expression vector (Qiagen). All of the variants were expressed and purified as described previously (24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar) with two modifications as outlined below. Firstly, an extra purification step was performed to aid removal of contaminating proteases. Following centrifugation of the sonicated cells, prior to binding to nickel-nitrilotriacetic acid affinity resin, the supernatant was mixed for 2 h at 4 °C with 2 ml of p-aminobenzamidine-agarose previously equilibrated with lysis buffer (50 mm NaH2PO4, 300 mm NaCl, 10 mm imidazole, pH 8.0). The unbound protein, containing the relevant ataxin-3 variant, was mixed with nickel-nitrilotriacetic acid resin. All other stages of the purification were the same as previously described, except that glycerol (10% v/v) and Triton X-100 (0.1%) were also present in all of the buffers prior to the gel filtration chromatography stage. All of the fluorescence measurements were performed in PBS (137 mm NaCl, 2.68 mm KCl, 10.1 mm NaH2PO4, 1.76 mm KH2PO4), pH 7.4. Fluorescence emission spectra were recorded on a PerkinElmer LS50B spectrofluorometer with a thermostated cuvette holder at 25 °C, using a 1-cm-path length quartz cuvette. An excitation wavelength of 280 nm was used, with measurement of spectra from 300 to 400 nm. Emission and excitation slit widths were set at 4.0 nm, and a scan speed of 25 nm/min was used. ThioT fluorescence was measured using an excitation wavelength of 445 nm, with the emission recorded at 480 nm. The excitation slit width used was 5.0 nm, and the emission slit width was 10.0 nm. The assay buffer contained PBS, pH 7.4. 15 μl of the relevant protein sample was mixed with 485 μl of assay buffer; these samples were analyzed immediately, with the fluorescence emission signal averaged over a 10-s period. CD spectra were measured on a Jasco-810 spectropolarimeter at 25 °C using a thermostatted cuvette with a path length of 0.1 cm. The spectra were recorded from 190 to 250 nm, using a scan speed of 20 nm/min, with 5 s/point signal averaging, and θ222 measurements were recorded with the signal averaged over 30 s. The CD spectra were analyzed by spectral deconvolution using the CONTINLL algorithm (31van Stokkum I.H. Spoelder H.J. Bloemendal M. van Grondelle R. Groen F.C. Anal. Biochem. 1990; 191: 110-118Crossref PubMed Scopus (437) Google Scholar, 32Provencher S.W. Glockner J. Biochemistry. 1981; 20: 33-37Crossref PubMed Scopus (1878) Google Scholar) as provided by the DICHROWEB on-line facility (33Lobley A. Wallace B.A. Biophys. J. 2001; 80: 373aGoogle Scholar, 34Lobley A. Whitmore L. Wallace B.A. Bioinformatics. 2002; 18: 211-212Crossref PubMed Scopus (645) Google Scholar). For the variant length comparative assay under native conditions, protein solutions at 65 μm were prepared at pH 7.4 using PBS buffer containing phenylmethylsulfonyl fluoride (final concentration, 2 mm), EDTA (final concentration, 5 mm), glycerol (10% v/v), and dithiothreitol (final concentration, 1 mm). The mixtures were incubated at a constant temperature of 37 °C. For monitoring potential fibrillogenesis under acidic conditions, the proteins were incubated at 25 °C in a 1:1 mixture of PBS (pH 7.4) with 50 mm HCl to give a final pH of 1.9; the final concentration of protein was 10.5 μm. For both fibrillogenesis assays, the samples were incubated in air tight containers, and at specific time points, a 15-μl aliquot of each protein sample was removed and added to 485 μl of 25 μm ThioT solution in PBS at pH 7.4. The change in fluorescence signal was then monitored as previously described (24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar). Transmission electron microscopy images were obtained using a Jeol JEM-200CX transmission electron microscope. The acceleration voltage was 100 kV. The samples were adsorbed onto a carbon-coated grid and stained with 1% (w/v) uranyl acetate. Different stock buffers, passed through a 0.22-μm filter, were used at different pH ranges in equilibrium unfolding experiments. 50 mm Tris-HCl was used between pH 7 and 8, 50 mm sodium phosphate was used between pH 6 and 7, 50 mm sodium acetate was used between pH 4 and 6, 50 mm sodium citrate was used between pH 2 and 4, and 50 mm HCl was used below pH 2. The protein, in PBS buffer, was diluted with equal part stock buffers of the desired pH. The reaction mixture was allowed to equilibrate for 5 min at 25 °C before fluorescence analysis, and the final pH was measured. More extensive equilibration for longer time periods up to 12 h showed no difference in results following spectroscopic analysis. The experiments were performed using final protein concentrations ranging from 1 to 5 μm. The samples were analyzed by recording the fluorescence emission spectra between 300 and 400 nm (λex = 280 nm) or monitoring the change in far-UV CD signal at 222 nm. For each sample, the absorbance of all solutions at the excitation wavelength was also measured and recorded. For the pH-induced fibrillogenesis assay, samples across a range of different pH values were prepared as for the equilibrium folding analysis, with a final protein concentration of 10.5 μm. Following overnight incubation at 25 °C, each sample was vortexed, and 50 μl was removed and incubated with ThioT; the change in fluorescence signal was then monitored as previously described (24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar). Experiments were performed on an Applied Photophysics SF.18MV stopped flow apparatus. Unfolding was monitored by measuring the changes in fluorescence intensity at 360 nm using an excitation wave-length of 278 nm. Unfolding at pH 1.9 was performed by rapidly mixing one volume of 0.1 mg/ml protein solution in PBS, pH 7.4, with an equal volume of 50 mm HCl buffer, pH 1.3, at 25 °C. Refolding experiments were performed by initially preincubating the protein for 30 min at 25 °C at a concentration of 2.5 μm in 50 mm HCl at pH 1.9. The protein was refolded by rapidly mixing one volume of the unfolded reaction mixture with an equal part of 20× PBS, pH 7.4; the final pH of refolding was 7.2. The data collected from unfolding and refolding experiments were collected from 10 experiments, which were averaged and fitted to either single or double exponential functions as appropriate. Characterization of Ataxin-3 Variants—We have successfully purified milligram quantities of ataxin-3 variants containing 15, 28, and 50 glutamine repeats, as shown in Fig. 1. In accordance with previous studies (22Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 25Shehi E. Fusi P. Secundo F. Pozzuolo S. Bairati A. Tortora P. Biochemistry. 2003; 42: 14626-14632Crossref PubMed Scopus (36) Google Scholar, 35Bennett M.J. Huey-Tubman K.E. Herr A.B. West Jr., A.P. Ross S.A. Bjorkman P.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11634-11639Crossref PubMed Scopus (116) Google Scholar, 36Trottier Y. Lutz Y. Stevanin G. Imbert G. Devys D. Cancel G. Saudou F. Weber C. David G. Tora L. Agid Y. Brice A. Mandel J.L. Nature. 1995; 378: 403-406Crossref PubMed Scopus (585) Google Scholar), we observed preferential binding of the polyglutamine-specific antibody 1C2 for the longer variant, which lies at the cusp of toxicity in Machado-Joseph disease, although nonpathological proteins could also be recognized (Fig. 1C). All three purified proteins were in a soluble monomeric form as judged by gel filtration analysis, and mass spectrometry showed that they contained the correct number of glutamine residues within their respective polyglutamine tracts (data not shown). The far-UV CD spectrum of Atax3(Q28) was concordant with previously reported data (24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar) (Fig. 2), and the spectra of all three variants were very similar to each other. This suggested that there were no major differences in secondary structure, an observation that was confirmed by spectral deconvolution of the far-UV spectra (Fig. 2, inset). Rapid Fibrillization of Ataxin-3 Containing an Expanded Glutamine Repeat—A key behavioral feature of polyglutamine-expanded proteins is the ready formation of fibrils, as shown for a range of different proteins (17Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar, 19Bevivino A.E. Loll P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11955-11960Crossref PubMed Scopus (151) Google Scholar, 20Chen S. Berthelier V. Hamilton J.B. O'Nuallain B. Wetzel R. Biochemistry. 2002; 41: 7391-7399Crossref PubMed Scopus (281) Google Scholar, 21Poirier M.A. Li H. Macosko J. Cai S. Amzel M. Ross C.A. J. Biol. Chem. 2002; 277: 41032-41037Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 22Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In the case of ataxin-3, it has been shown that a variant containing 78 glutamine residues will readily fibrillize under native conditions (19Bevivino A.E. Loll P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11955-11960Crossref PubMed Scopus (151) Google Scholar), whereas shorter variants are also capable of forming fibrillar aggregates under partially denaturing conditions (24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar, 25Shehi E. Fusi P. Secundo F. Pozzuolo S. Bairati A. Tortora P. Biochemistry. 2003; 42: 14626-14632Crossref PubMed Scopus (36) Google Scholar). In the current study, we examined the behavior and propensity of ataxin-3 of different lengths to form fibrils. The threshold of disease length polyglutamine tracts for ataxin-3 is ∼45 glutamine residues,1 and as such, it is not surprising to observe that under native conditions, Atax3(Q50) forms fibrils much more quickly than both Atax3(Q15) and Atax3(Q28). When incubated at 37 °C at 65 μm, following a lag phase of 3–4 h, Atax3(Q50) exhibits a significant increase in ThioT fluorescence, which continues to increase for up to and beyond 24 h. In contrast, Atax3(Q15) and Atax3(Q28) show no increase in ThioT fluorescence for up to 24 h (Fig. 3A). The faster fibrillization of longer variants is consistent with previously reported results for other polyglutamine-containing proteins and peptides (22Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 23Chen S. Berthelier V. Yang W. Wetzel R. J. Mol. Biol. 2001; 311: 173-182Crossref PubMed Scopus (280) Google Scholar, 26Scherzinger E. Sittler A. Schweiger K. Heiser V. Lurz R. Hasenbank R. Bates G.P. Lehrach H. Wanker E.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4604-4609Crossref PubMed Scopus (577) Google Scholar). Examination of the samples by electron microscopy after 24 h of incubation revealed that Atax3(Q50) formed filamentous aggregates with a fibrillar-like morphology, ranging from 20 to 45 nm in diameter (Fig. 3, B and C). Closer inspection of these fibrils suggested that they had a banded texture, suggestive of protofilament species that have assembled and aligned in a lateral manner (Fig. 3C). Inspection of Atax3(Q15) and Atax3(Q28) by electron microscopy at the same time point showed no formation of aggregates (data not shown). Acid Denaturation of Ataxin-3—The propensity of nonpathological length variants of ataxin-3 to aggregate under denaturing conditions has suggested that native state destabilization by an expanded polyglutamine tract plays a key role in polyglutamine aggregation and fibrillization (19Bevivino A.E. Loll P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11955-11960Crossref PubMed Scopus (151) Google Scholar, 24Chow M.K. Paulson H.L. Bottomley S.P. J. Mol. Biol. 2004; 335: 333-341Crossref PubMed Scopus (52) Google Scholar). Given our previous results wit" @default.
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• W1995989097 title "Polyglutamine Expansion in Ataxin-3 Does Not Affect Protein Stability" @default.
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