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• W2079771002 abstract "Huntington disease is an inherited neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats, resulting in a polyglutamine stretch of >37 on the N terminus of the protein huntingtin (htt). htt is a large (347 kDa), ubiquitously expressed protein. The precise functions of htt are not clear, but its importance is underscored by the embryonic lethal phenotype in htt knock-out mice. Despite the fact that the htt gene was cloned 13 years ago, little is known about the properties of the full-length protein. Here we report the expression and preliminary characterization of recombinant full-length wild-type human htt. Our results support a model of htt composed entirely of HEAT repeats that stack to form an elongated superhelix. Huntington disease is an inherited neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats, resulting in a polyglutamine stretch of >37 on the N terminus of the protein huntingtin (htt). htt is a large (347 kDa), ubiquitously expressed protein. The precise functions of htt are not clear, but its importance is underscored by the embryonic lethal phenotype in htt knock-out mice. Despite the fact that the htt gene was cloned 13 years ago, little is known about the properties of the full-length protein. Here we report the expression and preliminary characterization of recombinant full-length wild-type human htt. Our results support a model of htt composed entirely of HEAT repeats that stack to form an elongated superhelix. Huntington disease (HD) 3The abbreviations used are: HD, Huntington disease; htt, huntingtin; polyQ, polyglutamine; PBS, phosphate-buffered saline.3The abbreviations used are: HD, Huntington disease; htt, huntingtin; polyQ, polyglutamine; PBS, phosphate-buffered saline. is an autosomal-dominant neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats on the N terminus of the IT15 gene that encodes the protein huntingtin (htt) (1Huntington's Disease Collaborative Research GroupCell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7053) Google Scholar). HD occurs in individuals whose htt gene has more than 37 CAGs, resulting in a mutant protein with an abnormally extended polyglutamine (polyQ) tract. Symptoms can manifest at any age and typically involve movement disorders, including chorea, along with psychiatric and cognitive dysfunction. The median age at onset is 40 years, with death generally following 15–20 years after appearance of symptoms (2Harper P.S. Huntington's Disease. W.B. Saunders, Philadelphia1991Google Scholar, 3Bates G. Harper P.S. Jones L. Huntington's Disease. Oxford University Press, Oxford2002Google Scholar). Although the neurodegeneration caused by the HD mutation is particularly marked in the striatum and cortex, htt is widely expressed in many different tissues and its functions are critical for life (4Nasir J. Floresco S.B. O'Kusky J.R. Diewert V.M. Richman J.M. Zeisler J. Borowski A. Marth J.D. Phillips A.G. Hayden M.R. Cell. 1995; 81: 811-823Abstract Full Text PDF PubMed Scopus (675) Google Scholar, 5Zeitlin S. Liu J.P. Chapman D.L. Papaioannou V.E. Efstratiadis A. Nat. Genet. 1995; 11: 155-163Crossref PubMed Scopus (641) Google Scholar, 6Duyao M.P. Auerbach A.B. Ryan A. Persichetti F. Barnes G.T. McNeil S.M. Ge P. Vonsattel J.P. Gusella J.F. Joyner A.L. Science. 1995; 269: 407-410Crossref PubMed Scopus (571) Google Scholar). Mutant htt is prone to aggregation, and both cytosolic and nuclear inclusions have been observed (7Difiglia M. Sapp E. Chase K. Schwarz C. Meloni A. Young C. Martin E. Vonsattel J.P. Carraway R. Reeves S.A. Neuron. 1995; 14: 1075-1081Abstract Full Text PDF PubMed Scopus (618) Google Scholar, 8Graveland G.A. Williams R.S. Difiglia M. Science. 1985; 227: 770-773Crossref PubMed Scopus (494) Google Scholar, 9Sapp E. Ge P. Aizawa H. Bird E. Penney J. Young A.B. Vonsattel J.P. Difiglia M. Neuroscience. 1995; 64: 397-404Crossref PubMed Scopus (141) Google Scholar, 10Stine O.C. Li S.H. Pleasant N. Wagster M.V. Hedreen J.C. Ross C.A. Hum. Mol. Genet. 1995; 4: 15-18Crossref PubMed Scopus (25) Google Scholar). Extensive genetic and transgenic data suggest that the HD mutation causes disease primarily by conferring a toxic gain-of-function on the mutant protein (Ref. 4Nasir J. Floresco S.B. O'Kusky J.R. Diewert V.M. Richman J.M. Zeisler J. Borowski A. Marth J.D. Phillips A.G. Hayden M.R. Cell. 1995; 81: 811-823Abstract Full Text PDF PubMed Scopus (675) Google Scholar and reviewed in Ref. 11Rubinsztein D.C. Sci. Aging Knowledge Environ. 2003; 2003: E26Crossref Scopus (18) Google Scholar). However, it is possible that loss-of-function and/or dominant negative effects may also contribute to pathology (reviewed in Refs. 11Rubinsztein D.C. Sci. Aging Knowledge Environ. 2003; 2003: E26Crossref Scopus (18) Google Scholar, 12Cattaneo E. Zuccato C. Tartari M. Nat. Rev. Neurosci. 2005; 6: 919-930Crossref PubMed Scopus (512) Google Scholar). Proteolytic processing of htt is also likely to play an important role in HD pathogenesis. The toxicity of mutant htt may only be fully exposed after cleavage by proteases, including caspases, calpains, and a putative aspartic protease, to reveal a short, N-terminal polyQ-containing fragment of 100–150 residues (reviewed in Refs. 13Gafni J. Hermel E. Young J.E. Wellington C.L. Hayden M.R. Ellerby L.M. J. Biol. Chem. 2004; 279: 20211-20220Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 14Luo S. Vacher C. Davies J.E. Rubinsztein D.C. J. Cell Biol. 2005; 169: 647-656Crossref PubMed Scopus (142) Google Scholar, 15Wellington C.L. Ellerby L.M. Gutekunst C.A. Rogers D. Warby S. Graham R.K. Loubser O. van Raamsdonk J. Singaraja R. Yang Y.Z. Gafni J. Bredesen D. Hersch S.M. Leavitt B.R. Roy S. Nicholson D.W. Hayden M.R. J. Neurosci. 2002; 22: 7862-7872Crossref PubMed Google Scholar and references therein). N-terminal cleavage products have been found in inclusions from HD patients (16Lunkes A. Lindenberg K.S. Ben-Haiem L. Weber C. Devys D. Landwehrmeyer G.B. Mandel J.L. Trottier Y. Mol. Cell. 2002; 10: 259-269Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 17Goldberg Y.P. Nicholson D.W. Rasper D.M. Kalchman M.A. Koide H.B. Graham R.K. Bromm M. Kazemi-Esfarjani P. Thornberry N.A. Vaillancourt J.P. Hayden M.R. Nat. Genet. 1996; 13: 442-449Crossref PubMed Scopus (502) Google Scholar, 18Martindale D. Hackam A. Wieczorek A. Ellerby L. Wellington C. McCutcheon K. Singaraja R. Kazemi-Esfarjani P. Devon R. Kim S.U. Bredesen D.E. Tufaro F. Hayden M.R. Nat. Genet. 1998; 18: 150-154Crossref PubMed Scopus (422) Google Scholar), and N-terminal fragments with expanded polyQs readily form inclusions similar to those seen in HD patients. However, it is still unclear which combination of proteolytic events is required for generation of the toxic fragments. There are nine different inherited neurodegenerative diseases caused by CAG/polyQ expansion in the target proteins. Although all show inclusions containing the expanded polyQs, the parts of the brain affected by the different polyQ-expanded proteins differ, resulting in different symptom constellations. Therefore, the host protein itself and its distinct interactions with other proteins probably play key roles in determining the disease pathology (19La Spada A.R. Taylor J.P. Neuron. 2003; 38: 681-684Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Furthermore, recent data suggest that wild-type htt may have a beneficial effect on the gain-of-function toxicity of the mutant protein (20Van Raamsdonk J.M. Pearson J. Rogers D.A. Bissada N. Vogl A.W. Hayden M.R. Leavitt B.R. Hum. Mol. Genet. 2005; 14: 1379-1392Crossref PubMed Scopus (127) Google Scholar, 21Ho L.W. Brown R. Maxwell M. Wyttenbach A. Rubinsztein D.C. J. Med. Genet. 2001; 38: 450-452Crossref PubMed Scopus (72) Google Scholar). It is therefore important to determine the normal functions of htt in order to fully understand the pathogenesis of HD. Many htt-interacting proteins have been reported, and wild-type htt has been proposed to have roles in a wide range of activities, including neurotransmission, axonal transport, and neuronal positioning (22Harjes P. Wanker E.E. Trends Biochem. Sci. 2003; 28: 425-433Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 23Goehler H. Lalowski M. Stelzl U. Waelter S. Stroedicke M. Worm U. Droege A. Lindenberg K.S. Knoblich M. Haenig C. Herbst M. Suopanki J. Scherzinger E. Abraham C. Bauer B. Hasenbank R. Fritzsche A. Ludewig A.H. Bussow K. Coleman S.H. Gutekunst C.A. Landwehrmeyer B.G. Lehrach H. Wanker E.E. Mol. Cell. 2004; 15: 853-865Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Htt is a 347-kDa protein with no sequence homology to any other known protein. However, sequence analysis revealed that htt contains multiple HEAT repeat sequences (24Takano H. Gusella J.F. BMC Neurosci. 2002; 3: 15Crossref PubMed Scopus (117) Google Scholar). A HEAT (Htt, Elongation factor 3, the PR65/A subunit of protein phosphatase 2A, and the lipid kinase TOR) repeat is a degenerate ∼50-amino acid motif consisting of two anti-parallel α-helices forming a helical hairpin (see Fig. 5). HEAT repeat proteins generally mediate important protein-protein interactions involved in cytoplasmic and nuclear transport, microtubule dynamics, and chromosome segregation (25Neuwald A.F. Hirano T. Genome Res. 2000; 10: 1445-1452Crossref PubMed Scopus (231) Google Scholar). To date, there are four HEAT repeat proteins with known three-dimensional structures: importin-β, karyopherin-β2, the PR65/A subunit of PP2A, and Cand1 (26Cingolani G. Petosa C. Weis K. Muller C.W. Nature. 1999; 399: 221-229Crossref PubMed Scopus (449) Google Scholar, 27Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (291) Google Scholar, 28Groves M.R. Hanlon N. Turowski P. Hemmings B.A. Barford D. Cell. 1999; 96: 99-110Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 29Goldenberg S.J. Cascio T.C. Shumway S.D. Garbutt K.C. Liu J. Xiong Y. Zheng N. Cell. 2004; 119: 517-528Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). HEAT repeat proteins have very high helical content (≥50%) and often form superhelical structures with continuous hydrophobic cores (see Fig. 5) (30Perry J. Kleckner N. Cell. 2003; 112: 151-155Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 31Andrade M.A. Petosa C. O'Donoghue S.I. Muller C.W. Bork P. J. Mol. Biol. 2001; 309: 1-18Crossref PubMed Scopus (396) Google Scholar). Using cross-species comparative analysis, Takano and Gusella (24Takano H. Gusella J.F. BMC Neurosci. 2002; 3: 15Crossref PubMed Scopus (117) Google Scholar) predicted that vertebrate htt contains 28–36 HEAT repeats that span the entire protein. To understand the normal functions of wild-type htt, it is useful to know its structural features. All in vitro biochemical work on htt has been limited to polyQ-containing N-terminal fragments, such as exon 1. The aim of this study is to express and purify full-length wild-type human htt to allow characterization of its biochemical, biophysical, and structural properties and to provide a tool for addressing the molecular basis of its manifold interactions. Here we report the expression and purification of recombinant full-length human htt to over 95% homogeneity. Preliminary characterization by circular dichroism, dynamic light scattering, electron microscopy, and limited proteolysis supports a model of htt predominantly composed of HEAT repeats that stack into a rod-like superhelical structure. Materials—pFastBacHTb vector, DH10Bac competent cells, and the insect cell lines (Sf9 and Hi-Five) were all purchased from Invitrogen. Anti-htt monoclonal antibodies MAB2166, 2168, and 2170 were purchased from Chemicon International. Anti-FLAG M2 antibody and its antibody gel were purchased from Sigma-Aldrich. Horseradish peroxidase-conjugated anti-mouse antibody was purchased from DAKO A/S. Mark12 from Invitrogen was used for SDS-PAGE, and rainbow molecular weight marker from Amersham Biosciences was used for Western blot. HMW Native Marker kit was used for native-PAGE (Amersham Biosciences). Cloning, Expression, and Purification of Full-length Human htt—Full-length human htt cDNA (IT15) with N-terminal His6 and 3xFLAG tags was subcloned from pTre2Hyg:3xFLAG:htt17QFL construct (32Sugars K.L. Brown R. Cook L.J. Swartz J. Rubinsztein D.C. J. Biol. Chem. 2004; 279: 4988-4999Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) into Bac-to-Bac vector pFastBacHTb between the BamHI and NotI sites. The bacmid was generated according to Invitrogen's Bac-to-Bac manual, and the transfection of Sf9 cells was carried out using FuGENE (Roche Diagnostics Ltd.) following the manufacturer's instructions. The transfection supernatant containing the recombinant virus was amplified over three rounds and then used to infect Hi-Five cells for protein production. Cells were harvested 72 h after infection, and expression of full-length htt was initially verified by immunoblotting. Cell pellets were resuspended in buffer A (50 mm Tris, pH 7.4, 100 mm NaCl, and 10% glycerol) containing a protease inhibitor mixture (Complete, EDTA-free; Roche Diagnostics). Cells were broken by sonication, and cell debris was spun down by centrifugation. The supernatant was collected and passed through a 5-ml Hi-Trap metal-chelating column (Amersham Biosciences) precharged with Ni2SO4. After washing with buffer A containing 5 mm imidazole, bound proteins were eluted using a 5–200-mm imidazole gradient. Fractions were subjected to SDS-PAGE, and those containing full-length htt were pooled and concentrated using a 30-kDa cut-off spin concentrator (Viva Science). The semipurified htt was then passed through a Superose 6 16/60 gel filtration column pre-equilibrated in buffer A. Fractions containing htt were pooled and further purified using the ANTI-FLAG M2 affinity gel (Sigma-Aldrich) following the manufacturer's instructions. Briefly, htt (in buffer A) was incubated with the affinity gel at 4 °C overnight, followed by two washes with 5 resin-volumes of buffer A. The major contaminant, identified as tubulin by N-terminal sequencing, did not bind to the resin (data not shown). The protein that bound to the resin was eluted with 3xFLAG peptide (0.3 mg/ml in buffer A). htt purity was judged by SDS-PAGE and concentration determined using the Bradford assay. htt expression levels and purification yields were estimated from Coomassie-stained SDS-PAGE, using bovine serum albumin as the standard. PAGE and Western Blots—SDS- and native-PAGE were carried out using 3–8% Tris acetate gels from Invitrogen, following the manufacturer's instructions. Western blotting was performed using a Bio-Rad semidry transfer system and developed with the ECL system (Amersham Biosciences). Coomassie staining of native and SDS gels was conducted using SimplyBlue Safestain (Invitrogen), which has a reported sensitivity of 7 ng of bovine serum albumin. Circular Dichroism Spectroscopy—Purified full-length human htt (0.2 mg/ml) was dialyzed into 30 mm Tris, SO4 pH 7.4, overnight before CD analysis. CD spectra were measured at 20 °C on a JASCO 810 spectropolarimeter using a 0.5-mm cuvette, from 180 to 260 nm, and 20 scans were averaged. The spectra were analyzed using the CD Spectra Deconvolution program (CDNN, version 2.1) (33Bohm G. Biophys. Chem. 1996; 59: 1-32Crossref PubMed Scopus (54) Google Scholar). The experiment was repeated twice with material judged by SDS-PAGE to be 70 and 90% un-nicked, and identical traces were obtained. Negative Stain Electron Microscopy—A 4-μl droplet of each isolated fraction of htt from the Superose 6 column was placed on carbon-coated copper 400-mesh grids (Agar Scientific). The solution was blotted away and washed once with 4 μl of milliQ-filtered water and blotted. The grid was then stained using 4 μl of 2% uranyl acetate, blotted, and allowed to air dry. The grid was examined using a Hitachi 7100 transmission electron microscope operated at 100 kV, and images were taken using a Gatan Ultrascan 1000 CCD camera at magnifications of ×15,000–50,000. Images were converted to tif or jpg format using Digital micrograph software. Immunogold Electron Microscopy—5 μl of htt from fraction 4, which eluted at 77 ml off the Superose 6 column, was placed on a carbon-coated grid and incubated for 2 min. The grid was then placed sample side down onto a droplet of milk (1% w/v in PBS) for 10 min and then lifted off and blotted. The grid was placed onto a droplet of primary antibody (MAB2166, MAB2168, or MAB2170), diluted 1/1000 in 1% w/v milk in PBS, covered, and incubated for 60 min. The grid was washed ten times with filtered PBS and blotted and then incubated for 60 min on a droplet of secondary antibody (goat anti-mouse 10 nm or 5 nm conjugated-gold; Sigma) diluted 1/20 in PBS. Finally, the grid was washed three times for 5 min with PBS and three times for 5 min in water and blotted. The grid was then stained as described above with 2% uranyl acetate. Controls were done using the same protocol with water instead of protein solution. Dynamic Light Scattering—Dynamic light scattering was carried out at 20 °C on a DynaPro-801 molecular sizing instrument from Protein Solutions. htt at 0.4 mg/ml was passed through a 0.1-μm filter before loading into a 45-μl cuvette. 26 counts were taken and averaged for measuring the dynamic radius and the polydispersion. Limited Trypsin Digestion—Purified htt was treated with 0.04, 0.08, 0.12, and 0.24% trypsin (by weight) at room temperature for 40 min. The reactions were stopped by adding a 10-fold excess of soybean trypsin inhibitor (Sigma), and samples were run on SDS- and native-PAGE. The experiment shown in Fig. 4 was conducted on material judged to be 40% nicked during purification, but similar results were obtained from starting material judged to be only 10% nicked (data not shown). Molecular Modeling—A model of a HEAT repeat protein of 2880 residues was generated using the coordinates of importin-β (A chain from 1UKL) (34Lee S.J. Sekimoto T. Yamashita E. Nagoshi E. Nakagawa A. Imamoto N. Yoshimura M. Sakai H. Chong K.T. Tsukihara T. Yoneda Y. Science. 2003; 302: 1571-1575Crossref PubMed Scopus (173) Google Scholar). A continuous superhelix composed of four A chains was created by superimposing a C-terminal helix starting at residue 855 on the N-terminal helix of another monomer starting at residue 187. Overlapping residues were removed, and the resulting model was energy minimized using CNS (35Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). A ribbon diagram was generated using programs Molscript (36Esnouf R.M. J. Mol. Graph. Model. 1997; 15: 132-133Crossref PubMed Scopus (1794) Google Scholar) and Raster3D (37Merritt E.A. Murphy M.E. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar), and the surface representation was created using the program Spock. Expression and Purification of Full-length htt—The first step in determining the structural and functional properties of htt is to obtain milligram quantities of pure full-length protein. Purification of naturally expressed htt from human cells could never yield enough protein of sufficient quality, making it necessary to develop a recombinant system. We initially attempted expression in bacteria, but expression levels were very low and htt could only be detected in whole cell extracts. This was presumably due to the size of htt and the absence in Escherichia coli of the necessary protein folding machinery. We chose insect cells for expression of human htt because they have an htt homologue (38Li Z. Karlovich C.A. Fish M.P. Scott M.P. Myers R.M. Hum. Mol. Genet. 1999; 8: 1807-1815Crossref PubMed Scopus (52) Google Scholar) and are a well established eukaryotic expression system for cytosolic proteins (39Kost T.A. Condreay J.P. Jarvis D.L. Nat. Biotechnol. 2005; 23: 567-575Crossref PubMed Scopus (773) Google Scholar). Full-length human htt cDNA was cloned into pFastbacHTb vector with an N-terminal His6 tag followed by a 3xFLAG tag (Fig. 1A). The vector was transformed into DH10Bac cells to generate the bacmid that was used to transfect the insect cells. The full-length htt band was readily detected on a Coomassie-stained SDS gel from the transfected total cell lysate (Fig. 1B), and its identity was confirmed by Western blotting using anti-htt monoclonal antibodies (Fig. 1C, MAB2166, MAB2168, and MAB2170). After nickel affinity chromatography, full-length htt was ∼70% pure (Fig. 1D, lane 1). Further purification on a Superose 6 gel filtration column and anti-FLAG tag resin generated ∼95% pure htt (Fig. 1D, lane 2). The majority of the htt eluted from the Superose 6 column in high molecular mass complexes of either homo- or hetero-oligomers (Fig. 2). Interestingly, the last peak to elute corresponded to an approximate molecular mass of 500 kDa, whereas full-length htt is 347 kDa, suggestive of a non-globular protein fold. This peak was shown by native PAGE to be composed primarily of monomeric htt that again ran between the molecular mass markers corresponding to 440 and 669kDa and a small amount of dimer (Fig. 2B). The native gel was blotted with the full complement of anti-htt antibodies MAB2166, MAB2168, and MAB2170 to confirm the product was full-length htt (only MAB2166 shown in Fig. 2C). It is evident from the Western blot that fraction 4 is predominantly monomeric, with only a small fraction of dimer and no higher ordered species. Although yields varied somewhat, we typically obtained 0.5 mg of pure htt/liter of cell culture. This figure, 0.5 mg/liter, represents only ∼10% of the total expressed htt due to the association of at least half of the protein with the insoluble cell components and the aggregation of a large fraction of the soluble htt (as seen in A). Despite the low yields, we were successful in expressing and purifying sufficient quantities of full-length monomeric htt to undertake preliminary biophysical studies. Varying degrees of proteolytic nicking were observed in preps of htt. We observed that the extent of nicking ranged from 5 to 40% depending on whether EDTA was present in the cell lysis buffer. Implications of the proteolytic nicking of purified htt are dealt with in detail in the sections below.FIGURE 2Fractionation of semipurified htt on a gel filtration column. A, elution profile of nickel column-purified htt from a Superose 6 column. The column was calibrated with the high molecular mass calibration kit from Amersham Bioscience, and the elution positions of the standards are indicated. Four fractions were chosen for further analysis (numbers 1–4), and the position determined to correspond to monomeric htt is indicated. B, a Coomassie-stained non-denaturing native gel of the same fractions shows that early fractions contain large aggregates and later fractions contain monomeric htt and a small amount of dimer. C, the same native gel as in panel C, blotted with anti-htt antibody MAB2166 (similar results were obtained with MAB2168 and 2170). D, negative-stained electron microscopy pictures, with sections 1–4 corresponding to the fractions. Large aggregates are seen in fractions 1 and 2, and small particles (probably corresponding to monomeric htt) are seen in fraction 4. E, the same sample as in panel E, section 4, but with immunogold labeling (black) using anti-htt antibody MAB2166. This confirms that the distinct particles are htt.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Electron Microscopy Shows Monomeric htt Particles—Fractions isolated from the Superose 6 purification step were examined by negative stain electron microscopy (Fig. 2D) to confirm the presence of aggregates in the early peaks and to see whether any regular structure could be observed. Fraction 1 (void from Superose 6) showed large deposits of aggregated protein, whereas fractions 2 and 3 showed a mixture of nonspecific protein deposits with increasing numbers of small individual particles. The small particles were particularly noticeable in fraction 4. To determine whether the distinct particles were composed of htt, a sample of fraction 4 was labeled using monoclonal antibodies MAB2166, MAB2168, or MAB2170 and visualized using immunogold (Fig. 2E). All three antibodies specifically labeled the particles (only MAB2166 is shown in the figure), suggesting that the small particles observed in the negative stain electron microscopy correspond to monomeric full-length htt. htt Is an Elongated Molecule with High Helical Content—To see whether the purified material was homogeneous and whether htt was indeed elongated, we carried out a dynamic light-scattering experiment. htt concentrated to 0.4 mg/ml gave a single peak, which when analyzed as monomodal yielded a hydrodynamic radius of 13.4 nm with a polydispersion of 31.6%. Polydispersity above 20% normally suggests the presence of some higher ordered species; as we knew our solution contained a small amount of dimer (by native-PAGE, Fig. 2, B and C), this was not unexpected. A bimodal analysis yielded hydrodynamic radii of 9.5 and 21.0 nm, likely corresponding to monomeric and dimeric htt. Using a standard curve containing transferrin, enolase, alcohol dehydrogenase, aldolase, and apoferritin, we calculated a molecular mass of 522 kDa for a hydrodynamic radius of 9.5 nm, similar to what we obtain for monomeric htt by size-exclusion chromatography and native-PAGE. Thus, the hydrodynamic radius observed by dynamic light scattering, along with the migration behavior on size exclusion chromatography and native-PAGE, suggests an elongated structure for htt. The crystal structures of four other HEAT repeat proteins reveal a high helical content (>50%) (30Perry J. Kleckner N. Cell. 2003; 112: 151-155Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Because it is predicted that HEAT repeats span the entire htt gene (24Takano H. Gusella J.F. BMC Neurosci. 2002; 3: 15Crossref PubMed Scopus (117) Google Scholar), it is expected that human htt would have a similar high helical content with multiple solvent-exposed turns and coils connecting the helices. Indeed, the CD spectrum showed that purified recombinant full-length htt was predominantly composed of α-helical secondary structure (Fig. 3A). Deconvolution of the CD spectrum gave rise to 58.1% α-helix, 5.4% β-strand, and 27.7% turns and coils. This is in good agreement with Neural Network (40Combet C. Blanchet C. Geourjon C. Deleage G. Trends Biochem. Sci. 2000; 25: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1432) Google Scholar) secondary structure predictions (51.9% α-helix, 8.2% β-strand, and 39.8% coils). Thermal denaturation of recombinant htt was carried out by monitoring change to CD signal at 222 nm with increasing temperature. We observed a single broad transition with a midpoint at ∼52 °C (Fig. 3B), indicating that full-length htt does not unfold in the cooperative manner associated with globular proteins. To determine whether the apparent non-cooperativity was due to the proteolytic nicking of htt, we repeated the scans and melts with 70 and 90% intact material and observed no difference. Similar broad melting profiles have been observed for other helical repeat proteins that form non-globular structures (41Beddoe T. Bushell S.R. Perugini M.A. Lithgow T. Mulhern T.D. Bottomley S.P. Rossjohn J. J. Biol. Chem. 2004; 279: 46448-46454Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), whereas others appear more cooperative in nature (42Zweifel M.E. Barrick D. Biochemistry. 2001; 40: 14357-14367Crossref PubMed Scopus (88) Google Scholar). It is possible that the cooperativity of the melting transitions of helical repeat proteins is related to the size of the repeat regions. The most cooperative transition is observed for the smallest protein, the repeat portion of the Notch receptor, which contains ∼200 amino acids, whereas a broad non-cooperative transition is observed for the repeat segment of TOM70, which is ∼550 amino acids in size. htt contains over 3,000 amino acids and may have several segments that unfold semi-independently. htt Is Susceptible to Proteolysis but Remains Associated when Cleaved at Multiple Sites—During purification we noticed that htt was particularly prone to proteolysis even in the presence of a protease inhibitor mixture. In some preparations, a 220-kDa degradation product was the major band observed in the nickel column eluate, and some smaller fragments were still associated with full-length htt after gel filtration (Fig. 4A, lane 1). The degree of proteolysis during htt purification was found to depend on whether EDTA was present in the cell lysis buffer. Thus, the apparent purity of htt in Fig. 4A differs from that in Fig. 1D (where EDTA was present). Because the construct has a 3xFLAG tag at the N terminus, the monomeric htt fractions from the Superose 6 column were further purified on an ANTI-FLAG M2 affinity gel (Fig. 4A). However, after FLAG tag purification, all of the ∼220 kDa and most of the smaller bands still appeared in the eluate. The apparent association of htt fragments was not due to the presence of artifactual disulfide bonds, because we obtained similar results in the presence of 10 mm dithiothreitol (e.g. Fig. 4D). Using the three anti-htt monoclonal antibodies and the anti-FLAG an" @default.
• W2079771002 created "2016-06-24" @default.
• W2079771002 creator A5000432967 @default.
• W2079771002 creator A5008236816 @default.
• W2079771002 creator A5047905539 @default.
• W2079771002 creator A5057914646 @default.
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• W2079771002 date "2006-06-01" @default.
• W2079771002 modified "2023-10-10" @default.
• W2079771002 title "Expression and Characterization of Full-length Human Huntingtin, an Elongated HEAT Repeat Protein" @default.
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