The Machado-Joseph disease-associated expanded form of ataxin-3: Overexpression, purification, and preliminary biophysical and structural characterization

Autor(es)

Contessotto, Miriam G. G. ^[1] ; Rosselli-Murai, Luciana K. ^[2] ; Garcia, Maria Cristina C. ^[1] ; Oliveira, Cristiano L. P. ^{[3, 4]} ; Torriani, Iris L. ^{[4, 5]} ; Lopes-Cendes, Iscia ^{[1, 6]} ; Murai, Marcelo J. ^{[1, 2]}

Afiliação do(s) autor(es)

^[1] Univ Estadual Campinas, Sch Med Sci, Dept Med Genet, Campinas, SP - Brazil

^[2] Univ Michigan, Dept Pharmacol, Med Sch, Ann Arbor, MI 48109 - USA

^[3] Univ Sao Paulo, Inst Phys, Sao Paulo, SP - Brazil

^[4] Univ Estadual Campinas, Gleb Wataghin Inst Phys, Dept Condensed Matter Phys, Campinas, SP - Brazil

^[5] Brazilian Biosci Natl Lab, Ctr Res Energy & Mat, Campinas, SP - Brazil

^[6] Brazilian Inst Neurosci & Neurotechnol, Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 152, p. 40-45, DEC 2018.
• Citações Web of Science: 2

Resumo

An expansion of the polyglutamine (polyQ) tract within the deubiquitinase ataxin-3 protein is believed to play a role in a neurodegenerative disorder. Ataxin-3 contains a Josephin catalytic domain and a polyQ tract that renders it intrinsically prone to aggregate, and thus full-length protein is difficult to characterize structurally by high-resolution methods. We established a robust protocol for expression and purification of wild-type and expanded ataxin-3, presenting 19Q and 74Q, respectively. Both proteins are monodisperse as assessed by analytical size exclusion chromatography. Initial biophysical characterization was performed, with apparent transition melting temperature of expanded ataxin-3 lower than the wild-type counterpart. We further characterize the molecular envelope of wild-type and expanded polyQ tract in ataxin-3 using small angle X-ray scattering (SAXS). Characterization of protein-protein interactions between ataxin-3 and newly identified binding partners will benefit from our protocol. (AU)

• Processo FAPESP: 01/07542-6 - Fapesp structural genomics program.
• Beneficiário: Iscia Teresinha Lopes Cendes
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 01/10658-6 - SMolBNet - Rede de Biologia Molecular Estrutural do estado de São Paulo
• Beneficiário: Maria Cristina Cabral Garcia
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 01/12346-1 - Analise molecular e tridimensional da proteina ataxina 3.
• Beneficiário: Miriam Gisele Gasparotto Contessotto
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/07559-3 - Instituto Brasileiro de Neurociência e Neurotecnologia - BRAINN
• Beneficiário: Fernando Cendes
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs