title: Structural instability and fibrillar aggregation of non-expanded human ataxin-3 revealed under high pressure and temperature.
abstract: Protein misfolding and formation of structured aggregates are considered to be the earliest events in the development of neurodegenerative diseases, but the mechanism of these biological phenomena remains to be elucidated. Here, we report a study of heat- and pressure-induced unfolding of human Q26 and murine Q6 ataxin-3 using spectroscopic methods. UV absorbance and fluorescence revealed that heat and pressure induced a structural transition of both proteins to a molten globule conformation. The unfolding pathway was partly irreversible and led to a protein conformation where tryptophans were more exposed to water. Furthermore, the use of fluorescent probes (8-anilino-1-naphthalenesulfonate and thioflavin T) allowed the identification of different intermediates during the process of pressure-induced unfolding. At high temperature and pressure, human Q26, but not murine Q6, underwent concentration-dependent aggregation. Fourier transform infrared and circular dichroism spectroscopy revealed that these aggregates are characterized by an increased beta-sheet content. As revealed by electron microscopy, heat- and pressure-induced aggregates were different; high temperature treatment led to fibrillar microaggregates (8-10-nm length), whereas high pressure induced oligomeric structures of globular shape (100 nm in diameter), which sometimes aligned to higher order suprastructures. Several intermediate structures were detected in this process. Two factors appear to govern ataxin unfolding and aggregation, the length of the polyglutamine tract and its protein context.
authors.affiliation: INSERM U128, IFR 122, 1919 route de Mende, F-34293 Montpellier Cédex 5, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Marchal
authors.forename: Erlet
authors.initials: E
authors.lastname: Shehi
authors.forename: Marie-Cécile
authors.initials: MC
authors.lastname: Harricane
authors.forename: Paola
authors.initials: P
authors.lastname: Fusi
authors.forename: Frédéric
authors.initials: F
authors.lastname: Heitz
authors.forename: Paolo
authors.initials: P
authors.lastname: Tortora
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lange
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000067699:Ataxin-3; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins; D011312:Pressure; D011487:Protein Conformation; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014157:Transcription Factors
chemical_list: D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C092341:ATXN3 protein, human; D000067699:Ataxin-3; C511594:Atxn3 protein, mouse