title: A major role for side-chain polyglutamine hydrogen bonding in irreversible ataxin-3 aggregation.
abstract: The protein ataxin-3 consists of an N-terminal globular Josephin domain (JD) and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers the neurodegenerative disorder spinocerebellar ataxia type 3, when it is expanded beyond a critical threshold. The disease results from misfolding and aggregation, although the pathway and structure of the aggregation intermediates are not fully understood. In order to provide insight into the mechanism of the process, we monitored the aggregation of a normal (AT3Q24) ataxin-3, an expanded (AT3Q55) ataxin-3, and the JD in isolation. We observed that all of them aggregated, although the latter did so at a much slower rate. Furthermore, the expanded AT3Q55 displayed a substantially different behavior with respect to the two other variants in that at the latest stages of the process it was the only one that did the following: i) lost its reactivity towards an anti-oligomer antibody, ii) generated SDS-insoluble aggregates, iii) gave rise to bundles of elongated fibrils, and iv) displayed two additional bands at 1604 and 1656 cm(-1) in FTIR spectroscopy. Although these were previously observed in other aggregated polyglutamine proteins, no one has assigned them unambiguously, yet. By H/D exchange experiments we show for the first time that they can be ascribed to glutamine side-chain hydrogen bonding, which is therefore the hallmark of irreversibly SDS-insoluble aggregated protein. FTIR spectra also showed that main-chain intermolecular hydrogen bonding preceded that of glutamine side-chains, which suggests that the former favors the latter by reorganizing backbone geometry.
authors.affiliation: Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Milan, Italy.
authors.forename: Antonino
authors.initials: A
authors.lastname: Natalello
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Frana
authors.forename: Annalisa
authors.initials: A
authors.lastname: Relini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Apicella
authors.forename: Gaetano
authors.initials: G
authors.lastname: Invernizzi
authors.forename: Carlo
authors.initials: C
authors.lastname: Casari
authors.forename: Alessandra
authors.initials: A
authors.lastname: Gliozzi
authors.forename: Silvia Maria
authors.initials: SM
authors.lastname: Doglia
authors.forename: Paolo
authors.initials: P
authors.lastname: Tortora
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Regonesi
mesh_terms: D000067699:Ataxin-3; D003001:Cloning, Molecular; D006801:Humans; D006860:Hydrogen Bonding; D018625:Microscopy, Atomic Force; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D012097:Repressor Proteins; C097188:polyglutamine; C092341:ATXN3 protein, human; D000067699:Ataxin-3