title: Examination of Ataxin-3 (atx-3) Aggregation by Structural Mass Spectrometry Techniques: A Rationale for Expedited Aggregation upon Polyglutamine (polyQ) Expansion.
abstract: Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73-96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided.
authors.affiliation: From the ‡Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK;
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Scarff
authors.affiliation: §IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-4180 Porto, Portugal.
authors.forename: Bruno
authors.initials: B
authors.lastname: Almeida
authors.affiliation: §IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-4180 Porto, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Fraga
authors.affiliation: §IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-4180 Porto, Portugal.
authors.forename: Sandra
authors.initials: S
authors.lastname: Macedo-Ribeiro
authors.affiliation: From the ‡Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK; a.e.ashcroft@leeds.ac.uk s.e.radford@leeds.ac.uk.
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
authors.affiliation: From the ‡Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK; a.e.ashcroft@leeds.ac.uk s.e.radford@leeds.ac.uk.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
mesh_terms: D000067699:Ataxin-3 / Q000737:chemistry* / Q000235:genetics; D052160:Benzothiazoles; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D066329:Protein Aggregates; D066263:Protein Aggregation, Pathological / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013844:Thiazoles
chemical_list: D052160:Benzothiazoles; D005456:Fluorescent Dyes; D010455:Peptides; D066329:Protein Aggregates; D011994:Recombinant Proteins; D012097:Repressor Proteins; D013844:Thiazoles; C009462:thioflavin T; C097188:polyglutamine; C092341:ATXN3 protein, human; D000067699:Ataxin-3