title: Interactions of ataxin-3 with its molecular partners in the protein machinery that sorts protein aggregates to the aggresome.
abstract: Ataxin-3 (AT3) is the protein that triggers the inherited neurodegenerative disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 consists of the N-terminal globular Josephin domain (JD) and the C-terminal disordered one. It cleaves isopeptide bonds between ubiquitin monomers, an event involved in protein quality control mechanisms. AT3 has been implicated in the pathway that sorts aggregated protein to aggresomes via microtubules, in which dynein and histone deacetylase 6 (HDAC6) also seem to be involved. By taking advantage of small angle X-ray scattering (SAXS) and surface plasmon resonance (SPR), we have investigated the interaction of AT3 with tubulin and HDAC6. Based on SAXS results, the AT3 oligomer, consisting of 6-7 subunits, tightly binds to the tubulin hexameric oligomer in a "parallel" fashion. By SPR analysis we have demonstrated that AT3 binds to tubulin dimer with a 50nM affinity. Binding fits with a Langmuir 1:1 model and involves a single binding interface. Nevertheless, the interaction surface consists of three distinct, discontinuous tubulin-binding regions (TBR), one located in the JD, and the two others in the disordered domain, upstream and downstream of the polyQ stretch. In the absence of any of the three TBRs, the affinity is drastically reduced. By SPR we have also provided the first evidence of direct binding of AT3 to HDAC6, with affinity in the range 0.1-1μM. These results shed light on the interactions among the components of the transport machinery that sorts aggregate protein to the aggresome, and pave the way to in vivo studies aimed at further clarifying their roles.
authors.affiliation: Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Bonanomi
authors.affiliation: Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Mazzucchelli
authors.affiliation: Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: D'Urzo
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milano, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Nardini
authors.affiliation: European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany.
authors.forename: Petr V
authors.initials: PV
authors.lastname: Konarev
authors.affiliation: Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy.
authors.forename: Gaetano
authors.initials: G
authors.lastname: Invernizzi
authors.affiliation: European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany.
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.affiliation: Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy; SysBio Centre for Systems Biology, Milan and Rome, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Vanoni
authors.affiliation: Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy. Electronic address: mariaelena.regonesi@unimib.it.
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Regonesi
authors.affiliation: Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Tortora
mesh_terms: D000067699:Ataxin-3; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D066329:Protein Aggregates; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism
chemical_list: D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D066329:Protein Aggregates; D012097:Repressor Proteins; D014404:Tubulin; C092341:ATXN3 protein, human; D000067699:Ataxin-3; C433811:HDAC6 protein, human; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases
keywords: Ataxin-3; HDAC6; Small angle X-ray scattering; Surface plasmon resonance; Tubulin dimer