title: Proteomic and biochemical analyses unveil tight interaction of ataxin-3 with tubulin.
abstract: Ataxin-3 consists of an N-terminal globular Josephin domain and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers an inherited neurodegenerative disorder, spinocerebellar ataxia type 3, when its length exceeds a critical threshold. The pathology results from protein misfolding and intracellular accumulation of fibrillar amyloid-like aggregates. Plenty of work has been carried out to elucidate the protein's physiological role(s), which has shown that ataxin-3 is multifunctional; it acts as a transcriptional repressor, and also has polyubiquitin-binding/ubiquitin-hydrolase activity. In addition, a recent report shows that it participates in sorting misfolded protein to aggresomes, close to the microtubule-organizing center. Since a thorough understanding of the protein's physiological role(s) requires the identification of all the molecular partners interacting with ataxin-3, we pursued this goal by taking advantage of two-dimensional chromatography coupled to tandem mass spectrometry. We found that different ataxin-3 constructs, including the sole Josephin domain, bound alpha- and beta-tubulin from soluble rat brain extracts. Coimmunoprecipitation experiments confirmed this interaction. Also, normal ataxin-3 overexpressed in COS7 cultured cells partially colocalized with microtubules, whereas an expanded variant only occasionally did so, probably due to aggregation. Furthermore, by surface plasmon resonance we determined a dissociation constant of 50-70nM between ataxin-3 and tubulin dimer, which strongly supports the hypothesis of a direct interaction of this protein with microtubules in vivo. These findings suggest an involvement of ataxin-3 in directing aggregated protein to aggresomes, and shed light on the mode of interaction among the different molecular partners participating in the process.
authors: Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, I-20126 Milano, Italy.
authors: Serena
authors: S
authors: Mazzucchelli
authors: Antonella
authors: A
authors: De Palma
authors: Matteo
authors: M
authors: Riva
authors: Annalisa
authors: A
authors: D'Urzo
authors: Chiara
authors: C
authors: Pozzi
authors: Valentina
authors: V
authors: Pastori
authors: Francesca
authors: F
authors: Comelli
authors: Paola
authors: P
authors: Fusi
authors: Marco
authors: M
authors: Vanoni
authors: Paolo
authors: P
authors: Tortora
authors: Pierluigi
authors: P
authors: Mauri
authors: Maria Elena
authors: ME
authors: Regonesi
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006801:Humans; D017827:Machado-Joseph Disease / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008870:Microtubules; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D055503:Protein Multimerization; D021381:Protein Transport; D051381:Rats; D019108:Tight Junctions / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics; D014404:Tubulin / Q000378:metabolism*
chemical_list: D009419:Nerve Tissue Proteins; D014404:Tubulin