Proteomic and biochemical analyses unveil tight interaction of ataxin-3 with tubulin

Serena Mazzucchelli, Antonella De Palma, Matteo Riva, Annalisa D'Urzo, Chiara Pozzi, Valentina Pastori, Francesca Comelli, Paola Fusi, Marco Vanoni, Paolo Tortora, Pierluigi Mauri, Maria Elena Regonesi

Research output: Contribution to journalArticle

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 α- and β-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 50-70 nM 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.

Original languageEnglish
Pages (from-to)2485-2492
Number of pages8
JournalInternational Journal of Biochemistry and Cell Biology
Volume41
Issue number12
DOIs
Publication statusPublished - Dec 2009

Keywords

  • Aggresomes
  • Ataxin-3
  • Mass spectrometry
  • Microtubules
  • Surface plasmon resonance

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

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    Mazzucchelli, S., De Palma, A., Riva, M., D'Urzo, A., Pozzi, C., Pastori, V., Comelli, F., Fusi, P., Vanoni, M., Tortora, P., Mauri, P., & Regonesi, M. E. (2009). Proteomic and biochemical analyses unveil tight interaction of ataxin-3 with tubulin. International Journal of Biochemistry and Cell Biology, 41(12), 2485-2492. https://doi.org/10.1016/j.biocel.2009.08.003