Normal and pathogenic ataxin-3: biological roles, toxicity and fibrillogenesis

Ataxin-3 (AT3) is a deubiquitinating enzyme that triggers the inherited neurodegenerative disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. It consists of the N-terminal globular Josephin domain (JD) and the C-terminal disordered one. Regarding its physiological role, it has ubiquitin hydrolase activity implicated in the function of the ubiquitin-proteasome system, but also plays a role in the pathway that sorts aggregated protein to aggresomes via microtubules. In the first part of this work, we further investigated its function(s) by taking advantage of Small Angle X-ray Scattering (SAXS) and Surface Plasmon Resonance (SPR). We demonstrated that an AT3 oligomer consisting of 6-7 subunits tightly binds to the tubulin hexameric oligomer at the level of three distinct tubulin-binding regions, one located in the JD, and the two others in the disordered domain, upstream and downstream of the polyQ stretch. By SPR we have also provided the first evidence of direct binding of AT3 to HDAC6, one of the components of the transport machinery that sorts protein to the aggresome. In the second part of this work, we have investigated the mechanisms of AT3 cytotoxicity triggered by expanded variants. For this purpose, we used Saccharomyces cerevisiae as a eukaryotic cellular model. We expressed a wild type (Q26), a pathogenic (Q85) and a truncated (291Δ) variant of the protein. The expanded form caused reduction in viability, accumulation of reactive oxygen species, imbalance of the antioxidant defense system and loss in cell membrane integrity. An AT3 variant truncated upstream of the polyQ also exerted a detrimental effect on cell growth and similar cytotoxicity, although to a lesser extent, which points to the involvement of also non-polyQ regions in cytotoxicity. Finally, we sought to evaluate the effects of tetracycline and epigallocatechin-3-gallate (EGCG), two well-known inhibitors of amyloid aggregation, on AT3 fibrillogenesis and cytotoxicity. We observed that tetracycline does not apparently change the aggregation mode, as supported by Fourier Transform Infrared spectroscopy and Atomic Force Microscopy data, but slightly retards further aggregation of the earliest soluble oligomers. In contrast, EGCG apparently increases the aggregation rate but also leads to the formation of off-pathway, non-amyloid, final aggregates. Despite these different effects, co-incubation of the AT3 with either compounds resulted in significantly lower cytotoxicity during AT3 aggregation.