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Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with ataxin-3 with only Lys-117 present, ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interact-ing protein (CHIP), an E3 ubiquitin ligase that ubiquitinates ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability. [ABSTRACT FROM AUTHOR]

Machado-Joseph disease (MJD) is a dominantly inherited ataxia caused by a polyglutamine-coding expansion in the ATXN3 gene. Suppressing expression of the toxic gene product represents a promising approach to therapy for MJD and other polyglutamine diseases. We performed an extended therapeutic trial of RNA interference (RNAi) targeting ATXN3 in a mouse model expressing the full human disease gene and recapitulating key disease features. Adeno-associated virus (AAV) encoding a microRNA (miRNA)-like molecule, miRATXN3, was delivered bilaterally into the cerebellum of 6- to 8-week-old MJD mice, which were then followed up to end-stage disease to assess the safety and efficacy of anti-ATXN3 RNAi. Despite effective, lifelong suppression of ATXN3 in the cerebellum and the apparent safety of miRATXN3, motor impairment was not ameliorated in treated MJD mice and survival was not prolonged. These results with an otherwise effective RNAi agent suggest that targeting a large extent of the cerebellum alone may not be sufficient for effective human therapy. Artificial miRNAs or other nucleotide-based suppression strategies targeting ATXN3 more widely in the brain should be considered in future preclinical tests.Molecular Therapy (2013); 21 10, 1898-1908. doi:10.1038/mt.2013.144 [ABSTRACT FROM AUTHOR]