POST-DOCTORAL FELLOWSHIP Award

Thomas, M. Durcan, PhD

Montreal Neurological Institute, Quebec, Canada
Investigate the DUB activity of Ataxin-3 and its effect on parkin stability and activity

Machado Joseph disease (MJD)/Spinocerebellar ataxia-3 (SCA3) is the most common dominantly inherited ataxia worldwide. Screening for the disease causing gene led to the identification of ataxin-3, a gene identified as containing an expanded polyQ tract (>50Q) in MJD. Further studies identified ataxin-3 as a deubiquitinating enzyme, with this activity to remove Ub chains from a substrate protein residing in its N-terminal Josephin domain. Moreover, ataxin-3 contains 3 ubiquitin interacting motifs (UIMs), permitting ataxin-3 to bind Ub and ubiquitinated proteins. Both the UIMs and the josephin domain indicate that ataxin-3 plays a role within the Ub proteasome system (UPS). Yet, until recently little has been determined about the role of ataxin-3 in the UPS and if the presence of the polyQ expansion in MJD could affect the normal function of ataxin-3 in the UPS. To first understand the role of ataxin-3 within the UPS, it was critical to first identify a substrate that ataxin-3 could deubiquitinate. Recent studies in our lab identified parkin, an E3 ligase, as a novel substrate for ataxin-3 mediated deubiquitination. Parkin plays an opposing role to ataxin-3 in the UPS, possessing the ability to form Ub conjugates on substrate proteins and itself. Moreover, mutations in parkin account for a common familial form of PD. Identification of parkin as a substrate for ataxin-3 mediated deubiquitination was of interest to us as a result of previous studies, in which patients with MJD presented with overlapping symptoms of PD, suggesting a potential overlap between both diseases. For this study, we propose to examine further how ataxin-3 and parkin functionally interact and how this could potentially lead to parkinsonian symptoms in MJD patients. Previous studies observed ataxin-3 interacting directly with parkin through two interaction domains, facilitating direct deubiquitination of self-ubiquitinated parkin. Moreover, ataxin-3 was observed to deubiquitinate parkin in real-time, targeting Ub linkages as they are forming on parkin. Furthermore, by targeting Ub conjugates on parkin, ataxin-3 appears to regulate parkin levels with over expression of ataxin-3 causing a decrease in parkin levels and vice versa. Yet, what remains unclear is what Ub linkages are targeted by ataxin-3 to regulate parkin levels. The best characterized linkages studied are K48 linkages, observed to target substrate proteins for degradation and K63 linkages, observed to protect proteins from being targeted for degradation. In this study, we hypothesize that parkin self-ubiquitinates using primarily K63 Ub linkages, consistent with other studies. Moreover, we speculate that ataxin-3 targets K63 linkages leading to a shift in the ratio of Ub linkages on parkin towards K48 linkages, with increased targeting of parkin for degradation. To examine this in detail, a combination of in vitro ubiquitination assays and mass spectrometry analysis will be performed. In addition to providing the first insight into how ataxin-3 deubiquitinates a substrate protein and the linkages targeted by ataxin-3, we propose to examine how ataxin-3 mediated deubiquitination of parkin can affect its stability and activity towards other substrate proteins using both in vitro and in vivo assays. By examining the effect of ataxin-3 on aspects of parkin function and stability, this study will help us understand how parkin and ataxin-3 functionally interact for the first time. Given that both proteins play
opposing roles in the UPS and play a role in PD and MJD respectively, it is imperative to understand how the disease form of ataxin-3 could affect parkin function. Such findings could possibly provide an explanation for the overlapping parkinsonian symptoms observed in many MJD patients and delineate a novel mechanism in the pathogenesis of MJD.