Abstract
The pathology of spinocerebellar ataxia type 3, also known as Machado‐Joseph disease, is triggered by aggregation of toxic ataxin‐3 (ATXN3) variants containing expanded polyglutamine repeats. The physiological role of this deubiquitylase, however, remains largely unclear. Our recent work showed that ATX‐3, the nematode orthologue of ATXN3, together with the ubiquitin‐directed segregase CDC‐48, regulates longevity in Caenorhabditis elegans. Here, we demonstrate that the long‐lived cdc‐48.1; atx‐3 double mutant displays reduced viability under prolonged starvation conditions that can be attributed to the loss of catalytically active ATX‐3. Reducing the levels of the autophagy protein BEC‐1 sensitized worms to the effect of ATX‐3 deficiency, suggesting a role of ATX‐3 in autophagy. In support of this conclusion, the depletion of ATXN3 in human cells caused a reduction in autophagosomal degradation of proteins. Surprisingly, reduced degradation in ATXN3‐depleted cells coincided with an increase in the number of autophagosomes while levels of lipidated LC3 remained unaffected. We identified two conserved LIR domains in the catalytic Josephin domain of ATXN3 that directly interacted with the autophagy adaptors LC3C and GABARAP in vitro. While ATXN3 localized to early autophagosomes, it was not subject to lysosomal degradation, suggesting a transient regulatory interaction early in the autophagic pathway. We propose that the deubiquitylase ATX‐3/ATXN3 stimulates autophagic degradation by preventing superfluous initiation of autophagosomes, thereby promoting an efficient autophagic flux important to survive starvation.
Highlights
Ataxin‐3 (ATXN3), which is the founding member of the family of Josephin domain‐containing deubiquitylases (DUBs), plays a critical role in the maintenance of protein homeostasis and stress resistance (Burnett & Pittman, 2005; Durcan et al, 2011; Scaglione et al, 2011; Wang, Li, & Ye, 2006; Zhong & Pittman, 2006)
We demonstrate that the long‐lived cdc‐48.1; atx‐3 double mutant displays reduced viability under prolonged starvation conditions that can be attributed to the loss of catalytically active ATX‐3
In agreement with a new physiological role of ATXN3 in autophagy, we show that ATXN3 is an LC3C/GABARAP‐interacting DUB that regulates the formation of autophagosomes independent from its stabilizing effect on beclin‐1 (Ashkenazi et al, 2017)
Summary
Ataxin‐3 (ATXN3), which is the founding member of the family of Josephin domain‐containing deubiquitylases (DUBs), plays a critical role in the maintenance of protein homeostasis and stress resistance (Burnett & Pittman, 2005; Durcan et al, 2011; Scaglione et al, 2011; Wang, Li, & Ye, 2006; Zhong & Pittman, 2006). We observed that the partial depletion of unc‐51, the nematode orthologue of ULK1, which is the upstream Ser/Thr protein kinase involved in the activation of autophagy in C. elegans, further aggravated the mortality of atx‐3 mutant worms (Figure S1A, Table S5) Another process that strongly depends on functional autophagy and that is of direct relevance for polyQ diseases is the clearance of aggregation‐prone proteins (Ravikumar et al, 2004). The depletion of ATXN3 with either siRNA resulted in a significant increase in GFP‐LC3 puncta when lysosomal degradation was blocked by bafilomycin A1 treatment under basal and nutrient‐starved conditions (Figure 2d,e) This increase suggests that more autophagic structures were formed in ATXN3‐depleted cells during the treatment. We conclude that ATXN3 is an LC3C/GABARAP‐interacting protein that is recruited to autophagosomes in a transient fashion, where it regulates their formation and stimulates the autophagic flux
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