From quality control to neurodegeneration : regulation of autophagy and the DNA damage response by ubiquitin-modifying enzymes

Protein homeostasis and genome integrity are safeguarded by a variety of cellular quality control pathways. While protein quality is controlled by a delicate balance between protein biosynthesis, folding and degradation, the DNA is subject to tightly regulated surveillance processes that sense, signal and repair DNA lesions. Many of these processes are regulated by the conjugation of the small modifier ubiquitin to substrates and effector proteins. A polyglutamine-repeat expansion in the gene encoding the deubiquitylating enzyme ataxin-3 is the underlying cause of the neurodegenerative disorder Machado-Joseph disease. Similar to other polyglutamine disorders, the disease is characterized by intracellular inclusions of the mutant protein. Interestingly, wild-type ataxin-3, which disassembles both K63-linked and K48-linked ubiquitin chains, has been reported to be neuroprotective. By means of its catalytic activity, it suppresses toxic aggregation of polyglutamine proteins, including its own mutant counterpart. Ataxin-3 has further been implicated in cellular pathways that regulate protein quality control, transcription and DNA repair. The work presented in this thesis identified additional functions of ataxin-3 in cellular quality control. In paper I, we identified ataxin-3 as a novel interactor of the ubiquitin-like autophagy proteins LC3C and GABARAP and demonstrated that ataxin-3 is required for efficient autophagic degradation in both the nematode Caenorhabditis elegans and mammalian cells. Loss of ataxin-3 did not only result in aberrant accumulation of autophagic vesicles in mammalian cells but also decreased survival of nematodes upon nutrient deprivation, a condition that relies on functional autophagy for survival, and aggravated the accumulation of protein aggregates and aggregate-related motility defects. In paper II, we engineered an inducible, K63-specific ubiquitin ligase and an associated reference substrate, which allow studying the signaling capacity of K63-linked ubiquitin chains in different cellular contexts. This chain type has frequently been linked to inclusion bodies, autophagic degradation and neurodegeneration. Using a mitochondria-localized substrate, we demonstrate that K63-linked ubiquitylation is sufficient to induce perinuclear clustering of the mitochondria, even in the absence of mitochondrial damage, a phenotype that has previously only been described during clearance of damaged mitochondria by mitophagy. In paper III we identified a novel role for ataxin-3 in the regulation of the cellular response to DNA double-strand breaks. We demonstrate that ataxin-3 is recruited to DNA lesions and consolidates the DNA damage response by preventing premature chromatin-extraction of DNA repair proteins by the SUMO-targeted E3 ubiquitin ligase RNF4. We show that ataxin3 counteracts RNF4-mediated MDC1-ubiquitylation and chromatin-extraction, promoting recruitment of RNF8 and RNF168 and subsequent damage-induced ubiquitin signaling. Consequently, loss of ataxin-3 impaired recruitment of the repair factors 53BP1 and BRCA1 and therefore both non-homologous end-joining and homologous recombination. Similar to the recruitment of RNF4, recruitment of ataxin-3 to DNA lesions was dependent on damageinduced SUMOylation. In paper IV we show that ataxin-3 recruitment, in contrast to RNF4, additionally depends on DNA damage-induced poly(ADP-ribos)ylation, thereby restricting the actions of ataxin-3 to the early phase of the DNA damage response. Differential recruitment of ataxin-3 and RNF4 to DNA double-strand breaks thereby promotes the sequential actions of these enzymes on shared substrates and explains how both proteins promote efficient DNA repair despite their opposing activities. Collectively, the studies presented in this thesis corroborate the importance of ataxin-3 for maintaining genome and protein quality control and furthermore underline the importance of considering the homeostatic functions of wild-type ataxin-3 in the design of therapeutic strategies for Machado-Joseph disease. Additionally, the data presented in this thesis demonstrate the significance of K63-linked ubiquitylation in mediating mitochondrial clustering, not unlike its function in inclusion body formation, and provide a tool to further study the cellular function of these ubiquitin chains. LIST OF SCIENTIFIC PAPERS I. Herzog LK, Kevei É, Marchante R, Böttcher C, Bindesbøll C, Lystad AH, Pfeiffer A, Gierisch ME, Salomons FA, Simonsen A, Hoppe T and Dantuma NP. (2020). The Machado-Joseph disease deubiquitylase ataxin-3 interacts with LC3C/GABARAP and promotes autophagy. Aging Cell, 19:e13051 II. Richard TJC, Herzog LK, Suryo Rahmanto A, Sangfelt O, Salomons FA and Dantuma NP. An inducible, engineered K63-ubiquitin ligase mimics Parkin-mediated sequestration of mitochondria in the absence of mitochondrial damage. Manuscript (submitted) III. Pfeiffer A*, Luijsterburg MS*, Acs K, Wiegant WW, Helfricht A, Herzog LK, Minoia M, Böttcher C, Salomons FA, van Attikum H and Dantuma NP. (2017). Ataxin-3 consolidates the MDC1-dependent DNA double-strand break response by counteracting the SUMO-targeted ubiquitin ligase RNF4. The EMBO Journal, 36, 1066-1083 IV. Pfeiffer A, Herzog LK, Luijsterburg MS, Shah RG, Stoy H, Kühbacher U, van Attikum H, Shah GM and Dantuma NP. Poly(ADP-ribos)ylation limits SUMOdependent ataxin-3 recruitment to DNA double-strand breaks to the early phase of the DNA damage response. Manuscript (submitted) * these authors contributed equally PUBLICATIONS NOT INCLUDED IN THIS THESIS I. Dantuma NP and Herzog LK. (2020). Machado-Joseph Disease: A StressCombating Deubiquitylating Enzyme Changing Sides. In: Barrio R. , Sutherland J., Rodriguez M. (eds) Proteostasis and Disease. Advances in Experimental Medicine and Biology, vol 1233: 237–260. Springer, Cham CONTENTS