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Ubiquitin controls many cellular processes via its post-translational conjugation onto substrates. Its use is highly variable due to its ability to form poly-ubiquitin with various topologies. Among them, linear chains have emerged as important regulators of immune responses and protein degradation. Previous studies in Drosophila melanogaster found that expression of linear poly-ubiquitin that cannot be dismantled into single moieties leads to their own ubiquitination and degradation or, alternatively, to their conjugation onto proteins. However, it remains largely unknown which proteins are sensitive to linear poly-ubiquitin. To address this question, here we expanded the toolkit to modulate linear chains and conducted ultra-deep coverage proteomics from flies that express non-cleavable, linear chains comprising 2, 4, or 6 moieties. We found that these chains regulate shared and distinct cellular processes in Drosophila by impacting hundreds of proteins. Our results provide key insight into the proteome subsets and cellular pathways that are influenced by linear poly-ubiquitin with distinct lengths and suggest that the ubiquitin system is exceedingly pliable.

Spinocerebellar ataxia type 7 (SCA7) is a progressive neurodegenerative disorder resulting from abnormal expansion of polyglutamine (polyQ) in its disease protein, ataxin-7 (ATXN7). ATXN7 is part of Spt-Ada-Gcn5 acetyltransferase (SAGA), an evolutionarily conserved transcriptional coactivation complex with critical roles in chromatin remodeling, cell signaling, neurodifferentiation, mitochondrial health and autophagy. SCA7 is dominantly inherited and characterized by genetic anticipation and high repeat-length instability. Patients with SCA7 experience progressive ataxia, atrophy, spasticity, and blindness. There is currently no cure for SCA7, and therapies are aimed at alleviating symptoms to increase quality of life. Here, we report novel Drosophila lines of SCA7 with polyQ repeats in wild-type and human disease patient range. We find that ATXN7 expression has age- and polyQ repeat length-dependent reduction in survival and retinal instability, concomitant with increased ATXN7 protein aggregation. These new lines will provide important insight on disease progression that can be used in the future to identify therapeutic targets for SCA7 patients.

Ataxin-3 (Atxn3) is a deubiquitinase with a polyglutamine (polyQ) repeat tract whose abnormal expansion causes the neurodegenerative disease, Spinocerebellar Ataxia Type 3 (SCA3; also known as Machado-Joseph Disease). The ubiquitin chain cleavage properties of Atxn3 are enhanced when it is ubiquitinated at lysine (K) at position 117. K117-ubiqutinated Atxn3 cleaves poly-ubiquitin more rapidly in vitro compared to its unmodified counterpart and this residue is also important for Atxn3 roles in cell culture and in Drosophila melanogaster . How polyQ expansion causes SCA3 remains unclear. To gather insight into the biology of disease of SCA3, here we posited the question: is K117 important for toxicity caused by Atxn3? We generated transgenic Drosophila lines that express full-length, human, pathogenic Atxn3 with 80 polyQ with an intact or mutated K117. We found that K117 mutation mildly enhances the toxicity and aggregation of pathogenic Atxn3 in Drosophila . An additional transgenic line that expresses Atxn3 without any K residues confirms increased aggregation of pathogenic Atxn3 whose ubiquitination is perturbed. These findings suggest Atxn3 ubiquitination as a regulatory step of SCA3, in part by modulating its aggregation.

Spinocerebellar ataxia type 3 (SCA3), also known as Machadoâ€"Joseph disease, is the most common dominantly inherited ataxia. SCA3 is caused by a CAG repeat expansion in the ATXN3 gene that encodes an expanded tract of polyglutamine (polyQ) in the disease protein ataxin-3 (ATXN3). As a deubiquitinating enzyme, ATXN3 regulates numerous cellular processes including proteasome- and autophagy-mediated protein degradation. In SCA3 disease brain, polyQ-expanded ATXN3 accumulates with other cellular constituents, including ubiquitin (Ub)-modified proteins, in select areas like the cerebellum and the brainstem, but whether pathogenic ATXN3 affects the abundance of ubiquitinated species is unknown. Here, in mouse and cellular models of SCA3, we investigated whether elimination of murine Atxn3 or expression of wild-type or polyQ-expanded human ATXN3 alters soluble levels of overall ubiquitination, as well as K48-linked (K48-Ub) and K63-linked (K63-Ub) chains. Levels of ubiquitination were assessed in the cerebellum and brainstem of 7- and 47-week-old Atxn3 knockout and SCA3 transgenic mice, and also in relevant mouse and human cell lines. In older mice, we observed that wild-type ATXN3 impacts the cerebellar levels of K48-Ub proteins. In contrast, pathogenic ATXN3 leads to decreased brainstem abundance of K48-Ub species in younger mice and changes in both cerebellar and brainstem K63-Ub levels in an age-dependent manner: younger SCA3 mice have higher levels of K63-Ub while older mice have lower levels of K63-Ub compared to controls. Human SCA3 neuronal progenitor cells also show a relative increase in K63-Ub proteins upon autophagy inhibition. We conclude that wild-type and mutant ATXN3 differentially impact K48-Ub- and K63-Ub-modified proteins in the brain in a region- and age-dependent manner.