Characterizing the role of the polyglutamine neurodegenerative protein ataxin-3 in the ubiquitin-proteasome pathway

Spinocerebellar ataxia type 3/Machado Joseph disease is a dominantly inherited neurodegenerative disease caused by expansion of a polyglutamine domain in the protein ataxin-3 (AT3). In normal individuals the gene contains 12--40 CAG repeats expanding to 55--86 repeats in clinically diagnosed MJD patients. Expansion of the glutamine domain in AT3 as well as in at least 8 other members of the polyglutamine neurodegenerative disease family increases protein misfolding resulting in aggregation and formation of nuclear inclusions. At the start of this thesis the normal physiological function of AT3 was unknown. A bioinformatic study revealed that AT3 contained multiple ubiquitin interacting motifs (UIMs) providing a potential link between AT3 and the ubiquitin-proteasome pathway. The objective of my thesis was to determine if AT3 UIMs were functional and then determine if AT3 had a role in the ubiquitin-proteasome pathway. Studies presented in this thesis show that AT3 is a deubiquitylating enzyme that may be involved in protein quality control. AT3 has several properties characteristic of deubiquitylating enzymes including removing ubiquitin from polyubiquitin chains, on 125I-lysozyme, and cellular proteins, cleaving a ubiquitin protease substrate, and binding the specific ubiquitin protease inhibitor, ubiquitin-aldehyde. Mutating the predicted catalytic cysteine in AT3 inhibits each of these ubiquitin protease activities. The ability to bind and cleave ubiquitylated proteins is consistent with AT3 playing a role in the ubiquitin-proteasome system. Cellular studies show that endogenous AT3 co-localizes with aggresomes and preaggresome particles of misfolded cystic fibrosis transmembrane regulator mutant, CFTRDeltaF508, and associates with histone deacetylase 6 (HDAC6) and dynein, proteins required for aggresome formation and transport of misfolded protein. SiRNA knockdown of AT3 greatly reduces aggresomes formed by CFTRDeltaF508 demonstrating a critical role of AT3 in this process. These and other data indicate that both the deubiquitylating activity of AT3 as well as its UIMs play essential roles in CFTRDeltaF508 aggresome formation and raises the possibility that pathological AT3, which tends to misfold and aggregate, may be exposed to aggregate-prone misfolded/denatured proteins as part of its normal function.