| dc.description.abstract | Autosomal Recessive Cerebellar Ataxia (ARCA) develops as a result of inefficient protein turnover and further accumulation of damaged proteins inside the cells. Some of mutations associated with ARCA are identified in the STUB1 gene which encodes CHIP (C-terminus of Heat shock protein 70-Interacting Protein) as a dimeric co-chaperone and E3 ubiquitin ligase protein. Currently, there is limited information available regarding the role of these mutations in the pathogenesis of ARCA. This study aims to characterize the effects of six selected mutations on the structure and function of CHIP, mainly by the use of in vitro methods. Mutant CHIP constructs were created by site-directed mutagenesis, and recombinant proteins were subjected to different assays including ubiquitination activity assay, limited proteolysis, oligomerization analysis, and circular dichroism spectroscopy. Results indicated that mutations N65S and T246M were associated with more severe consequences compared to the rest (E28K, K145Q, M 211I, and S236T). Impaired Hsc70-ubiquitination activity was observed for both N65S and T246M. The N65S mutation resulted in more stabilized and compact protein structure, while the T246M mutant presented more flexible and loose structures with a high tendency for aggregation. The other mutations investigated were associated with intact ubiquitination activity, but showed lower protein stability and loss of some secondary structures, although to a lesser extent when compared to T246M. Altogether, this study provides insights into the development of ARCA through demonstrating the effect of STUB1 mutations in the expression of damaged CHIP proteins unable to participate efficiently in the protein turnover system, which may lead to toxic accumulation of abnormal proteins and cell death. | en_US |
