Polyglutamine Expansion in Ataxin-3 Does Not Affect Protein Stability

Abstract

Polyglutamine proteins that cause neurodegenerative disease are known to form proteinaceous aggregates, such as nuclear inclusions, in the neurons of affected patients. Although polyglutamine proteins have been shown to form fibrillar aggregates in a variety of contexts, the mechanisms underlying the aberrant conformational changes and aggregation are still not well understood. In this study, we have investigated the hypothesis that polyglutamine expansion in the protein ataxin-3 destabilizes the native protein, leading to the accumulation of a partially unfolded, aggregation-prone intermediate. To examine the relationship between polyglutamine length and native state stability, we produced and analyzed three ataxin-3 variants containing 15, 28, and 50 residues in their respective glutamine tracts. At pH 7.4 and 37°C, Atax3(Q50), which lies within the pathological range, formed fibrils significantly faster than the other proteins. Somewhat surprisingly, we observed no difference in the acid-induced equilibrium and kinetic un/folding transitions of all three proteins, which indicates that the stability of the native conformation was not affected by polyglutamine tract extension. This has led us to reconsider the mechanisms and factors involved in ataxin-3 misfolding, and we have developed a new model for the aggregation process in which the pathways of un/folding and misfolding are distinct and separate. Furthermore, given that native state stability is unaffected by polyglutamine length, we consider the possible role and influence of other factors in the fibrillization of ataxin-3.

The Two-stage Pathway of Ataxin-3 Fibrillogenesis Involves a Polyglutamine-independent Step

“…2B). Even upon incubation times of up to 200 h, at3(QHQ) and at3(Q15) did not form SDS-resistant aggregates, suggesting that under physiologically relevant conditions ataxin-3 forms SDS-resistant aggregates in a poly(Q) length-dependent manner similar to that seen in other poly(Q) proteins (15)(16)(17)(18)(19).…”

“…Characterization of Ataxin-3 Variants-We expressed and purified three ataxin-3 variants from Escherichia coli as previously described (19) (Fig. 1A).…”

“…Expression and Purification of Ataxin-3 Variants-All ataxin-3 variants were expressed and purified as described previously (19). Protein was stored at Ϫ80°C and run on a Superose 12 column prior to each experiment to confirm it was monomeric.…”

“…We and others have demonstrated that under physiologically relevant conditions expanded ataxin-3 can form amyloid-like fibrils (19,28). Furthermore, under certain conditions non-expanded ataxin-3 can also form amyloid-like fibrils and their precursors (29 -33).…”

“…In vitro studies have shown that poly(Q) peptides, fragments, and proteins can form amyloid-like fibrillar aggregates and that the aggregation rate increases with increasing glutamine tract length (15)(16)(17)(18)(19). Various types of intraneuronal aggregates, including nuclear inclusions, of the poly(Q) protein in question are also evident upon post-mortem analysis (20 -22).…”