Impact of the Acidic C-Terminal Region Comprising Amino Acids 109−140 on α-Synuclein Aggregation in Vitro

Abstract

The aggregation of α-synuclein, involved in the pathogenesis of several neurodegenerative disorders such as Parkinson's disease, is enhanced in vitro by biogenic polyamines binding to the highly charged C-terminal region aa109−140. In this study, we investigated the influence of this region on the aggregation kinetics, monitored by thioflavin T binding and static light scattering, and morphology, assessed by electron microscopy, fluorescence microscopy, and turbidity, by comparing the effect of various solution conditions on the wild-type protein, the disease related mutants A53T and A30P, and two truncated variants, syn(1−108) and syn(1−124), lacking the complete or the C-terminal half of the polyamine binding site. In the presence of the intact C-terminus, aggregation was strongly retarded in physiological buffer. This inhibition of aggregation was overridden by (i) addition of spermine or MgCl₂ or lowering of pH, leading to strong charge shielding in the C-terminus or (ii) by truncation of aa125−140 or aa109−140. Addition of MgCl₂ or spermine or acidification were not effective in promoting aggregation of syn(1−108). The impact of the disease-related mutations on the aggregation kinetics was dependent on the solution conditions, with the aggregation propensity order A53T ∼ wt > A30P at low ionic strength, but A53T > wt ∼ A30P at high ionic strength, with exceedingly potent promotion of aggregation by the A53T mutation in the presence of spermine. In contrast to full-length α-synuclein aggregates, those formed from syn(1−108) did not exhibit a pronounced polymorphism. The effects of the C-terminus on aggregation cannot be rationalized merely by a contribution to the protein net charge, but rather suggest a specific role of aa109−140 in the regulation of aggregation, presumably involving formation of intramolecular contacts.