Effects of Familial Mutation and C-Terminal Truncation on Nucleation and Fibril Elongation of α-Synuclein

Abstract

Aggregation of α-synuclein (α-syn) into amyloid fibrils is closely associated with Parkinson's disease (PD). Familial mutations or posttranslational truncations in α-syn are known as risk factor for PD. An A53T mutation is most frequent and links to early-onset PD. in human brain with PD, α-syn is specifically truncated at N103 by asparagine endopeptidase, and the resulting C-terminal-truncated fragment Δ104-140 has high aggregation propensity and neurotoxicity. Here, we examined the physicochemical mechanism of aggregation of PD-related α-syn variants, A53T and Δ104-140, using kinetic and thermodynamic analyses.α-Syn aggregation was monitored by amyloid-specific dye, thioflavin T (ThT), and the kinetics of the ThT fluorescence increase were analyzed by Finke-Watzky 2-step model of a homogeneous nucleation followed by autocatalytic heterogeneous fibril elongation. At physiological condition (20 μM α-syn, pH 7.4, 37 oC), both A53T and Δ104-140 variants aggregated faster than WT α-syn, in which the A53T mutation markedly increases nucleation rate whereas the C-terminal truncation significantly increases both nucleation and fibril elongation rates. Analysis of the dependence of aggregation reaction of the variants on the monomer concentration suggested that the A53T mutation enhances conversion of monomers to amyloid nuclei whereas the C-terminal truncation enhances autocatalytic aggregation on existing fibrils. in addition, thermodynamic analysis of the kinetics of nucleation and fibril elongation of the variants indicated that both nucleation and fibril elongation of WT α-syn are enthalpically and entropically unfavorable. interestingly, the unfavorable activation enthalpy of nucleation is greatly reduced by the A53T mutation, and becomes reversed in sign for the Δ104-140 variant.Taken together, our results indicate that the A53T mutation and the C-terminal truncation enhance α-syn aggregation by reducing unfavorable activation enthalpy of nucleation, and suggest that the C-terminal truncation further triggers the autocatalytic fibril elongation on the fibril surfaces.