Impact of N-Terminal Acetylation on α-Synuclein Amyloid Formation

Abstract

The accumulation of α-synuclein (αSyn) as β-sheet-rich amyloid in Lewy bodies is one of the hallmarks of Parkinson's disease. Recent data have demonstrated that αSyn is constitutively N-terminally acetylated in mammalian cells, a co-translational modification that has been shown to increase α-helical propensity in the N-terminal region of the protein and modulate membrane-binding behavior. The results of in vitro experiments comparing the amyloid formation kinetics of acetylated (Ac-αSyn) and the more widely studied non-acetylated a-synuclein (NH3-αSyn) have been contradictory. However, these assays largely rely on the extrinsic dye thioflavin T (ThT), which displays a dramatic increase in quantum yield upon binding to the cross-β sheet structure in amyloids. We find that the ThT response is approximately tenfold lower in Ac-αSyn, and that Ac-αSyn aggregates more slowly than NH3-αSyn at physiological pH. Intrinsic fluorescence of Trp mutants confirms that ThT emission accurately reports on the kinetics of amyloid formation for both forms of the protein. Seeding experiments indicate that the ThT fluorescence intensity is determined by the final fibrillar structure templated from the seeds rather than the presence or absence of the N-terminal acetyl group. Structural differences of the de novo and seeded fibrils are corroborated by Raman spectroscopy, limited proteolysis experiments, and transmission electron microscopy. These results indicate that N-terminal acetylation has important consequences for the aggregation propensity and amyloid structure of αSyn, and that nonacetylated αSyn is not a substitute for the biological form of the protein.