Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Aggregates of α-synuclein (αS) are hallmarks of synucleinopathies, including Parkinson’s Disease (PD) and Multiple System Atrophy (MSA). We have recently shown that αS lysine acetylation in the soluble monomer pool varies between healthy controls, PD, and MSA patients. To study the effects of lysine acetylation at all disease-relevant sites of αS, we first compared production of acetylated αS through either native chemical ligation or non-canonical amino acid (ncAA) mutagenesis. Since yields were comparable, ncAA mutagenesis was deemed superior for scanning many acetylation sites. We expressed and purified 12 disease-relevant variants and studied their binding to membranes as well as their aggregation propensities, and found that acetylation of lysine 12, 43, and 80 had particularly strong effects. To understand the implications for acetylation of monomeric αS found in healthy cells, we performed NMR experiments to study protein conformation and fluorescence correlation spectroscopy experiments to quantify lipid binding. We also investigated the effects of acetylation at lysine 12, 43, and 80 on fibril seeding in neurons. Collectively, our biochemical and cell biological investigations indicated that acetylation of K ₈₀ could inhibit aggregation without conferring negative effects on monomer function in healthy cells. Therefore, we studied the structures of fibrils with K ₈₀ acetylation through cryo-electron microscopy to uncover the structural basis for these effects. Finally, we identified inhibition of HDAC8 as a way of potentially increasing acetylation at K ₈₀ and other inhibitory sites for therapeutic benefit.