Characterization of semisynthetic and naturally Nα-acetylated α-synuclein in vitro and in intact cells: implications for aggregation and cellular properties of α-synuclein

摘要: N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), that has been linked to the pathogenesis of Parkinson disease, constitutively Nα-acetylated vivo. Nevertheless, most biochemical biophysical studies on structure, aggregation, α-syn vitro utilize recombinant from Escherichia coli, which not N-terminally acetylated. To elucidate effect Nα-acetylation biological α-syn, we produced first using semisynthetic methodology based expressed ligation (Berrade, L., Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909–3922) then expression strategy, compare unacetylated α-syn. We demonstrate both WT share similar secondary structure oligomeric state purified preparations in-cell NMR E. coli overexpressing The two proteins have close aggregation propensities as shown by thioflavin T binding sedimentation assays. Furthermore, exhibited ability bind synaptosomal membranes HeLa cells, where internalized prominent cytosolic subcellular distribution. determined attenuating living nonacetylable mutant silencing enzyme responsible for Nα-acetylation. Both approaches revealed distribution membrane suggesting does significantly affect intact cells.