Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells IMPLICATIONS FOR AGGREGATION AND CELLULAR PROPERTIES OF α-SYNUCLEIN

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