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

Bruno Fauvet, Mohamed Bilal Fares, Filsy Samuel, Igor Dikiy, Anurag Tandon, David Eliezer, Hilal A. Lashuel

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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), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively N α-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of N α- acetylation on the biophysical and biological properties of α-syn, we produced N α-acetylated α-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 α-syn. We demonstrate that both WT and N α-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing N α- acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both N α-acetylated and WT α-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 α- acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn N α- acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells.

Original languageEnglish
Pages (from-to)28243-28262
Number of pages20
JournalJournal of Biological Chemistry
Issue number34
Publication statusPublished - 17 Aug 2012


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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