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

Research output: Contribution to journalArticle

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Abstract

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
Volume287
Issue number34
DOIs
Publication statusPublished - 17 Aug 2012
Externally publishedYes

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Synucleins
Cell Aggregation
Acetylation
Agglomeration
Proteins
Escherichia coli
Membranes
Post Translational Protein Processing
HeLa Cells
Sedimentation
Ligation
Parkinson Disease
In Vitro Techniques
Assays
Physical properties
Cells
Nuclear magnetic resonance
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

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

In: Journal of Biological Chemistry, Vol. 287, No. 34, 17.08.2012, p. 28243-28262.

Research output: Contribution to journalArticle

Fauvet, Bruno ; Fares, Mohamed Bilal ; Samuel, Filsy ; Dikiy, Igor ; Tandon, Anurag ; Eliezer, David ; Lashuel, Hilal A. / 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. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 34. pp. 28243-28262.
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AU - Fares, Mohamed Bilal

AU - Samuel, Filsy

AU - Dikiy, Igor

AU - Tandon, Anurag

AU - Eliezer, David

AU - Lashuel, Hilal A.

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