title: Detection of S-Acylated CD95 by Acyl-Biotin Exchange.
abstract: S-acylation is the covalent addition of a fatty acid, most generally palmitate onto cysteine residues of proteins through a labile thioester linkage. The death receptor CD95 is S-palmitoylated and this post-translational modification plays a crucial role on CD95 organization in cellular membranes and thus on CD95-mediated signaling. Here, we describe the nonradioactive detection of CD95 S-acylation by acyl-biotin exchange chemistry in which a biotin is substituted for the CD95-linked fatty acid. This sensitive technique, which depends on the ability of hydroxylamine to specifically cleave the thioester linkage between fatty acids and proteins, relies on three chemical steps: (1) blockage of free thiols of non-modified cysteine residues, (2) hydroxylamine-mediated cleavage of thioester-linked fatty acids to restore free thiols and (3) biotinylation of free thiols with a thiol reactive biotinylation agent. Resulting biotinylated proteins can be easily purified by an avidin capture and analyzed by SDS-PAGE and immunoblotting.
authors.affiliation: Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR 1091, Université Côte d'Azur, Parc Valrose, Bâtiment des Sciences Naturelles, 06108, Nice, France.
authors.forename: Aurelie
authors.initials: A
authors.lastname: Rossin
authors.affiliation: Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR 1091,Université de Nice, Nice, France. hueber@unice.fr.
authors.forename: Anne-Odile
authors.initials: AO
authors.lastname: Hueber
mesh_terms: D000215:Acylation; D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006898:Hydroxylamines; D054878:Lipoylation; D019014:fas Receptor / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006898:Hydroxylamines; D019014:fas Receptor; D001710:Biotin
keywords: Acyl-biotin exchange; Hydroxylamine; S-acylation; S-palmitoylation