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.
Rossin A, Derouet M, Abdel-Sater F, Hueber AO (2009) Palmitoylation of the TRAIL receptor DR4 confers an efficient TRAIL-induced cell death signalling. Biochem J 419(1):185–192, 182 p following 192CrossRefPubMedGoogle Scholar
Berg V et al (2015) miRs-138 and -424 control palmitoylation-dependent CD95-mediated cell death by targeting acyl protein thioesterases 1 and 2 in CLL. Blood 125(19):2948–2957CrossRefPubMedPubMedCentralGoogle Scholar
Rossin A et al (2015) Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability. Cell Death Differ 22(4):643–653CrossRefPubMedGoogle Scholar
Leon-Bollotte L et al (2011) S-nitrosylation of the death receptor fas promotes fas ligand-mediated apoptosis in cancer cells. Gastroenterology 140(7):2009–2018, 2018.e2001–2004CrossRefPubMedGoogle Scholar
Jaffrey SR, Snyder SH (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 2001(86):pl1PubMedGoogle Scholar