CD95 pp 189-198 | Cite as

Detection of S-Acylated CD95 by Acyl-Biotin Exchange

  • Aurelie Rossin
  • Anne-Odile HueberEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1557)


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.

Key words

S-acylation S-palmitoylation Hydroxylamine Acyl-biotin exchange 


  1. 1.
    Linder ME, Deschenes RJ (2007) Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol 8(1):74–84CrossRefPubMedGoogle Scholar
  2. 2.
    Chamberlain LH, Shipston MJ (2015) The physiology of protein S-acylation. Physiol Rev 95(2):341–376CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Tsutsumi R, Fukata Y, Fukata M (2008) Discovery of protein-palmitoylating enzymes. Pflugers Arch 456(6):1199–1206CrossRefPubMedGoogle Scholar
  4. 4.
    Greaves J, Chamberlain LH (2011) DHHC palmitoyl transferases: substrate interactions and (patho)physiology. Trends Biochem Sci 36(5):245–253CrossRefPubMedGoogle Scholar
  5. 5.
    Drisdel RC, Green WN (2004) Labeling and quantifying sites of protein palmitoylation. Biotechniques 36(2):276–285PubMedGoogle Scholar
  6. 6.
    Martin BR, Wang C, Adibekian A, Tully SE, Cravatt BF (2012) Global profiling of dynamic protein palmitoylation. Nat Methods 9(1):84–89CrossRefGoogle Scholar
  7. 7.
    Wan J, Roth AF, Bailey AO, Davis NG (2007) Palmitoylated proteins: purification and identification. Nat Protoc 2(7):1573–1584CrossRefPubMedGoogle Scholar
  8. 8.
    Chakrabandhu K et al (2007) Palmitoylation is required for efficient Fas cell death signaling. EMBO J 26(1):209–220CrossRefPubMedGoogle Scholar
  9. 9.
    Feig C, Tchikov V, Schütze S, Peter ME (2007) Palmitoylation of CD95 facilitates formation of SDS-stable receptor aggregates that initiate apoptosis signaling. EMBO J 26(1):221–231CrossRefPubMedGoogle Scholar
  10. 10.
    Guardiola-Serrano F et al (2010) Palmitoylation of human FasL modulates its cell death-inducing function. Cell Death Dis 1:e88CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    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
  12. 12.
    Utsumi T et al (2001) Transmembrane TNF (pro-TNF) is palmitoylated. FEBS Lett 500(1–2):1–6CrossRefPubMedGoogle Scholar
  13. 13.
    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
  14. 14.
    Peter ME et al (2007) The CD95 receptor: apoptosis revisited. Cell 129(3):447–450CrossRefPubMedGoogle Scholar
  15. 15.
    Rossin A et al (2015) Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability. Cell Death Differ 22(4):643–653CrossRefPubMedGoogle Scholar
  16. 16.
    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
  17. 17.
    Jaffrey SR, Snyder SH (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 2001(86):pl1PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR 1091Université Côte d’AzurNiceFrance
  2. 2.Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR 1091,Université de NiceNiceFrance

Personalised recommendations