Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry

Megan C. Yap, Morris A. Kostiuk, Dale D.O. Martin, Maneka A. Perinpanayagam, Pieter G. Hak, Anjaiah Siddam, Janaki R. Majjigapu, Gurram Rajaiah, Bernd O. Keller, Jennifer A. Prescher, Peng Wu, Carolyn R. Bertozzi, John R. Falck, Luc G. Berthiaume

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Abstract

Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identification methods. We first report that a synthetic ω-alkynyl-palmitate analog can be readily and specifically incorporated into GAPDH or mitochondrial 3-hydroxyl-3- methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fl uorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or flat bed fluorescence scanning. The acylated cysteine residues were confirmed by MS. Second, ω-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with ω-alkynyl-myristate or ω-alkynyl-stearate, via an alkali sensitive thioester bond. Third, ω-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of ω-alkynylpalmitate and ω-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with ω-alkynyl- palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H- and N-Ras could be easily detected using ω-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The ω-alkynylmyristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo.

Original languageEnglish (US)
Pages (from-to)1566-1580
Number of pages15
JournalJournal of Lipid Research
Volume51
Issue number6
DOIs
StatePublished - Jun 1 2010

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Keywords

  • Enzymes
  • Membranes
  • Mitochondria
  • Myristoylation
  • Palmitoylation

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

Cite this

Yap, M. C., Kostiuk, M. A., Martin, D. D. O., Perinpanayagam, M. A., Hak, P. G., Siddam, A., Majjigapu, J. R., Rajaiah, G., Keller, B. O., Prescher, J. A., Wu, P., Bertozzi, C. R., Falck, J. R., & Berthiaume, L. G. (2010). Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry. Journal of Lipid Research, 51(6), 1566-1580. https://doi.org/10.1194/jlr.D002790