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

Authors :
Yap MC
Kostiuk MA
Martin DD
Perinpanayagam MA
Hak PG
Siddam A
Majjigapu JR
Rajaiah G
Keller BO
Prescher JA
Wu P
Bertozzi CR
Falck JR
Berthiaume LG
Source :
Journal of lipid research [J Lipid Res] 2010 Jun; Vol. 51 (6), pp. 1566-80. Date of Electronic Publication: 2009 Dec 21.
Publication Year :
2010

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 omega-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 fluorogenic 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, omega-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with omega-alkynyl-myristate or omega-alkynyl-stearate, via an alkali sensitive thioester bond. Third, omega-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of omega-alkynyl-palmitate and omega-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with omega-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 omega-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The omega-alkynyl-myristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo.

Details

Language :
English
ISSN :
1539-7262
Volume :
51
Issue :
6
Database :
MEDLINE
Journal :
Journal of lipid research
Publication Type :
Academic Journal
Accession number :
20028662
Full Text :
https://doi.org/10.1194/jlr.D002790