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Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry.
- 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
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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 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.
- Subjects :
- Acetylation
Amino Acid Sequence
Animals
COS Cells
Chlorocebus aethiops
Enzymes metabolism
Humans
Intracellular Space metabolism
Jurkat Cells
Lipoylation
Mice
Molecular Sequence Data
Reproducibility of Results
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Substrate Specificity
Time Factors
ras Proteins chemistry
ras Proteins metabolism
Alkynes chemistry
Fatty Acids chemistry
Fatty Acids metabolism
Proteins chemistry
Proteins metabolism
Subjects
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