Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry.
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.
Yap
Megan C
MC
Department of Cell Biology, Faculty of MedicineDentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Kostiuk
Morris A
MA
Martin
Dale D O
DD
Perinpanayagam
Maneka A
MA
Hak
Pieter G
PG
Siddam
Anjaiah
A
Majjigapu
Janaki R
JR
Rajaiah
Gurram
G
Keller
Bernd O
BO
Prescher
Jennifer A
JA
Wu
Peng
P
Bertozzi
Carolyn R
CR
Falck
John R
JR
Berthiaume
Luc G
LG
D000107:Acetylation; D000480:Alkynes / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004798:Enzymes / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D019169:Jurkat Cells; D054878:Lipoylation; D051379:Mice; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism
D000480:Alkynes; D004798:Enzymes; D005227:Fatty Acids; D011506:Proteins; D018631:ras Proteins