title: Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry.
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.
authors.affiliation: Department of Cell Biology, Faculty of MedicineDentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Megan C
authors.initials: MC
authors.lastname: Yap
authors.forename: Morris A
authors.initials: MA
authors.lastname: Kostiuk
authors.forename: Dale D O
authors.initials: DD
authors.lastname: Martin
authors.forename: Maneka A
authors.initials: MA
authors.lastname: Perinpanayagam
authors.forename: Pieter G
authors.initials: PG
authors.lastname: Hak
authors.forename: Anjaiah
authors.initials: A
authors.lastname: Siddam
authors.forename: Janaki R
authors.initials: JR
authors.lastname: Majjigapu
authors.forename: Gurram
authors.initials: G
authors.lastname: Rajaiah
authors.forename: Bernd O
authors.initials: BO
authors.lastname: Keller
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Prescher
authors.forename: Peng
authors.initials: P
authors.lastname: Wu
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
authors.forename: John R
authors.initials: JR
authors.lastname: Falck
authors.forename: Luc G
authors.initials: LG
authors.lastname: Berthiaume
mesh_terms: 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
chemical_list: D000480:Alkynes; D004798:Enzymes; D005227:Fatty Acids; D011506:Proteins; D018631:ras Proteins