1.Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues.
Takamitsu E1, Fukunaga K1, Iio Y1, Moriya K1, Utsumi T2. Anal Biochem. 2014 Nov 1;464:83-93. doi: 10.1016/j.ab.2014.07.006. Epub 2014 Jul 18.
To establish a non-radioactive, cell-free detection system for protein N-myristoylation, metabolic labeling in a cell-free protein synthesis system using bioorthogonal myristic acid analogues was performed. After Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with a biotin tag, the tagged proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on a polyvinylidene fluoride (PVDF) membrane, and then protein N-myristoylation was detected by enhanced chemiluminescence (ECL) using horseradish peroxidase (HRP)-conjugated streptavidin. The results showed that metabolic labeling in an insect cell-free protein synthesis system using an azide analogue of myristic acid followed by CuAAC with alkynyl biotin was the most effective strategy for cell-free detection of protein N-myristoylation. To determine whether the newly developed detection method can be applied for the detection of novel N-myristoylated proteins from complementary DNA (cDNA) resources, four candidate cDNA clones were selected from a human cDNA resource and their susceptibility to protein N-myristoylation was evaluated using the newly developed strategy.
2.Global analysis of protein N-myristoylation and exploration of N-myristoyltransferase as a drug target in the neglected human pathogen Leishmania donovani.
Wright MH1, Paape D2, Storck EM3, Serwa RA3, Smith DF2, Tate EW4. Chem Biol. 2015 Mar 19;22(3):342-54. doi: 10.1016/j.chembiol.2015.01.003. Epub 2015 Feb 26.
N-Myristoyltransferase (NMT) modulates protein function through the attachment of the lipid myristate to the N terminus of target proteins, and is a promising drug target in eukaryotic parasites such as Leishmania donovani. Only a small number of NMT substrates have been characterized in Leishmania, and a global picture of N-myristoylation is lacking. Here, we use metabolic tagging with an alkyne-functionalized myristic acid mimetic in live parasites followed by downstream click chemistry and analysis to identify lipidated proteins in both the promastigote (extracellular) and amastigote (intracellular) life stages. Quantitative chemical proteomics is used to profile target engagement by NMT inhibitors, and to define the complement of N-myristoylated proteins. Our results provide new insight into the multiple pathways modulated by NMT and the pleiotropic effects of NMT inhibition. This work constitutes the first global experimental analysis of protein lipidation in Leishmania, and reveals the extent of NMT-related biology yet to be explored for this neglected human pathogen.
3.N-Myristoyl transferase-mediated protein labelling in vivo.
Heal WP1, Wickramasinghe SR, Leatherbarrow RJ, Tate EW. Org Biomol Chem. 2008 Jul 7;6(13):2308-15. doi: 10.1039/b803258k. Epub 2008 May 12.
N-Myristoyl transferase-mediated labelling using a substrate modified with an azide or alkyne tag is described as an efficient and site-selective method for the introduction of a bioorthogonal tag at the N-terminus of a recombinant protein. The procedure may be performed in vitro, or in a single over-expression/tagging step in vivo in bacteria; tagged proteins may then be captured using Staudinger-Bertozzi or 'click' chemistry protocols to introduce a secondary label for downstream analysis. The straightforward synthesis of the chemical and molecular biological tools described should enable their use in a wide range of N-terminal labelling applications.
4.Myristoylation profiling in human cells and zebrafish.
Broncel M1, Serwa RA1, Ciepla P1, Krause E2, Dallman MJ3, Magee AI4, Tate EW1. Data Brief. 2015 Jul 2;4:379-83. doi: 10.1016/j.dib.2015.06.010. eCollection 2015.
Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223-6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.