g. biotin) uniquely enables global quantitative analysis of protein lipidation by enrichment coupled to standard liquid chromatography-mass spectrometry. In this review we discuss the development buy CHIR-99021 of chemical
proteomics technologies that have resulted in the first quantitative whole-proteome studies of the known major classes of protein lipidation, and the first insights into their full scope in vivo. The most-well characterized form of protein N-acylation is N-terminal N-myristoylation, the irreversible attachment of a C14-fatty acid (myristate) to the N-terminal glycine of substrate proteins, which is catalyzed by N-myristoyltransferase (NMT) [ 4]. NMT is encoded by a single copy gene in lower eukaryotes, whereas in humans and SCH727965 molecular weight most other higher organisms two NMT genes (nmt1 and nmt2) have been identified. Protein N-myristoylation increases affinity for membranes and is required for viability and survival in every organism in which its essentiality has been studied. Dysregulation of myristoylated proteins has been linked to several diseases and NMT has been proposed as a potential drug target in viral, fungal, bacterial or parasitic infections, as well as in cancer [ 4]. Chemical tools have been developed to study N-myristoylation, including alkyne and azido-tagged analogues of the natural lipid
substrate (YnMyr and AzMyr, respectively) [ 5], as well as competitive inhibitors of the protein binding Ureohydrolase site of NMT. YnMyr is used in most recent studies as it is known to give minimal background labeling [ 6], and alkyne-tagged lipids appear to recapitulate endogenous lipid metabolism [ 7]. Potent NMT inhibitors have also been reported, for example against NMT from yeast [ 8] and from Trypanosoma brucei (the causative agent of human sleeping sickness) [ 9 and 10], although these had variable selectivity against NMT from various species. More selective inhibitors
have been reported recently [ 11], and can be used as selective chemical tools to pharmacologically knockdown N-myristoylation in different organisms. However, metabolism (e.g. chain elongation) of N-myristoylation probes can result in trafficking into unrelated lipidation pathways including GPI anchors and S-palmitoylation (see following sections). In this context, the combination of NMT inhibitors with YnMyr and quantitative proteomic analysis has proven particularly powerful in establishing the N-myristoylated proteome in vivo, without interference by off-target protein labeling. In this approach, the response of YnMyr-tagged proteins to selective NMT inhibitors is quantified, and correlated with the identification of each protein as a substrate or non-substrate of NMT.