Abstract
Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, thus promoting protein-protein and protein-membrane interactions. NMT is essential for the viability of numerous human pathogens and is also up-regulated in several tumors. Here we describe a new, nonradioactive, ELISA-based method for measuring NMT activity. After the NMT-catalyzed reaction between a FLAG-tagged peptide and azido-dodecanoyl-CoA (analog of myristoyl-CoA), the resulting azido-dodecanoyl-peptide-FLAG was coupled to phosphine-biotin by Staudinger ligation, captured by plate-bound anti-FLAG antibodies and detected by streptavidin-peroxidase. The assay was validated with negative controls (including inhibitors), corroborated by HPLC analysis, and demonstrated to function with fresh or frozen tissues. Recombinant murine NMT1 and NMT2 were characterized using this new method. This versatile assay is applicable for exploring recombinant NMTs with regard to their activity, substrate specificity, and possible inhibitors as well as for measuring NMT-activity in tissues.
Highlights
Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid to the N-terminal glycine of proteins, promoting protein-protein and protein-membrane interactions
N-myristoylation refers to an irreversible protein modification involving the covalent attachment of n-tetradecanoic acid to the N-terminal glycine residue of proteins [1, 2]
Our aim was to develop an assay that would allow measurement of NMT activity in tissues and cell lysates as well as in experiments dealing with recombinant NMTs, enzyme kinetics, substrate specificity, and inhibitors
Summary
Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, promoting protein-protein and protein-membrane interactions. N-myristoylation refers to an irreversible protein modification involving the covalent attachment of n-tetradecanoic acid (myristic acid) to the N-terminal glycine residue of proteins [1, 2]. This process can occur co- or post-translationally [3,4,5,6] and helps to promote protein interactions with membranes or with hydrophobic domains of other proteins [7, 8]. Increased expression and activity of NMT has been demonstrated in human tumors (e.g., colorectal carcinoma, gallbladder carcinoma, oral squamous cell carcinoma, and brain tumors) [19,20,21,22,23]
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