N-Myristoyltransferase (NMT) catalyses the co-translational transfer of myristic acid from myristoyl-CoA in amide linkage to the NH2-terminal glycine residues of a number of viral and eukaryotic cellular proteins. N-Myristoylation is an essential cellular process which is required for the subcellular targeting, protein conformation, and/or biological activity of many proteins. The known involvement of N-myristoylproteins in several signal transduction and growth control pathways also suggest that NMT may be functional in aberrant cellular processes that account for progression to the malignant phenotype. Recent work in our laboratory has revealed new regulatory signals essential for in vivo N- myristoylation and which may present new oppportunities for chemotherapeutic interdiction. In contrast with the commonly held view of NMT as a soluble >>48 kDa monomer we have recently found that mammalian NMTs actually exists as several multiple high molecular mass species (i.e., 120- 670 kDa) resulting from multimerization of >>60 kDa NMT subunit(s) and/or complex formation with other cellular proteins. Also found are small amounts of a fully active NMT monomer which arises by proteolytic cleavage of the >>12 kDa N-terminus of the >>60 kDa subunit. Removal of this domain has no apparent affect on in vitro NMT activity suggesting that the N-terminus of NMT has non-catalytic regulatory function(s). We have now shown that human lymphoma cells also express a >>60 kDa NMT subunit which is converted to a >>48 kDa form by proteolysis. 5'-RACE analysis of a human cDNA library confirms the existence of an additional in-frame start-site upstream from the known human NMT gene (i.e., encoding a >>48 kDa enzyme) which accounts for an open reading frame encoding the larger >>60 kDa enzyme. Furthermore, >>80% of the NMT activity in fresh cell extracts is found to co-sediment with the major 260 nm absorption profile on isokinetic sucrose density gradients suggesting the enzyme's specific association with cellular ribosomes. Recombinant >>60 kDa and >>48 kDa NMTs which have been expressed and purified from bacteria elute from gel filtration columns as >>105 kDa and >>60 kDa forms suggesting that one function of the N- terminal domain likely facilitates NMT subunit dimerization. Purified recombinant >>60 kDa or >>48 kDa NMTs added to cell homogenates were found to fractionate with the ribosomal or cytosolic fractions, respectively, supporting the suggestion that the N-terminus of NMT also regulates ribosomal targeting. We have also found that a subpannel of 6 leukemic cell lines are selectively growth inhibited by myristoyl hydroxymate and 1-bromo-2-pentadecanone in the NCI in vitro anti-cancer screen. Both compounds were also found to be in vitro inhibitors of NMT and inhibited in vivo radiolabeling of N-myristoylproteins with [3H]myristate but not protein radiolabeling with [3H]palmitate. Our experiments thus identify two new in vitro inhibitors of NMT which also block protein N-myristoylation in vivo and selectively inhibit the growth of human leukemic/lymphoma cells.
