N-myristoyltransferase (Nmt) covalently links the 14 carbon fatty acid, myristate, to the N-terminal glycine of nascent eukaryotic and viral proteins. This grant has supported our efforts to examine the enzymology and biological significance of protein N-myristoylation in S. cerevisiae and Cryptococcus neoformans. Genetic studies established that NMT is essential for the viability of C. neoformans. We found that purified fungal and human Nmts have divergent peptide substrate specificities and that these differences can be used to develop a class of peptidomimetic inhibitors that are fungicidal. The structural basis for the differences in peptide substrate specificities between orthologous Nmts needs to be defined to guide design of additional classes of more potent, biologically active inhibitors. We have used X-ray crystallography to determine, at 2.9 Angstrom units resolution, the structure of a ternary complex of S. cerevisiae Nmt1p with a nonhydrolyzable myristoylCoA analog and peptidomimetic inhibitor.
Our specific aim 1 will be to compare the structures of ternary complexes of S. cerevisiae, C. neoformans and human Nmts with bound peptide substrates and to test structure/activity relationships by site-directed mutagenesis. The factors that allow fungal pathogens to survive during stationary phase are poorly understood and may have an important impact on pathogenesis. Using S. cerevisiae as a model, we found that defects in protein N-myristoylation impair survival during stationary phase and also accelerate aging. Deletion of 48 genes encoding known or putative Nmt1p substrates in a wild type strain disclosed that starvation sensitivity and rapid aging can be recapitulated by removing Sip2p, a N-myristoylprotein associated with a kinase (Snf1p) involved in regulating global cellular responses to glucose starvation.
Our specific aim 2 will be to characterize the mechanisms by which N-myristoylproteins regulate resistance to nutrient deprivation and aging. The Sip2p pathway will be dissected genetically in S. cerevisiae. An expression cloning strategy will be used to identify C. neoformans cDNAs that can complement the stationary phase (and other) phenotypes produced by sip2delta in S. cerevisiae. The C. neoformans ortholog of SIP2 will be recovered and a null allele generated. The impact of the gene deletion on C. neoformans' ability to withstand periods of nutrient deprivation will be examined in culture and in vivo. These studies may yield therapeutic targets for limiting the ability of fungal pathogens to survive in host compartments where nutrients are scarce. They should also provide molecular insights about the relationship between resistance to nutrient deprivation and aging that are applicable to other organisms.
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