MyristoylCoA:protein N- myristoyltransferase (Nmt) transfers myristate (C14:0) from myristoylCoA to the N-terminal Gly residue of eukaryotic proteins with diverse biological functions. Knowledge of the regulation of protein N-myristoylation comes from genetic and biochemical studies in S.cerevisiae. Substrate specificities and kinetic mechanisms of Nmt1p have been defined. MyristoylCoA is derived from fatty acid synthetase and from activation of imported and endogenous fatty acids by cellular long chain acylCoA synthetase (Faas). Deletion of the single copy NMT1 gene causes recessive lethality. Conditional lethal nmt1 alleles have been used to identify other gene products that participate in the regulation of protein N-myristoylation. Cryptococcus neoformans var. neoformans is a haploid opportunistic pathogen that causes systemic fungal infection in AIDS patients. Persistent C. neoformans infections are common after completion of primary therapy with amphotericin B or fluconazole for meningitis. The search for alternative therapeutic targets in this organism has been hampered by the inability to use targeted gene disruption to determine whether a given gene product is essential for viability. The investigators have used homologous recombination to replace the wild type NMT locus with a conditional lethal allele and establish that Nmt is an essential enzyme in C.neoformans. Sufficient differences exist in the peptide substrate specificities of the fungal and human Nmts to make an attractive target for development of fungal agents. This grant application is a continuation of the specific aims described in AI30188. Studies for Aim 1 are focused on S. cerevisae.
The Aim 1 is to perform genetic and biochemical studies in S. cerevisae to better understand the regulation of protein N-myristoylation. This will include the following: (i) continued evaluation of Nmt1p structure/activity relationships using titration calorimetry to evaluate the interactions between wild type and mutant Nmts and their substrates and the use of random PCR mutagenesis combined with a colony sectoring assay to identify residues critical for substrates recognition and catalysis, (ii) analyses of how myristoylCoA metabolism is regulated by studying the phenotypes of isogenic strains having (a) wild type NMT1 or conditional lethal nmt1 alleles; (b) various combinations of wild type and mutant FAA (acylCoA synthetases) (c) an active Fas (fatty acid synthetases) complex or one inactivated by cerulenin; and (d) peroxisomal assembly (PAS) mutants (iii) synthetic lethality screens to identify additional gene products that affect the specificity/efficiency of protein N-myristoylation, (iv) an evaluation of the regulation of protein N-myristoylation during stationary phase.
The Aim 2 is to conduct genetic and biochemical studies to further evaluate the regulation and functional significance of protein N- myristoylation in C.neoformans. These studies will entail: (i) kinetic and calorimetric analyses of the substrate specifications of C. neoformans Nmt, plus definition of enzyme structure using x-ray crystallography (ii) further phenotypic analyses of isogenic strains with wild type and conditional lethal NMT alleles, (iii) isolation of C. neoformans genes involved in regulating protein N-myristoylation by complementation of S.cerevisae strains containing various mutant alleles and by suppression of the conditional lethality produced by C. neoformans nmt487D in C. neoformans.
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