The 14 carbon saturated fatty acid myristate is linked via an amide bond to the NH2-terminal glycine residues of a number of viral proteins including the P17gag and F/3'orf of HIV-I. Site directed mutagenesis of the NH2-terminal Gly of the Mason-Pfizer Monkey viral Pr78gag to an Ala has been shown to prevent myristoylation, inhibit its subsequent proteolytic processing, and result in accumulation of intracytoplasmic A-type particles due to a presumed blockade in capsid transport to the plasma membrane where budding and release occur. We have isolated the enzyme responsible for this co- or early post-translational protein modification from lower (yeast, wheat germ) and higher (rat and human) eukaryotes. The fatty acid and peptide substrate specificities of Myristoyl CoA:Protein N-Myristoyltransferase (NMT) has been defined using an in vitro acylation assay. During the course of these studies we have synthesized oxy- and thio-substituted analogs of myristate which have remarkable reductions in their hydrophobicity yet exhibit similar kinetic properties to myristoyl CoA. These analogs compete with myristate for the acylation of cellular proteins but are not apparently toxic to cells.
The specific aims of this multicenter study are (i) to extend our evaluation of acylCoA-NMT interactions through the continued synthesis of saturated and unsaturated fatty acids with one or more heteroatoms, (ii) to evaluate the ability of these analogs to inhibit retroviral - specifically HIV-I-replication in cultured cell lines, and (iii) to examine the atomic details of NMT-ligand interactions by cloning NMT, expressing the protein in E. coli and to crystallize it with and without its ligands.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI027179-03
Application #
3547164
Study Section
Special Emphasis Panel (SRC (34))
Project Start
1988-09-30
Project End
1991-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Knoll, L J; Johnson, D R; Bryant, M L et al. (1995) Functional significance of myristoyl moiety in N-myristoyl proteins. Methods Enzymol 250:405-35
Bhatnagar, R S; Gordon, J I (1995) Thermodynamic studies of myristoyl-CoA: protein N-myristoyltransferase using isothermal titration calorimetry. Methods Enzymol 250:467-86
Knoll, L J; Schall, O F; Suzuki, I et al. (1995) Comparison of the reactivity of tetradecenoic acids, a triacsin, and unsaturated oximes with four purified Saccharomyces cerevisiae fatty acid activation proteins. J Biol Chem 270:20090-7
Knoll, L J; Johnson, D R; Gordon, J I (1995) Complementation of Saccharomyces cerevisiae strains containing fatty acid activation gene (FAA) deletions with a mammalian acyl-CoA synthetase. J Biol Chem 270:10861-7
Johnson, D R; Bhatnagar, R S; Knoll, L J et al. (1994) Genetic and biochemical studies of protein N-myristoylation. Annu Rev Biochem 63:869-914
Lodge, J K; Jackson-Machelski, E; Toffaletti, D L et al. (1994) Targeted gene replacement demonstrates that myristoyl-CoA: protein N-myristoyltransferase is essential for viability of Cryptococcus neoformans. Proc Natl Acad Sci U S A 91:12008-12
Johnson, D R; Knoll, L J; Levin, D E et al. (1994) Saccharomyces cerevisiae contains four fatty acid activation (FAA) genes: an assessment of their role in regulating protein N-myristoylation and cellular lipid metabolism. J Cell Biol 127:751-62
Johnson, D R; Cok, S J; Feldmann, H et al. (1994) Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation. Proc Natl Acad Sci U S A 91:10158-62
Johnson, D R; Knoll, L J; Rowley, N et al. (1994) Genetic analysis of the role of Saccharomyces cerevisiae acyl-CoA synthetase genes in regulating protein N-myristoylation. J Biol Chem 269:18037-46
Doering, T L; Lu, T; Werbovetz, K A et al. (1994) Toxicity of myristic acid analogs toward African trypanosomes. Proc Natl Acad Sci U S A 91:9735-9

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