title: N-Myristoyltransferase isozymes exhibit differential specificity for human immunodeficiency virus type 1 Gag and Nef.
abstract: Myristoylation of the human immunodeficiency virus type 1 (HIV-1) proteins Gag and Nef by N-myristoyltransferase (NMT) is a key process in retroviral replication and virulence, yet remains incompletely characterized. Therefore, the roles of the two isozymes, NMT1 and NMT2, in myristoylating Gag and Nef were examined using biochemical and molecular approaches. Fluorescently labelled peptides corresponding to the N terminus of HIV-1 Gag or Nef were myristoylated by recombinant human NMT1 and NMT2. Kinetic analyses indicated that NMT1 and NMT2 had 30- and 130-fold lower K(m )values for Nef than Gag, respectively. Values for K(cat) indicated that, once Gag or Nef binds to the enzyme, myristoylation by NMT1 and NMT2 proceeds at comparable rates. Furthermore, the catalytic efficiencies for the processing of Gag by NMT1 and NMT2 were equivalent. In contrast, NMT2 had approximately 5-fold higher catalytic efficiency for the myristoylation of Nef than NMT1. Competition experiments confirmed that the Nef peptide acts as a competitive inhibitor for the myristoylation of Gag. Experiments using full-length recombinant Nef protein also indicated a lower K(m) for Nef myristoylation by NMT2 than NMT1. Small interfering RNAs were used to selectively deplete NMT1 and/or NMT2 from HEK293T cells expressing a recombinant Nef-sgGFP fusion protein. Depletion of NMT1 had minimal effect on the intracellular distribution of Nef-sgGFP, whereas depletion of NMT2 altered distribution to a diffuse, widespread pattern, mimicking that of a myristoylation-deficient mutant of Nef-sgGFP. Together, these findings indicate that Nef is preferentially myristoylated by NMT2, suggesting that selective inhibition of NMT2 may provide a novel means of blocking HIV virulence.
authors: Department of Pharmacology, Penn State College of Medicine, Hershey, PA, USA.
authors: Kelly E
authors: KE
authors: Seaton
authors: Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC, USA.|Department of Pharmacology, Penn State College of Medicine, Hershey, PA, USA.
authors: Charles D
authors: CD
authors: Smith
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D005982:Glutathione Transferase