ANALOGS OF MYRISTIC ACID TO MODULATE HIV-I ASSEMBLY

14碳饱和脂肪酸肉豆蔻酸酯通过酰胺连接 与一些病毒的NH2末端甘氨酸残基结合 蛋白质包括HIV-I的P17gag和F/3‘orf。站点定向 梅森-辉瑞猴氨基端甘氨酸的诱变 病毒Pr78gag到ALA已被证明可以防止肉豆蔻酰化， 抑制其后续的蛋白质分解过程，并导致 胞质内A型颗粒积聚的原因可能是 阻止衣壳运输到萌芽处的质膜 和释放发生了。我们已经分离出了负责 这种共同或早期的翻译后蛋白质修饰来自 低等(酵母、小麦胚芽)和高等(大鼠和人)真核生物。 肉豆蔻的脂肪酸和多肽底物专一性 CoA：蛋白质N-肉豆蔻酰基转移酶(NMT)已被定义为 体外酰化试验。在这些研究过程中，我们 合成了肉豆蔻酸的氧基和硫基取代的类似物 它们的疏水性已经显著降低 表现出与肉豆蔻酰辅酶A相似的动力学性质。这些类比 与肉豆蔻酸盐竞争细胞蛋白质的酰化，但 对细胞没有明显的毒性。这样做的具体目的是 多中心研究：(I)扩展我们对酰化辅酶A-NMT的评价 通过继续合成饱和的和非饱和的 含有一个或多个杂原子的不饱和脂肪酸，(Ii)至 评估这些类似物抑制逆转录病毒的能力- 特别是HIV-I-在培养细胞系中的复制，以及(Iii) 用克隆技术研究NMT-配体相互作用的原子细节 NMT，在大肠杆菌中表达该蛋白，并用 而且没有它的配体。

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.