MODULATION OF PROTEIN N-MYRISTOYLATION IN MYCO PATHOGENS

14碳饱和脂肪酸-肉豆蔻酸- 许多细胞和病毒蛋白质的NH 2-末端Gly残基 对它们的功能至关重要。 我们的团队一直在使用生物化学， 有机化学和分子遗传学的方法来理解 底物特异性和酶催化的作用机制 这种修饰-肉豆蔻酰CoA：蛋白N-肉豆蔻酰转移酶（NMT，E.C. 2.3.1.97）-以及肉豆蔻酸的物理化学性质 这是重要的履行其生物功能，在N- 肉豆蔻酰化蛋白质。 在这些研究过程中，我们 具有改变的肉豆蔻酸酯的合成的含杂原子类似物 疏水性，其（i）是哺乳动物（人） NMT;（ii）选择性地掺入细胞和病毒的亚群中， N-肉豆蔻酰蛋白（包括HIV-I Pr 55 gag）;和（iii）抑制HIV 在急性H9 T细胞测定中， 毒性 我们还从S.酿酒酵母和显示 从细胞中去除NMT（通过基因破坏或缺失）， 对生物体是致命的 最后，基质的差异 已经鉴定了酵母和哺乳动物NMT的特异性。 的 这项拨款提案的总体目的是开发化合物， 表现出对真菌NMT的物种特异性抑制。 这些目标 从C.白色念珠菌-一种常见的人类病原体在HIV-I感染 个体 C. 将比较白色念珠菌和人NMT。 我们将分离C。白色 基因并在E.大肠杆菌中使用的宿主载体系统已经用于 表达S。酿酒酵母NMT。 这将提供一个持续的 大量的材料用于酶和结构研究。 一 将采用快速、良好表征的体外测定系统来筛选 几种不同类型的化合物作为C. 白色念珠菌NMT。 这些包括（i）一大组肉豆蔻酸类似物 具有系统变化的结构特征;（ii）过渡态 化合物;（iii）合成肽和（iv）天然产物。 的 拟议的项目涉及一个多学科小组，由以下成员组成： 两所大学和孟山都公司， 在过去的三年里，蛋白质N-豆蔻酰化。

Covalent attachment of the 14 carbon saturated fatty acid - myristic acid - to the NH2-terminal Gly residue of a number of cellular and viral proteins is critical to their function. Our group has been using biochemical, organic and chemical and molecular genetic approaches to understand the substrate specificity and mechanism of action of the enzyme which catalyzes this modification - myristoyl CoA:Protein N-myristoyltranserase (NMT, E.C. 2.3.1.97) - as well as the physical chemical properties of myristic acid which are important for fulfilling its biological function in N- myristoylated proteins. During the course of these studies we have synthesized heteroatom containing analogs of myristate with altered hydrophobicity which (i) are alternative substrates for mammalian (human) NMTs; (ii) are selectively incorporated into subsets of cellular and viral N-myristoyl proteins (including the HIV-I Pr55gag); and (iii) inhibit HIV replication in an acute H9 T cell assay without accompanying cellular toxicity. We have also cloned the MNT gene from S. cerevisiae and shown that removing NMT from the cell (through gene disruption or deletion) is lethal to the organism. Finally, differences in the substrate specificities of yeast and mammalian NMTs have been identified. The overall purpose of this grant proposal is to develop compounds which will exhibit species specific inhibition of fungal NMTs. The goals are to isolate NMT from C. albicans - a common human pathogen in HIV-I infected individuals. The acyl CoA and peptide substrate specificities of C. albicans and human NMTs will be compared. We will isolate the C. albicans gene and express it in E. coli using a host vector system already used for expression of S. cerevisiae NMT. This will provide a continuing source of large amounts of material for enzymatic as well as structural studies. A rapid, well characterized in vitro assay system will be employed to screen several different classes of compounds as specific inhibitors of C. albicans NMT. These include (i) a large panel of analogs of myristic acid with systematically varied structural features; (ii) transition state compounds; (iii) synthetic peptides and (iv) natural products. The proposed project involves a multidisciplinary team, composed of members of two universities and the Monsanto Company that has worked with one another over the past 3 years on protein N-myristoylation.