MODULATION OF PROTEIN N-MYRISTOYLATION IN MYCO PATHOGENS

14碳饱和脂肪酸-肉豆蔻酸--的共价结合 到一些细胞和病毒蛋白的NH2末端甘氨酸残基 对它们的功能至关重要。我们小组一直在使用生化， 有机、化学和分子遗传学方法来理解 催化酶的底物专一性及其作用机制 这种修饰-肉豆蔻酰辅酶A：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 对于在N-细胞中实现其生物学功能是重要的 肉豆蔻酰化的蛋白质。在这些研究过程中，我们有 人工合成的含肉豆蔻酸酯类似物的杂原子 疏水性：(I)哺乳动物(人)的替代底物 NMT；(Ii)选择性地结合到细胞和病毒的亚群中 N-肉豆蔻基蛋白(包括HIV-I Pr55gag)；和(Iii)抑制HIV 无伴随细胞的急性H9T细胞复制试验 毒性。我们还从酿酒酵母中克隆了mnt基因，并展示了 将NMT从细胞中移除(通过基因破坏或缺失)是 对生物体是致命的。最后，衬底的差异 酵母和哺乳动物NMT的特异性已经被鉴定。这个 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。我们的目标是 从人类感染HIV-I的常见病原体--白色念珠菌中分离NMT 个人。结果表明，C. 白念珠菌和人类NMT将被比较。我们将分离出白色念珠菌 利用已经使用的宿主载体系统在大肠杆菌中表达 酿酒酵母NMT的表达。这将提供一个持续的来源 大量用于酶和结构研究的材料。一个 将采用快速、良好的体外检测系统进行筛选 几种不同种类的化合物作为C. 白色念珠菌NMT。这些包括(I)肉豆蔻酸类似物的一大组 具有系统变化的结构特征；(二)过渡态 化合物；(Iii)合成肽和(Iv)天然产物。这个 拟议的项目涉及一个多学科团队，由以下成员组成 两所大学和孟山都公司相互合作 在过去的3年里，关于蛋白质N-肉豆蔻酰化的研究。

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.