STRUCTURE AND MECHANISM OF PROTEIN PRENYL TRANSFERASES

异戊二烯类脂的添加(异戊二烯基化)是活性的关键 在信号转导中起关键作用的许多酶 途径或膜运输。三种蛋白质戊烯基转移酶 一种法尼基转移酶(FTase)，它增加了15个碳 和两个香叶酰转移酶(GTase-I和-II)，它们 加入20碳的类异戊二烯。FTase和GTase-I修饰一个保守的半胱氨酸 位于修饰后的C端四肽(CAAX基序)中的残基 蛋白。一些小分子调节性G蛋白的预基化被激活 并将它们定向到它们的细胞膜上。具有特殊的医学关联性 是最近观察到的RAS癌基因蛋白法尼化。这 对于此转换活动，绝对需要修改 蛋白。FTase Has抑制剂对RAS转化细胞的作用 已被证明导致组织中转化的表型逆转 培养细胞。目前认为抑制RAS法尼化是一种 最有希望的抗癌靶点之一。大约30%的人类癌症 与RAS的致癌形式有关。三个方面的澄清 因此，FTase的空间结构对两者都很重要 了解信号转导的基本过程，并了解 开发抗癌药物或现有药物的衍生品。 这项建议的目标是了解其机制和基础。 蛋白质戊烯基转移酶蛋白的三种特异性 空间结构。具体目标概括如下： 1.法呢基三维结构的确定和提纯 转移酶与合适底物络合物结合的X射线分析 结晶学。哺乳动物FTase和结合了FTase的晶体 已经生长出衍射率优于2.5A的多肽底物 分辨率和本地日期集已收集。据我们所知，这是 是第一个结晶的戊烯基转移酶。该结构将 为理解和解释生化和生物化学提供了基础 蛋白质预烯基化的生物物理数据。 2.确定FTase三元络合物的共晶结构， 多肽底物和法尼基二磷酸类似物。要确定 FTase与合适底物形成的其他络合物的晶体结构 和抑制剂：多肽、法尼基二磷酸、多肽模拟物等 抑制剂。这些结构对于理解催化剂是必不可少的 原子细节中的机理与衬底性质的确定 专一性。这些结构共同提供了一个结构基础 改进的抗癌疗法的设计。 3.远程目标也是获得晶体并确定晶体 GGTase-1的结构和适当的底物复合体。

Addition of isoprenoid lipids (prenylation) is critical from the activity of a number of enzymes that play essential roles in signal transduction pathways or membrane trafficking. Three protein prenyl transferases have been identified; a farnesyl transferase (FTase) which adds a 15-carbon isoprenoid, and two geranylgeranyl transferases (GTase-I, and -II) which add a 20-carbon isoprenoid. FTases and GTase-I modify a conserved cysteine residue located in a C-terminal tetrapeptide ("CAAX" motif) of the modified protein. Prenylation of a number of small regulatory G proteins activate and target these to their cell membranes. Of particular medical relevance is the recent observation that Ras oncogen proteins are farnesylated. This modification is absolutely required for the transforming activity this protein. Treatment of Ras transformed cells with inhibitors to FTase has been shown to result in reversion of the transformed phenotype in tissue culture cells. Inhibition of Ras farnesylation is currently considered one of the most promising anti-cancer targets. Roughly 30% of human carcinomas are associated with oncogenic forms of Ras. Elucidation of the three dimensional structure of FTase is therefore important both for understanding fundamental processes in signal transduction and for the development of anti-cancer drugs or derivatives of existing drugs. The goal of this proposal is to understand the mechanism and substrate specificity of protein prenyl transferase proteins in terms of their three- dimensional structure. The specific aims are summarized as follows: 1. To determine and refine the three-dimensional structures of farnesyl transferase together with appropriate substrate complexes by X-ray crystallography. Crystals of mammalian FTase and FTase with a bound peptide substrate have been grown that diffract to better than 2.5 A resolution and a native date set has been collected. To our knowledge this is the first prenyl transferase enzyme crystallized. The structure will provide a basis for understanding and interpreting biochemical and biophysical data on protein prenylation. 2. To determine the co-crystal structure of a ternary compllex of FTase, peptide substrate, and a farnesyl diphosphate analog. To determine the crystal structures of other complexes of FTase with appropriate substrates and inhibitors: peptides, farnesyl diphosphate, peptidomimetic and other inhibitors. These structures are essential for understanding the catalytic mechanism in atomic detail and determining the nature of substrate specificity. Together these structures provide a structural foundation for the design of improved anti-cancer therapeutics. 3. A long range goal is also to obtain crystals and determine the crystal structure of the GGTase-1 together with appropriate substrate complexes.