STRUCTURE AND MECHANISM OF PROTEIN PRENYL TRANSFERASES

添加类异戊二烯脂质（异戊二烯化）对活性至关重要 在信号传导中起重要作用的酶 途径或膜运输。 三种蛋白质异戊二烯基转移酶具有 法呢基转移酶（FTase），它添加了一个15-碳 类异戊二烯和两种香叶基香叶基转移酶（GT酶-I和-II）， 添加20碳类异戊二烯。 FTases和GTase-I修饰保守的半胱氨酸 位于修饰的多肽的C-末端四肽（“CAAX”基序）中的残基 蛋白 一些小的调节性G蛋白的异戊烯化激活 并将其定位在细胞膜上。 具有特殊的医学相关性 是最近观察到Ras癌基因蛋白被法尼基化。 这 对于这种转化活性， 蛋白 用FTase抑制剂处理Ras转化细胞， 已显示导致组织中转化表型的逆转 培养细胞。 Ras法尼基化的抑制目前被认为是一种 最有希望的抗癌靶点。 大约30%的人类癌症 与Ras的致癌形式有关。 三个解释 因此，FTase的三维结构对于 了解信号转导的基本过程， 开发抗癌药物或现有药物的衍生物。 本提案的目标是了解机制和底物 蛋白质异戊烯基转移酶蛋白质在它们的三个方面的特异性- 空间结构 具体目标概述如下： 1. 确定和完善法呢基的三维结构 转移酶与适当的底物复合物，通过X射线 结晶学 哺乳动物FTase和具有结合的FTase的晶体 已经生长了肽底物，其活性优于2.5 已收集分辨率和本地日期集。 据我们所知， 是第一种结晶的异戊二烯基转移酶 该结构将 为理解和解释生物化学和 蛋白质异戊二烯化的生物物理数据。 2. 为了确定FTase三元复合物的共晶结构， 肽底物和法呢基二磷酸类似物。 确定 FTase与适当底物的其他复合物的晶体结构 和抑制剂：肽，法呢基二磷酸，肽模拟物和其他 抑制剂的 这些结构对于理解催化反应是必不可少的。 在原子细节和确定衬底的性质机制 的特异性 这些结构共同为以下方面提供了结构基础： 改进抗癌疗法的设计。 3. 一个长期的目标也是获得晶体和确定晶体 GGTase-1的结构以及适当的底物复合物。

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.