Structure/Function Analysis of Icmt

Ras是最常与人类癌症相关的癌基因。因此，Ras是一个有吸引力的抗肿瘤靶点。 癌症药物发现Ras蛋白是只有当与细胞内的蛋白质结合时才具有生物活性的GTP酶。 膜。Ras是一个蛋白质大家族的创始成员，该蛋白质大家族的第二目标是细胞凋亡， 通过C-末端CAAX基序的翻译后修饰来修饰细胞膜。CAAX序列被修改 通过异戊烯化、蛋白质水解和羧基甲基化，分别由法呢基或香叶基催化的反应， 香叶基转移酶、Ras转化酶1（Reel）和异戊二烯基半胱氨酸羧基甲基转移酶（Icmt）。 法尼基转移酶抑制剂（FTIs）是近年来发展起来的一种抗癌药物。最近的证据表明，细胞缺乏 在Reel或Icmt中对Ras转化具有抗性，这引发了人们对这些酶作为药物的高度兴趣 目标的在我们的实验室克隆，并显示是一个内在的ER膜蛋白，很少有人知道， ICMT结构、酶学、调控和生物学功能。我们建立了一个ICMT突变体库， 并发展了研究ICMT结构和功能的方法。ICMT的结构/功能分析是 这个提议的主题。具体目标是：1。ICMT的生化分析。使用点和截断 突变体，光亲和性和化学交联的底物和共免疫沉淀，我们将映射催化 和ICMT的监管领域。使用传统的和新的方法，我们将映射的拓扑结构，这个多 跨膜酶2. ICMT的体外功能分析：在小GT3信号传导中的作用。使用 我们将研究Icmt基因缺陷的小鼠成纤维细胞和Icmt基因沉默的人细胞的作用， Icmt在Ras、Ral、Rho和Rab GTP酶的功能和稳定性中的作用。我们还将在蛋白质水平上研究 ICMT在正常和肿瘤细胞中的表达3. Icmt在体内的功能分析：在H-Ras驱动的 小鼠乳腺肿瘤模型。使用Icmtnox/n <$x小鼠，我们将测试Icmt是必需的假设， 使用Ras驱动的乳腺癌的小鼠模型在体内进行肿瘤发生和肿瘤维持， 组织特异性和时间可控性。本申请中提出的研究将阐明生物化学 和生物学的一种酶，修改了主机的信号GTP酶，并将告知正在进行的努力， 作用于Ras运输途径的抗癌药物。“你好”-1-“

Ras is the oncogene most often associated with human cancer. Accordingly, Ras is an attractive target for anti- cancer drug discovery. Ras proteins are GTPases that are biologically active only when associated with cellular membranes. Ras is the founding member of a large family of proteins that are targeted secondarily to cellular membranes by the posttranslational modification of a C-terminal CAAX motif. CAAX sequences are modified by prenylation, proteolysis and carboxyl methylation, reactions catalyzed respectively by farnesyl or geranyl- geranyltransferases, Ras converting enzyme 1 (Reel) and isoprenylcysteine carboxyl methyltransferase (Icmt). Farnesyl transferase inhibitors (FTIs) have been developed as anti-cancerdrugs. Recent evidence that cells deficient in Reel or Icmt are resistant to transformation by Ras has sparked heightened interest in these enzymes as drug targets. Cloned in our laboratory and shown to be an intrinsic ER membrane protein, little is known about the structure, enzymology, regulation and biological function of Icmt. We have generated a library of Icmt mutants and developed methods to study the structure and function of Icmt. A structure/function analysis of Icmt is the subject of this proposal. The Specific Aims are: 1. Biochemical analysis of Icmt. Using point and truncation mutants, photoaffinity and chemical crosslinking of substrates and co-immunoprecipitation we will map catalytic and regulatory domains of Icmt. Using conventional and novel approaches we will map the topology of thismultiple membrane spanning enzyme. 2. Functional analysis of Icmt in vitro: role in small GTPase signaling. Using mouse fibroblasts deficient in Icmt and human cells in which the Icmt gene is silenced we will study the role of Icmt in the function and stability of Ras, Ral, Rho and Rab GTPases. We will also study, at the protein level, the expression of Icmt in normal and tumor cells 3. Functional analysis of Icmt in vivo: role in an H-Ras driven mouse mammary tumor model. Using Icmtnox/n¿x mice we will test the hypothesis that Icmt is required for oncogenesis and tumor maintenance in vivo using a murine model of Ras-driven mammary carcinoma that is both tissue specific and temporally controllable. The studies proposed in this application will elucidate the biochemistry and biology of an enzyme that modifies a host of signaling GTPases and will inform the ongoing effort to develop anti-cancer drugs that act on the Ras trafficking pathway. '¿]'¿ '"¿ '¿ ¿ ' "¿ ¿ ' -1-' Ir