Structure/Function Analysis of Icmt

描述（由申请人提供）：Ras是最常与人类癌症相关的致癌基因。因此，Ras是抗癌药物发现的一个有吸引力的靶点。Ras蛋白是一种gtp酶，只有在与细胞膜结合时才具有生物活性。Ras是一大家族蛋白的创始成员，通过c端CAAX基序的翻译后修饰二级靶向细胞膜。CAAX序列被戊烯酰化、蛋白水解和羧基甲基化修饰，这些反应分别由法尼基或香叶基-香叶基转移酶、Ras转换酶1 （Reel）和异戊酰半胱氨酸羧基甲基化转移酶（Icmt）催化。法尼基转移酶抑制剂（FTIs）作为抗癌药物得到了广泛的应用。最近有证据表明，缺乏Reel或Icmt的细胞对Ras的转化具有抗性，这引起了人们对这些酶作为药物靶点的兴趣。Icmt是一种内源性内质网膜蛋白，目前对其结构、酶学、调控和生物学功能等方面的研究还很少。我们已经建立了一个Icmt突变体库，并开发了研究Icmt结构和功能的方法。本提案的主题是Icmt的结构/功能分析。具体目标是：1。Icmt的生化分析。利用点和截短突变体，光亲和和化学交联底物和共免疫沉淀，我们将绘制Icmt的催化和调控结构域。使用传统和新颖的方法，我们将绘制这种多膜跨越酶的拓扑结构。2. 体外Icmt的功能分析：在小GTPase信号传导中的作用。利用缺乏Icmt基因的小鼠成纤维细胞和Icmt基因沉默的人类细胞，我们将研究Icmt在Ras、Ral、Rho和Rab gtpase的功能和稳定性中的作用。我们还将在蛋白水平上研究Icmt在正常细胞和肿瘤细胞中的表达。体内Icmt的功能分析：在H-Ras驱动的小鼠乳腺肿瘤模型中的作用。使用Icmtflox/flox小鼠，我们将使用具有组织特异性和时间可控性的ras驱动乳腺癌小鼠模型来验证Icmt是体内肿瘤发生和肿瘤维持所必需的假设。本申请中提出的研究将阐明一种酶的生物化学和生物学特性，这种酶可以修饰一系列信号gtpase，并将为开发作用于Ras运输途径的抗癌药物提供信息。

DESCRIPTION (provided by applicant): 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-cancer drugs. 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 this multiple 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 Icmtflox/flox 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.