Structure/Function Analysis of Icmt

Icmt的结构/功能分析

• 批准号: 7021878
• 负责人: MARK Reid PHILIPS
• 金额: $ 30万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7021878
• 关键词: catalyst; drug discovery /isolation; enzymes; human; methylation; methyltransferase; model; motivation; mutant; neoplasm /cancer; prenylation; proteins; role

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.

描述（由申请人提供）：Ras是最常与人类癌症相关的癌基因。因此，Ras是抗癌药物发现的有吸引力的靶标。Ras蛋白是仅当与细胞膜结合时才具有生物活性的GTP酶。Ras是一个蛋白质大家族的创始成员，其通过C-末端CAAX基序的翻译后修饰而次级靶向细胞膜。CAAX序列通过异戊烯化、蛋白水解和羧基甲基化进行修饰，这些反应分别由法尼基或香叶基-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）催化。法尼基转移酶抑制剂（FTIs）已被开发为抗癌药物。最近的证据表明，缺乏Reel或Icmt的细胞对Ras的转化具有抗性，这引发了人们对这些酶作为药物靶点的高度兴趣。ICMT是在我们实验室克隆的，并被证明是一种内在的ER膜蛋白，但人们对ICMT的结构、酶学、调节和生物学功能知之甚少。我们已经产生了一个Icmt突变体库，并开发了研究Icmt结构和功能的方法。ICMT的结构/功能分析是本提案的主题。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基板和共免疫沉淀，我们将映射ICMT的催化和调节域。使用传统的和新的方法，我们将映射这种多跨膜酶的拓扑结构。2. ICMT的体外功能分析：在小GT3信号传导中的作用。使用Icmt缺陷的小鼠成纤维细胞和Icmt基因沉默的人细胞，我们将研究Icmt在Ras、Ral、Rho和Rab GTP酶的功能和稳定性中的作用。我们还将在蛋白水平上研究Icmt在正常和肿瘤细胞中的表达。Icmt在体内的功能分析：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Ras驱动的乳腺癌的小鼠模型（组织特异性和时间可控性）来检验Icmt是体内肿瘤发生和肿瘤维持所需的假设。本申请中提出的研究将阐明修饰大量信号转导GTP酶的酶的生物化学和生物学，并将为开发作用于Ras运输途径的抗癌药物的持续努力提供信息。