Structure/Function Analysis of Icmt

RAS是最常与人类癌症有关的癌基因。因此，RAS是一个有吸引力的反 抗癌药物的发现。RAS蛋白是只有在与细胞结合时才具有生物活性的GTP酶 膜。RAS是一个大的蛋白质家族的创始成员，这些蛋白质是细胞内的次级靶点。 通过C-末端CAAX基序的翻译后修饰。修改了CAAX序列 通过戊烯基化、蛋白质降解和羧甲基化，分别由法尼基和香叶基催化的反应. 香叶酰基转移酶、RAS转换酶1(REEL)和异丙半胱氨酸羧甲基转移酶(ICMT)。 法尼基转移酶抑制剂(FTI)已被开发为抗癌药物。最近的证据表明，细胞缺乏 在Reel或ICMT中抵抗RAS的转化引发了人们对这些酶作为药物的浓厚兴趣 目标。我们实验室克隆了一种内质网固有的膜蛋白，目前对它知之甚少。 ICMT的结构、酶学、调节和生物学功能。我们已经建立了一个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 这项提案的主题。具体目标为：1.ICMT生化分析。使用点和截断 突变体、底物的光亲和力和化学交联以及免疫共沉淀，我们将绘制催化图谱 和ICMT的监管领域。使用传统和新的方法，我们将绘制出这多个 膜跨越酶。2.ICMT的体外功能分析：在小GTP酶信号转导中的作用vbl.使用 ICMT缺失的小鼠成纤维细胞和ICMT基因沉默的人细胞中，我们将研究ICMT的作用 ICMT对Ras、Ral、Rho和Rab GTP酶的功能和稳定性的影响。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.体内ICMT的功能分析：在H-RAS驱动中的作用 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 利用RAS驱动的小鼠乳腺癌模型进行体内致癌和肿瘤维持 具有组织特异性和时间可控性。在本申请中提出的研究将阐明生物化学。 以及一种酶的生物学，这种酶可以改变一系列信号GTP酶，并将为正在进行的开发工作提供信息 作用于RAS转运途径的抗癌药物。‘’]‘’“‘”’‘-1-’IR

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