STEROL SENSING AND TRANSPORT IN A MODEL EUKARYOTE

真核生物模型中的甾醇传感和运输

• 批准号: 6745856
• 负责人: STEPHEN Laurence STURLEY
• 金额: $ 4.62万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6745856
• 关键词: Niemann Pick disease; Saccharomyces cerevisiae; biological signal transduction; gene expression; gene mutation; intracellular transport; molecular pathology; protein localization; protein protein interaction; site directed mutagenesis; sterols; yeast two hybrid system

The concentration of intracellular free sterol is strictly regulated to optimize the fluidity and function of all eukaryotic membranes. This homeostasis is imparted by several mechanisms, the crux of which is sensing of sterol and transport of this molecule from sites of synthesis or uptake to intracellular organelles where it can be metabolized or utilized. Aberrations in these processes lead to multiple disease pathologies, including atherosclerosis and Niemann Pick C (NPC) disease. In this project, genetic strategies in yeast will be applied to study sterol transport and sensing as it pertains to human NPC disease. Using a candidate gene approach based on sequence conservation between humans and yeast, we have identified an excellent yeast homolog to the gene defective in Niemann Pick type C disease (NPC1). There are two principle aims to this project. Aim 1 will define the role of the Niemann-Pick C type 1 gene family in intracellular sterol transport. This will test the hypothesis that NPC1 and its yeast homolog, Ncr1, mediate vesicle fusion events and/or act as sterol responsive signal transducers. Molecular, genetic and biochemical approaches including site-directed mutagenesis and sub-cellular protein localization, will be applied. Aim 2 has two components that target pathways that interact in NPC disease to mediate sterol transport and sensing. Firstly, to characterize components of sterol transport pathways by virtue of interactions with the NCR1 gene product that effect normal cell viability. The second component will isolate constituents of the NPC/Ncr-complex to test the hypothesis that key steps in sterol transport and its regulation involve protein-protein interactions. Factors that complex with NCR1 and NPC1 will be identified using the yeast two-hybrid system. The NPC2 gene may arise from this screen. These studies will likely elucidate NPC disease in particular and intracellular sterol transport in general.

细胞内游离甾醇的浓度受到严格调节 优化所有真核细胞膜的流动性和功能。 这 体内平衡是由多种机制实现的，其关键是 甾醇的传感和该分子从合成位点的运输 或摄取到细胞内细胞器，在那里它可以被代谢或 被利用。 这些过程的异常会导致多种疾病 病理学，包括动脉粥样硬化和尼曼皮克 C (NPC) 疾病。 在这个项目中，酵母的遗传策略将被应用于研究 甾醇转运和传感与人类鼻咽癌疾病有关。 使用 基于人类之间序列保守的候选基因方法 和酵母，我们已经鉴定出该基因的优秀酵母同源物 C 型尼曼皮克病 (NPC1) 有缺陷。 有两个 原则旨在该项目。 目标 1 将定义以下角色： Niemann-Pick C 1 型基因家族在细胞内甾醇运输中的作用。 这将检验 NPC1 及其酵母同源物 Ncr1 的假设， 介导囊泡融合事件和/或充当甾醇反应信号 换能器。 分子、遗传和生化方法，包括 定点突变和亚细胞蛋白定位，将 被应用。 目标 2 有两个部分，针对相互作用的途径 在鼻咽癌疾病中介导甾醇转运和传感。 首先，要 凭借以下特征来表征甾醇转运途径的组成部分 与影响正常细胞的 NCR1 基因产物的相互作用 生存能力。 第二个成分将隔离的成分 NPC/Ncr 复合物检验甾醇中关键步骤的假设 运输及其调节涉及蛋白质-蛋白质相互作用。 与 NCR1 和 NPC1 复合的因子将使用 酵母二杂交系统。 NPC2基因可能源自该筛选。 这些研究可能会特别阐明鼻咽癌疾病 一般细胞内甾醇转运。