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: 6517497
• 负责人: STEPHEN Laurence STURLEY
• 金额: $ 25.75万
• 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/6517497
• 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 体内平衡是由几种机制赋予的，其中的关键是 甾醇的传感和该分子从合成位点的转运 或吸收到细胞内细胞器，在那里它可以被代谢， 利用。 这些过程的异常导致多种疾病 病理学，包括动脉粥样硬化和尼曼匹克C（NPC）病。 本计画将利用酵母的遗传策略来研究 固醇转运和传感，因为它涉及人NPC疾病。 使用 基于人与人之间序列保守性的候选基因方法 和酵母，我们已经确定了一个很好的酵母同源基因， C型尼曼匹克病（Niemann Pick Type C Disease，NPC 1） 有两 原则是针对这个项目。 目标1将确定 细胞内固醇转运中的Niemann-Pick C 1型基因家族 这将检验NPC 1及其酵母同系物Ncr 1， 介导囊泡融合事件和/或充当甾醇响应信号 传感器 分子、遗传和生物化学方法，包括 定点诱变和亚细胞蛋白定位将 被应用。 AIM 2有两个组件，针对相互作用的途径 在NPC疾病中介导固醇转运和传感。 一是 通过以下方式表征固醇转运途径的组分： 与影响正常细胞的NCR 1基因产物的相互作用 生存能力。 第二个组成部分将分离的成分 NPC/Ncr-复合物来检验甾醇中的关键步骤 转运及其调节涉及蛋白质-蛋白质相互作用。 与NCR 1和NPC 1复合的因素将使用 酵母双杂交系统 NPC 2基因可能来自该筛选。 这些研究将可能阐明特别是NPC疾病， 细胞内固醇转运。