Sterol Sensing and Transport in Model Eukaryote

模型真核生物中的甾醇传感和运输

• 批准号: 7113220
• 负责人: STEPHEN Laurence STURLEY
• 金额: $ 28.77万
• 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 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7113220
• 关键词: SDS polyacrylamide gel electrophoresis; cell line; cytotoxicity; eukaryote; fungal genetics; gene expression; homeostasis; intracellular transport; ligands; lipid metabolism; lipid transport; polymerase chain reaction; protein structure function; site directed mutagenesis; sterols; tissue /cell culture; transport proteins; yeasts

DESCRIPTION (provided by applicant): The toxicity of sterols to cells is a significant component of several disease syndromes ranging from Alzheimer's disease to Atherosclerosis. We hypothesize that a pivotal component of obviating this threat is the removal of the offending metabolite to non-toxic environments, for neutralization by esterification or efflux to extracellular acceptors. The endoplasmic reticulum is a critical organelle with regard to sterol homeostasis. Sterol levels in this organelle are maintained at very low levels, relative to the plasma membrane, in part by anteriograde transport. We have identified an evolutionary conserved component of this transport, Arvlp that directs the egress of sterol from the endoplasmic reticulum to the plasma membrane. Furthermore, one consequence of loss of Arvlp is induction of the unfolded protein response. The association of this pathway with membrane related changes is unprecedented and will likely impact pathologies that arise from protein misfolding (e.g. prion disorders). Our studies will utilize genetic approaches to understand the mechanisms and consequences of ARVl-mediated sterol transport. In this revised proposal, we will continue to exploit genetic and cell biology approaches in yeast, and expand these approaches to understanding the role of Arvl in higher cells and multicellular organisms. Our specific aims and hypotheses are: 1. To define the mechanisms by which Arvlp mediates sterol transport in yeast. We hypothesize that the structure of ARV1 (Zn-binding motif and transmembrane nature) is required for sterol transport and that these domains mediate ligand (lipid and protein) binding. 2. To identify and characterize new components of sterol homeostasis. We hypothesize that ARV1 represents a decision point in terms of responses to sterol overloading of a cell. Factors that interact with ARV1 will be critical to sterol homeostasis and cell survival. One example of such a pathway is the Unfolded Protein response (UPR) which is induced by loss of ARVl. We will identify and analyze these pathways using genetic and biochemical approaches. 3. To define the role of Arvlp in mammalian cells. We hypothesize that the role of ARVl in human cells is also to transport sterol to the plasma membrane. We will test the ramifications of overexpression or knockdown of human ARVl in various tissue culture cell models. Lipid efflux to extracellular acceptors will be studied in detail.

描述（由申请人提供）：固醇对细胞的毒性是从阿尔茨海默病到动脉粥样硬化等多种疾病综合征的重要组成部分。我们假设，消除这种威胁的一个关键组成部分是将有害的代谢物转移到无毒的环境中，通过酯化或外排到细胞外受体进行中和。内质网是一个关键的细胞器，对于固醇的稳态。相对于质膜，该细胞器中的甾醇水平维持在非常低的水平，部分是通过顺行运输。我们已经确定了一个进化保守的组成部分，这种运输，Arvlp，指导出口的固醇从内质网到质膜。此外，Arvlp缺失的一个后果是诱导未折叠蛋白应答。这种途径与膜相关变化的关联是前所未有的，并且可能会影响由蛋白质错误折叠引起的病理（例如朊病毒疾病）。我们的研究将利用遗传学方法来理解ARV 1介导的固醇转运的机制和结果。在这个修订后的提案中，我们将继续利用酵母中的遗传和细胞生物学方法，并扩展这些方法以了解Arvl在高等细胞和多细胞生物中的作用。我们的具体目标和假设是：1。确定Arvlp介导酵母甾醇转运的机制。我们推测ARV1的结构（锌结合基序和跨膜性质）是固醇转运所需的，这些结构域介导配体（脂质和蛋白质）结合。2.确定和表征甾醇稳态的新组分。我们假设ARV1代表了细胞对固醇超载反应的一个决定点。与ARV1相互作用的因子对固醇稳态和细胞存活至关重要。这种途径的一个实例是由ARVl的损失诱导的未折叠蛋白质应答（UPR）。我们将使用遗传和生物化学方法识别和分析这些途径。3.明确Arvlp在哺乳动物细胞中的作用。我们假设ARV1在人细胞中的作用也是将固醇转运到质膜。我们将在各种组织培养细胞模型中测试人ARVl的过表达或敲低的后果。将详细研究脂质流出到细胞外受体。