Sterol Sensing and Transport in Model Eukaryote

描述（由申请人提供）：甾醇对细胞的毒性是多种疾病综合征的重要组成部分，从阿尔茨海默病到动脉粥样硬化。我们假设，消除这种威胁的一个关键组成部分是将有害代谢物去除到无毒环境中，通过酯化或外排到细胞外受体进行中和。内质网是固醇稳态的关键细胞器。相对于质膜，该细胞器中的固醇水平维持在非常低的水平，部分原因是通过顺行转运。我们已经确定了这种运输的一个进化保守成分，Arvlp，它指导甾醇从内质网出口到质膜。此外，Arvlp缺失的一个后果是未折叠蛋白反应的诱导。该途径与膜相关变化的关联是前所未有的，并可能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方法来了解arvl介导的固醇转运的机制和后果。在这个修订后的提案中，我们将继续利用酵母的遗传和细胞生物学方法，并扩展这些方法来理解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在酵母中介导甾醇转运的机制。我们假设ARV1（锌结合基元和跨膜性质）的结构是固醇转运所必需的，并且这些结构域介导配体（脂质和蛋白质）的结合。2. 鉴定和表征甾醇稳态的新成分。我们假设ARV1代表了细胞对固醇超载反应的一个决策点。与ARV1相互作用的因子对固醇稳态和细胞存活至关重要。这种途径的一个例子是未折叠蛋白反应（UPR），它是由ARVl的丢失引起的。我们将使用遗传和生化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。我们推测ARVl在人类细胞中的作用还包括将固醇转运到质膜。我们将在各种组织培养细胞模型中测试人类ARVl过表达或敲低的后果。脂质向细胞外受体的外排将被详细研究。

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.