Sterol Sensing and Transport in Model Eukaryote

描述(由申请人提供)：类固醇对细胞的毒性是从阿尔茨海默氏症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，避免这种威胁的一个关键成分是将有害的代谢物移至无毒环境中，以便通过酯化或排出到细胞外受体进行中和。内质网是维持类固醇稳态的重要细胞器。相对于质膜，这个细胞器中的甾醇水平维持在非常低的水平，部分是由于顺行运输。我们已经确定了这种运输的一个进化保守成分，Arvlp，它直接将固醇从内质网排出到质膜。此外，Arvlp缺失的一个后果是诱导未折叠的蛋白质反应。这一途径与膜相关变化的联系是史无前例的，可能会影响由蛋白质错误折叠引起的病理(例如，Pron紊乱)。我们的研究将利用遗传学方法来了解ARV1介导的类固醇运输的机制和后果。在这个修订后的提案中，我们将继续利用酵母中的遗传和细胞生物学方法，并扩展这些方法以了解Arv1在高等细胞和多细胞生物体中的作用。我们的具体目标和假设是：1.明确Arvlp在酵母中介导类固醇运输的机制。我们假设ARV1的结构(锌结合基序和跨膜性质)是固醇运输所必需的，这些结构域介导了配体(脂和蛋白质)的结合。2.鉴定和鉴定甾醇动态平衡的新成分。我们假设ARV1代表细胞对类固醇超载的反应的一个决策点。与ARV1相互作用的因子将对类固醇稳态和细胞生存至关重要。这种途径的一个例子是由ARV1的缺失诱导的未折叠蛋白反应(UPR)。我们将使用遗传和生化方法来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在人类细胞中的作用也是将类固醇运输到质膜。我们将在不同的组织培养细胞模型中测试人类ARV1过度表达或敲除的后果。脂类外流到细胞外受体将被详细研究。

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.