Mechanism of Endocytic Trafficking of Cholesterol

描述（由申请人提供）：细胞胆固醇需求通过响应细胞内胆固醇升高的负反馈回路进行调节。该途径的核心是固醇调节元件结合蛋白（SREBPs），它激活参与胆固醇合成和摄取的基因表达。在内质网中，胆固醇通过与SREBP裂解激活蛋白（SCAP）结合并诱导构象变化来调节SREBP的加工。调节内质网胆固醇池似乎是质膜（PM）衍生的，尽管PM胆固醇转移到内质网膜的机制尚不清楚。生化和遗传学研究表明，PM脂质在PM胆固醇向内质网的运动中起关键作用。我们的中心假设是，PM胆固醇转移到内质网膜的机制主要取决于PM脂质和PM结构的组织和组成。我们提出胆固醇转移的可能途径是通过直接PM-ER膜接触。氧化甾醇是固醇稳态的生理调节剂，可能通过与PM脂质直接相互作用，调节PM到内质网胆固醇的转移，导致PM胆固醇转移到内质网膜。另外，氧甾醇可能通过与内质网膜的直接相互作用，促进ER-PM融合，促进PM胆固醇向内质网的转移。该项目的目标是确定参与这一固醇运输途径的机制，并研究胆固醇在PM和ER之间转移的分子机制。我们将通过以下方法来验证我们的中心假设：1)使用基因筛选分离具有胆固醇转运缺陷的中国仓鼠卵巢（CHO）细胞突变体；2)表征CHO突变体中遗传缺陷对PM脂质组成和形态的影响，以及PM到内质网胆固醇转运的影响；3)使用独特的胆固醇和氧甾醇探针进行生物物理研究，以检验PM胆固醇转移到内质网膜的机制。本提案中概述的研究将有助于我们理解内质网胆固醇稳态调节的机制，并可能确定防止胆固醇过载的细胞毒性作用的策略。

DESCRIPTION (provided by applicant): Cellular cholesterol requirements are regulated through a negative feedback loop that responds to elevations in intracellular cholesterol. Central to this pathway are sterol regulatory element-binding proteins (SREBPs) that activate expression of genes involved in the synthesis and uptake of cholesterol. In the ER, cholesterol modulates SREBP processing through its binding to the SREBP cleavage activating protein (SCAP) and induction of conformational change. The regulatory ER cholesterol pool appears to be plasma membrane (PM)-derived, though the mechanism by which PM cholesterol is transferred to ER membranes is poorly understood. Biochemical and genetic studies suggest that PM lipids play a critical role in movement of PM cholesterol to the ER. Our central hypothesis is that the mechanism(s) governing the transfer of PM cholesterol to the ER membranes critically depend upon the organization and composition of PM lipids and PM structure. We propose that a possible route of cholesterol transfer is via direct PM-ER membrane contacts. Oxysterols, which are physiological regulators of sterol homeostasis, may modulate PM to ER cholesterol transfer through direct interaction with PM lipids, leading to displacement of PM cholesterol for transfer to ER membranes. Alternatively, oxysterols may facilitate transfer of PM cholesterol to the ER through direct interaction with ER membranes by promoting ER-PM fusion. The goals of this project are to identify the machinery involved in this sterol trafficking pathway and to investigate the molecular mechanisms involved in cholesterol transfer between PM and ER. We will test our central hypothesis by 1) using a genetic screen to isolate Chinese hamster ovary (CHO) cell mutants with cholesterol trafficking defects, 2) characterization of the effect of the genetic defects in the CHO mutants on PM lipid composition and morphology, and on PM to ER cholesterol trafficking, and 3) performing biophysical studies with unique cholesterol and oxysterol probes to examine the mechanism of PM cholesterol transfer to ER membranes. Studies outlined in this proposal will contribute to our understanding of the mechanisms involved in regulation of ER cholesterol homeostasis, and may identify strategies for protection from the cytotoxic effects of cholesterol overload.