Mechanism of Endocytic Trafficking of Cholesterol

描述（由申请方提供）：细胞胆固醇需求通过对细胞内胆固醇升高做出反应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（SREBP），其激活参与胆固醇合成和摄取的基因的表达。在ER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加工。调节ER胆固醇池似乎是质膜（PM）衍生的，但PM胆固醇转移到ER膜的机制知之甚少。生化和遗传学研究表明，PM脂质在PM胆固醇向ER的运动中起关键作用。我们的中心假设是，PM胆固醇转移到ER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。氧固醇是固醇体内平衡的生理调节剂，可以通过与PM脂质的直接相互作用调节PM至ER胆固醇转移，导致PM胆固醇转移至ER膜的置换。或者，氧固醇可以通过促进ER-PM融合，通过与ER膜的直接相互作用促进PM胆固醇转移到ER。本项目的目标是确定参与这一固醇运输途径的机制，并研究参与PM和ER之间胆固醇转移的分子机制。我们将通过以下方式检验我们的中心假设：1）使用遗传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脂质组成和形态以及对PM至ER胆固醇转运的影响，和3）用独特的胆固醇和氧固醇探针进行生物物理学研究，以检查PM胆固醇转移到ER膜的机制。本提案中概述的研究将有助于我们了解ER胆固醇稳态调节的机制，并可能确定保护胆固醇过载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.