ROLE OF ORP6 IN INTRACELLULAR CHOLESTEROL TRAFFICKING

体内平衡，或由生命系统维持一个稳定状态的内部环境，是生命的核心原则。胆固醇稳态是一个保守的和必要的真核过程。在哺乳动物细胞内，细胞膜内胆固醇水平受到严格调节，通过复杂的细胞机制在不同的细胞内部位维持适当的胆固醇浓度。为此，细胞间的胆固醇运动需要囊泡性和非囊泡性胆固醇运输途径。尽管对这些途径进行了广泛的研究，但关于胆固醇在不同细胞间转移的机制以及这些过程是如何被调节的，仍然存在许多问题。氧甾醇结合（OSBP）相关蛋白（ORPs）是一个脂质转移蛋白（LTPs）家族，参与许多与氧甾醇、细胞器间接触、脂质代谢和非囊性固醇运输相关的细胞过程。我的NSERC研究项目的目标是研究ltp在维持细胞和全身胆固醇稳态中的作用。氧甾醇结合蛋白相关蛋白6 （ORP6）是ORP家族中一个特征不明确的成员，最近被发现是细胞内胆固醇水平的一种新的调节因子。我们之前的研究表明，ORP6调节胆固醇在细胞间（核内体和内质网）和质膜之间的运输以进行外排。然而，ORP6如何调节这些通路，以及它在大脑中表达最多的地方的功能，都是未知的。基因和蛋白表达数据表明，ORP6在大脑中的表达相对于其他组织，更具体地说，在中枢神经系统（CNS）的星形胶质细胞和神经元中的表达相对于其他细胞丰富。为了研究ORP6如何调节胆固醇转运并确定其在全身胆固醇稳态中的生理作用，我们提出：1 .研究ORP6在神经元和神经胶质胆固醇稳态中的作用。2。确定orp6介导的胆固醇转运机制。3。确定ORP6在胆固醇稳态中的生理作用。目前的提案结合了我在固醇结合蛋白，胆固醇运输和脂质代谢方面的独特专业知识。我们将采用实验室建立的技术组合来测试ORP6调节中枢神经系统胆固醇稳态的假设。由于运送脂质供给身体组织的脂蛋白不能穿过血脑屏障，因此大脑是研究脂质稳态的一个特别有趣的微环境，因为它必须受到更严格的控制。有了功能性生物学工具来研究ORP6，我们准备剖析这种独特的ORP家族成员在细胞胆固醇稳态中的基本作用机制，并阐明中枢神经系统和哺乳动物细胞中脂质转运的基本机制。

Homeostasis, or the maintenance of a steady-state internal environment by living systems, is a core principal of life. Cholesterol homeostasis is a conserved and essential eukaryotic process. Within mammalian cells, intracellular membrane cholesterol levels are tightly regulated, with complex cellular mechanisms in place to maintain proper cholesterol concentrations at distinct intracellular sites. For this, both vesicular and non-vesicular cholesterol trafficking pathways are required for cholesterol movement between cell compartments. Despite extensive study of these pathways, many questions remain about the mechanisms of cholesterol transfer between different cell compartments and how these processes are regulated. Oxysterol-binding (OSBP)-related proteins (ORPs) are a family of lipid transfer proteins (LTPs) implicated in many cellular processes related with oxysterols, inter-organelle contact, lipid metabolism and non-vesicular sterol transport. The goal of my NSERC research program is to investigate the role of LTPs in the maintenance of cellular and whole-body cholesterol homeostasis. Oxysterol-binding protein-Related Protein 6 (ORP6), a poorly characterized member of the ORP family, has recently emerged as a novel regulator of intracellular cholesterol levels. We previously showed that ORP6 regulates cholesterol trafficking between cell compartments (endosomes and the ER) and the plasma membrane for efflux. Yet, how ORP6 regulates these pathways, and its function in the brain where it is most highly expressed, are unknown. Gene and protein expression data provide evidence that ORP6 expression is enriched in the brain relative to other tissues, and more specifically in astrocytes and neurons relative to other cells of the central nervous system (CNS). To investigate how ORP6 regulates cholesterol transport and to define its physiological role in whole-body cholesterol homeostasis, we propose to: I.Investigate the role of ORP6 in neuronal and glial cholesterol homeostasis. II.Identify mechanisms of ORP6-mediated cholesterol trafficking. III.Determine the physiological role of ORP6 in cholesterol homeostasis. The current proposal combines my unique expertise on oxysterol-binding proteins, cholesterol trafficking, and lipid metabolism. We will employ a combination of techniques established in the lab to test the hypothesis that ORP6 regulates cholesterol homeostasis in the CNS. Because lipoproteins that shuttle lipid to supply tissues of the body cannot cross the blood-brain barrier, the brain constitutes a particularly interesting microenvironment to study lipid homeostasis as it must be more tightly controlled. With functional biological tools in-hand to study ORP6, we are poised to dissect the fundamental mechanisms of action of this unique ORP family member in cellular cholesterol homeostasis and shed light on fundamental mechanisms of lipid transport in the CNS and mammalian cells.