Selective uptake and hydrolysis of cholesteryl ester by SR-BI

本研究的长期目标是了解清道夫受体B I型的功能 （SR-BI）在将胆固醇酯（CE）从高密度脂蛋白（HDL）递送至血浆中的作用 膜，用于随后的代谢。SR-BI是调节HDL胆固醇的HDL受体 高密度脂蛋白（HDL）是高密度脂蛋白代谢的重要组成部分，与HDL的抗动脉粥样硬化能力直接相关。因此了解 SR-BI如何将HDL-CE递送到质膜中允许其有效代谢的位点是关键 开发预防心血管疾病的方法。该提案包括三个主要方面 目的是评价SR-BI介导的HDL-CE摄取和水解机制。要求1 将研究SR-BI在质膜上的结构组织。目标1将使用荧光 共振能量转移技术来扩展我们对SR-BI低聚物结构的理解 在完整的细胞中，在存在和不存在配体的情况下。此外，诱变研究将有助于描述 SR-BI内的低聚物特性所需的区域。目标2将检查功能 结构域的SR-BI的胞外结构域中使用色氨酸淬灭自旋标记的脂肪酸。目的2 旨在检验SR-BI改变膜磷脂组成以有利于 HDL-CE的选择性摄取。目标1将比较野生型小鼠的肝/肾上腺膜磷脂谱， 和SR-BI敲除小鼠。然后，我们将利用腺病毒介导的 SR-BI及其突变体的肝/肾上腺表达，以研究SR-BI的结构区域/功能， 是磷脂形态变化所必需的。目标2将检查SR-BI独立的影响 改变膜磷脂对选择性摄取效率的影响，无论是通过转染细胞， 去饱和酶/延伸酶或通过将SR-BI重构为具有不同比例的鞘磷脂： 胆固醇目的3将帮助我们了解HDL-CE的传递和水解机制， 质膜我们假设CE在质膜上的积累会阻止 进一步摄取HDL-CE。因此，在目标1，囊泡重建中，除了使用一种新的 荧光脂质，将使我们能够测量CE在质膜上的积累， 野生型或非功能性SR-BI突变体。目标2将检查对药物敏感的 脂肪酶与SR-BI在质膜上水解肾上腺细胞和组织中的HDL-CE：一起， 这些研究将为SR-BI在HDL-CE选择性摄取中的作用提供新的机制信息 和水解，并揭示了胆固醇代谢和预防动脉粥样硬化的新见解。

The long-term objective of this research is to understand the function of scavenger receptor class B type I (SR-BI) in the delivery of cholesteryl ester (CE) from high density lipoproteins (HDL) to the plasma membrane for its subsequent metabolism. SR-BI is the HDL receptor that regulates HDL cholesterol metabolism and is directly linked to the ability of HDL to be athero-protective. Therefore, understanding how SR-BI delivers HDL-CE to a site in the plasma membrane that allows for its efficient metabolism is key to developing methods for prevention of cardiovascular disease. This proposal consists of three primary objectives that will evaluate the mechanisms of SR-BI-mediated uptake and hydrolysis of HDL-CE. Aim 1 will investigate the structural organization of SR-BI at the plasma membrane. Goal 1 will use fluorescence resonance energy transfer techniques to extend our understanding of the oligomeric organization of SR-BI in intact cells in the presence and absence of ligands. Additionally, mutagenesis studies will help delineate the region within SR-BI that is required for its oligomeric properties. Goal 2 will examine the functional domains in the extracellular domain of SR-BI using tryptophan quenching by spin labeled fatty acids. Aim 2 is designed to test the hypothesis that SR-BI modifies the composition of membrane phospholipids to favour selective uptake of HDL-CE. Goal 1will compare liver/adrenal membrane phospholipid profiles in wild-type and SR-BI knock-out mice using tandem mass spectrometry. Then, we will exploit adenovirus-mediated liver/adrenal expression of SR-BI and its mutants to investigate Ijhe structural regions/functions of SR-BI that are required for changes in phospholipid speciation. Goal 2 will examine the effects of SR-BI-independent alterations in membrane phospholipids on selective uptake efficiency, either by transfection of cells with desaturases/elongases or by reconstitution of SR-BI into liposomes with different ratios of sphingomyelin: cholesterol. Aim 3 will help us understand the mechanisms of HDL-CE delivery and hydrolysis at the plasma membrane. We hypothesize that an accumulation of CE in the plasma membrane will prevent further uptake of HDL-CE. Therefore, in Goal 1, vesicle reconstitution, in addition to the use of a novel fluorescent lipid, will let us measure the accumulation of CE at the plasma membrane in the presence of wild-type or a non-functional SR-BI mutant. Goal 2 will examine the co-localization of hormone-sensitive lipase with SR-BI at the plasma membrane for hydrolysis of HDL-CE in adrenal cells and tissues: Together, these studies will provide new mechanistic information about the role of SR-BI in HDL-CE selective uptake and hydrolysis, and shed new insights into cholesterol metabolism and protection against atherosclerosis.