Selective uptake and hydrolysis of cholesteryl ester by SR-BI

本研究的长期目标是了解B型清道夫受体I的功能 (SR-BI)在将胆固醇酯(CE)从高密度脂蛋白(HDL)输送到血浆中 其后续新陈代谢所需的膜。SR-BI是调节高密度脂蛋白胆固醇的高密度脂蛋白受体 代谢，并与高密度脂蛋白具有动脉粥样硬化保护作用的能力直接相关。因此，理解 SR-BI如何将高密度脂蛋白-CE运送到质膜上允许其有效代谢的位置是关键 开发预防心血管疾病的方法。这项提案包括三个主要部分 目的探讨SR-BI介导的高密度脂蛋白-CE的摄取和降解机制。目标1 将研究SR-BI在质膜上的结构组织。目标1将使用荧光 共振能量转移技术扩展了我们对SR-BI寡聚结构的理解 在存在和不存在配体的情况下的完整细胞中。此外，突变研究将有助于描绘 SR-BI中低聚体特性所需的区域。目标2将检查功能 自旋标记脂肪酸对色氨酸猝灭SR-BI胞外区结构域的影响。目标2 旨在检验SR-BI修饰膜磷脂成分以有利于 选择性摄取高密度脂蛋白-CE。目标1将比较野生型动物的肝脏/肾上腺膜磷脂分布 和SR-BI基因敲除小鼠进行串联质谱分析。然后，我们将使用腺病毒介导的 SR-BI及其突变体的肝脏/肾上腺表达研究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 膜磷脂的改变对选择性摄取效率的影响 去饱和酶/延长酶或通过将SR-BI重组为具有不同比例的鞘磷脂的脂质体： 胆固醇。目标3将帮助我们理解高密度脂蛋白-CE的传递和水解的机制 质膜。我们假设CE在质膜中的积累将阻止 进一步摄取高密度脂蛋白-CE。因此，在目标1、囊泡重建方面，除了使用了一种新颖的 荧光脂质，会让我们测量CE在质膜上的积累量 野生型或非功能性SR-BI突变体。目标2将研究荷尔蒙敏感型的共同定位。 脂肪酶与SR-BI在质膜上对肾上腺细胞和组织中高密度脂蛋白-CE的水解酶：一起， 这些研究将为SR-BI在高密度脂蛋白-CE选择性摄取中的作用提供新的机制信息 并为胆固醇代谢和预防动脉粥样硬化提供了新的见解。

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.