Selective uptake and hydrolysis of cholesteryl ester by SR-BI

描述(申请人提供)：这项研究的长期目标是了解B型清道夫受体(SR-BI)在将胆固醇酯(CE)从高密度脂蛋白(HDL)运送到质膜以进行后续代谢的功能。SR-BI是调节高密度脂蛋白胆固醇代谢的高密度脂蛋白受体，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，了解SR-BI如何将高密度脂蛋白-CE输送到质膜上允许其有效代谢的位置是开发预防心血管疾病的方法的关键。这项建议由三个主要目标组成，将评估SR-BI介导的高密度脂蛋白-CE的摄取和水解机制。目的1研究SR-BI在质膜上的结构组成。目标1将使用荧光共振能量转移技术来扩展我们对在存在和不存在配体的情况下完整细胞中SR-BI的寡聚组织的理解。此外，突变研究将有助于描绘SR-BI内其寡聚体特性所需的区域。目标2将使用自旋标记脂肪酸的色氨酸猝灭来检查SR-BI细胞外域的功能域。目的2旨在验证SR-BI改变膜磷脂组成以有利于选择性摄取高密度脂蛋白-CE的假设。目标1将使用串联质谱仪比较野生型和SR-BI基因敲除小鼠的肝脏/肾上腺膜磷脂图谱。然后，我们将使用腺病毒介导的SR-BI及其突变体的肝脏/肾上腺表达来研究SR-BI的结构区域/功能，这些区域/功能是磷脂形态变化所必需的。目标2将考察膜磷脂中SR-BI非依赖性改变对选择性摄取效率的影响，方法是将脱饱和酶/延长酶的细胞转化为SR-BI，或者将SR-BI重组到具有不同鞘磷脂：胆固醇比例的脂质体中。目标3将帮助我们了解高密度脂蛋白-CE在质膜上的传递和水解的机制。我们假设，CE在质膜中的积累将阻止进一步摄取HDLCE。因此，在目标1中，除了使用一种新的荧光脂质外，囊泡重建还将使我们能够测量在野生型或无功能SR-BI突变体存在的情况下CE在质膜上的积累。目标2将研究激素敏感脂肪酶和SR-BI在肾上腺细胞和组织中高密度脂蛋白-CE的质膜上的共定位：这些研究将共同提供关于SR-BI在高密度脂蛋白-CE选择性摄取和水解中的作用的新的机制信息，并为胆固醇代谢和预防动脉粥样硬化提供新的见解。

DESCRIPTION (provided by applicant): 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 1 will 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 the 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.