SR-BI and PCPE2: Novel partners in bi-directional cholesterol transport

项目摘要 清道夫受体B I型（SR-BI）和高密度脂蛋白（HDL）的受体-配体复合物是 负责从体内清除胆固醇酯（CE）。这一进程对于防止 高胆固醇血症和动脉粥样硬化。最近，一项小鼠研究报告称，敲除（KO） 前胶原内肽酶增强子2（PCPE 2），一种细胞外基质蛋白，导致了一种矛盾的 发现血浆HDL升高与动脉粥样硬化程度增加相关。补充调查 表明，尽管PCPE 2 KO小鼠肝脏中SR-BI水平增加， 清除率和甾醇排泄减少。基于这些新的发现，我们认为PCPE 2 直接与HDL颗粒或SR-BI或两者的组合相互作用，以稳定HDL相互作用 和/或提供支架以将SR-BI保持在膜内的适当位置。在这个多PI Sorci-Thomas博士和Sahoo博士建议开展合作研究来验证这一假设 PCPE 2使细胞表面的SR-BI受体功能化， 运输在目标1中，我们研究了SR-BI如何与PCPE 2协同工作以维持细胞胆固醇 体内平衡新生成的CRISPR/Cas9 PCPE 2缺失的3 T3细胞系和新的组织特异性PCPE 2 敲除小鼠模型将用于阐明SR-BI之间伙伴关系的生理相关性。 和PCPE 2，并定义它们如何共同促进受体易位和双向胆固醇 运输初步数据已经支持了这种伙伴关系在小鼠脂肪中的独特生理作用 组织中，在分离的小鼠脂肪细胞和分化的3 T3细胞中，这两种蛋白质起作用， 在脂质储存中的重要作用。目标2将探索定义SR-BI如何与PCPE 2合作的机制， 增强从HDL摄取CE。具体地，我们将确定PCPE 2是否促进SR-BI在细胞上的寡聚化。 质膜，这是一个过程，假定使HDL-CE交付到细胞，也许是通过协调 HDL和SR-BI之间的相互作用。这将在新鲜分离的脂肪细胞以及我们的 最近使用CRISPR/Cas9方法创建了PCPE 2缺失的3 T3细胞系。在目标2中，利用以下组合 诱变，创新的生化和质谱策略，我们将确定特定的区域， PCPE 2与SR-BI或PCPE 2与HDL之间的相互作用。总之，这些创新的研究将利用 Sorci-Thomas和Sahoo实验室的互补专业知识，以确定调节SR的新机制， BI在胆固醇双向转运中的作用。这些研究的结果将有助于确定新的 用于预防高胆固醇血症及其相关病理的治疗策略， 动脉粥样硬化

PROJECT SUMMARY The receptor-ligand complex of scavenger receptor class B type I (SR-BI) and high density lipoprotein (HDL) is responsible for removal of cholesteryl ester (CE) from the body. This process is crucial to the prevention of hypercholesterolemia and atherosclerosis. Recently, a study in mice reported that knocking out (KO) procollagen endopeptidase enhancer 2 (PCPE2), an extracellular matrix protein, resulted in a paradoxical finding of increased plasma HDL associated with a greater extent of atherosclerosis. Additional investigation demonstrated that in spite of increased SR-BI levels in livers of PCPE2 KO mice, both plasma cholesterol clearance and sterol excretion were reduced. Based on these novel findings, we suggest that PCPE2 either directly interacts with HDL particles or with SR-BI, or a combination of the two, to stabilize HDL interaction(s) with the receptor and/or provide a scaffold to hold SR-BI in place within the membrane. In this multi-PI application Dr. Sorci-Thomas and Dr. Sahoo propose to carry out collaborative studies to test the hypothesis that PCPE2 functionalizes SR-BI receptors on the cell surface to facilitate bidirectional cholesterol transport. In Aim 1 we investigate how SR-BI works in synergy with PCPE2 to maintain cellular cholesterol homeostasis. A newly-generated CRISPR/Cas9 PCPE2-deleted 3T3 cell line and a new tissue-specific PCPE2 knockout mouse model will be used to elucidate the physiological relevance of the partnership between SR-BI and PCPE2, and define how they work together to promote receptor translocation and bidirectional cholesterol transport. Preliminary data already support a unique physiological role for this partnership in mouse adipose tissue, in isolated mouse adipocytes and in differentiated 3T3 cells, suggesting that these two proteins play a significant role in lipid storage. Aim 2 will explore the mechanism defining how SR-BI partners with PCPE2 to enhance CE uptake from HDL. Specifically, we will determine if PCPE2 facilitates SR-BI oligomerization on the plasma membrane, a process that is postulated to enable HDL-CE delivery into cells, perhaps by orchestrating interactions between HDL and SR-BI. This will be tested in both freshly isolated adipocytes as well as in our PCPE2-deleted 3T3 cell line recently created using CRISPR/Cas9 methods. In Aim 2, utilizing a combination of mutagenesis, and innovative biochemical and mass spectrometry strategies, we will identify specific regions of interaction between PCPE2 and SR-BI or PCPE2 and HDL. Together, these innovative studies will exploit the complementary expertise of the Sorci-Thomas and Sahoo labs to identify novel mechanisms that regulate SR- BI's function in bidirectional cholesterol transport. The findings from these studies will help identify new therapeutic strategies for preventing hypercholesterolemia and its associated pathologies such as atherosclerosis.