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

项目总结 清道夫受体BI型(SR-BI)和高密度脂蛋白(HDL)的受体-配体复合体是 负责清除体内的胆固醇酯(CE)。这一过程对于预防 高胆固醇血症和动脉粥样硬化。最近，一项对小鼠的研究报告称，敲除(KO) 前胶原内肽酶增强子2(PCPE2)是一种细胞外基质蛋白，导致一种矛盾的 发现血浆高密度脂蛋白升高与动脉粥样硬化程度更大有关。补充调查 研究表明，尽管PCPE2KO小鼠肝脏中SR-BI水平升高，但血浆胆固醇 清除量和类固醇排泄减少。基于这些新的发现，我们认为PCPE2 直接与高密度脂蛋白颗粒或SR-BI相互作用，或两者的组合，以稳定高密度脂蛋白相互作用(S) 与受体结合和/或提供支架以将SR-BI固定在膜内。在这个多PI中 应用索尔西-托马斯博士和萨胡博士建议开展合作研究来检验这一假说 PCPE2使细胞表面SR-BI受体功能化以促进双向胆固醇 运输。在目标1中，我们研究了SR-BI如何与PCPE2协同工作以维持细胞胆固醇 动态平衡。新构建的CRISPR/Cas9 PCPE2缺失的3T3细胞系和新的组织特异性PCPE2 基因敲除小鼠模型将被用来阐明SR-BI之间伙伴关系的生理相关性 和PCPE2，并确定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3T3细胞中，表明这两种蛋白发挥着 在脂肪储存方面具有重要的作用。AIM 2将探索定义SR-BI与PCPE2如何合作的机制 增强从高密度脂蛋白中摄取CE。具体地说，我们将确定PCPE2是否促进了SR-BI在 质膜，这一过程被认为能够将高密度脂蛋白-CE输送到细胞内，可能是通过编排 高密度脂蛋白与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 PCPE2-删除了最近使用CRISPR/Cas9方法创建的3T3细胞系。在目标2中，使用了 突变，以及创新的生化和质谱学策略，我们将确定 PCPE2与SR-BI或PCPE2与高密度脂蛋白的相互作用。总之，这些创新的研究将利用 索尔西-托马斯和萨胡实验室的互补专业知识，以确定监管SR- BI在胆固醇双向转运中的作用。这些研究的发现将有助于识别新的 预防高胆固醇血症及其相关病理的治疗策略，如 动脉硬化。

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.