Regulation of cholesterol transport in adipocytes by SR-BI and PCPE2

摘要 美国成年人和儿童的肥胖患病率每年都在上升。 肥胖是多种慢性疾病的危险因素，包括2型糖尿病和心血管疾病 (CVD)，以及其他。脂肪细胞是脂肪积累的主要细胞，含有脂滴， 对摄取过量循环甘油三酯（TG）的反应作为防止器官损害的保护机制， 脂毒性脂肪细胞含有大约四分之一的身体的游离胆固醇池，但具有减少的胆固醇。 合成自身胆固醇的能力。因此，脂肪细胞在很大程度上依赖于循环脂蛋白来获得 肥大性膨胀所需的胆固醇量。这也意味着胆固醇的运输是 是脂肪细胞功能的重要决定因素。然而，在以下方面仍然存在重大知识差距： 脂肪细胞胆固醇转运的机制和调控。在这个应用程序中，我们建立在令人兴奋的 初步数据表明，双向胆固醇运输功能的HDL受体，清道夫 受体B I型（SR-BI）在缺乏前胶原肽酶内肽酶的情况下受损 增强子2（PCPE 2），一种细胞外基质蛋白，在小鼠脂肪组织和从 小鼠胚胎成纤维细胞（MEFs）。基于这些发现，本研究金提案中描述的实验 将测试PCPE 2促进SR-BI的胆固醇转运功能以调节脂肪细胞的新假设。 胆固醇稳态目标1旨在确定PCPE 2可能促进的机制 脂肪细胞中的SR-BI寡聚化，这是胆固醇转运所需的过程。我们将使用全氟辛酸 酸性聚丙烯酰胺凝胶电泳[PFO-PAGE]，以检测存在或不存在SR-BI寡聚化的情况 PCPE2此外，我们将使用尖端的质谱技术验证SR-BI/PCPE 2 在脂肪细胞中的相互作用，并确定该复合物是否含有其他蛋白质伴侣。目标2旨在 研究PCPE 2可能影响SR-BI介导的信号传导和膜定位的机制。 首先，我们将测量已知触发HDL-胆固醇酯摄取的信号网络（例如MAPK）的激活 途径）。接下来，我们将采用不连续蔗糖梯度分离法分离脂肪细胞脂质 筏微结构域，以在PCPE 2不存在和存在下可视化SR-BI的膜定位。在一起， 这些研究将确定（1）SR-BI和PCPE 2可能发生物理相互作用的机制， 双向胆固醇流，以及（2）PCPE 2如何影响SR-BI在介导双向胆固醇流中的作用。 脂肪细胞中的胆固醇流量。我们预计，这些研究的结果将有助于确定新的 这些信息将帮助我们更好地了解导致肥胖、心血管疾病和其他疾病的机制。 这些情况引起的并发症。

ABSTRACT The prevalence of obesity in the United States continues to rise in both adults and children on an annual basis. Obesity is a risk factor for a multitude of chronic diseases, including type 2 diabetes and cardiovascular disease (CVD), among others. Adipocytes, the primary cells for fat accumulation, contain lipid droplets that expand in response to taking up excess circulating triglycerides (TG) as a protective mechanism to prevent organ lipotoxicity. Adipocytes contain approximately a quarter of the body’s pool of free cholesterol, but have a reduced ability to synthesize its own cholesterol. Therefore, adipocytes rely heavily on circulating lipoproteins to acquire the necessary amount of cholesterol for hypertrophic expansion. This also means that cholesterol transport is an essential determinant for adipocyte function. However, there remains a significant knowledge gap regarding the mechanism and regulation of cholesterol transport in adipocytes. In this application, we build on exciting preliminary data that shows that the bi-directional cholesterol transport functions of the HDL receptor, scavenger receptor class B type I (SR-BI), are compromised in the absence of procollagen peptidase endopeptidase enhancer 2 (PCPE2), an extracellular matrix protein, in mouse adipose tissue and adipocytes differentiated from mouse embryonic fibroblasts (MEFs). Based on these findings, experiments described in this fellowship proposal will test the novel hypothesis that PCPE2 facilitates SR-BI’s cholesterol transport functions to regulate adipocyte cholesterol homeostasis. Aim 1 is designed to determine the mechanism by which PCPE2 may be facilitating SR-BI oligomerization in adipocytes, a process required for cholesterol transport. We will use perfluorooctanoic acid polyacrylamide gel electrophoresis [PFO-PAGE] to test SR-BI oligomerization in the presence or absence of PCPE2. Further, using cutting-edge mass spectrometry technology, we will validate the SR-BI/PCPE2 interaction in adipocytes and determine if this complex contains other protein partners. Aim 2 is designed to investigate the mechanisms by which PCPE2 may impact SR-BI-mediated signaling and membrane localization. First, we will measure activation of signaling networks known to trigger HDL-cholesterol ester uptake (e.g. MAPK pathway). Next, we will perform discontinuous sucrose gradient fractionation methods to isolate adipocyte lipid raft microdomains to visualize SR-BI’s membrane localization in the absence and presence of PCPE2. Together, these studies will identify (1) the mechanism by which SR-BI and PCPE2 may physically interact to facilitate bidirectional cholesterol flux, and (2) how PCPE2 may be impacting SR-BI’s role in mediating bidirectional cholesterol flux in adipocytes. We anticipate that the findings from these studies will help identify novel information that will help us better understand the mechanisms driving obesity, cardiovascular disease, and other complications that arise from these conditions.