Phosphatidylcholine transfer protein interacts with PPARd to modulate activity

摘要： 据预测，到2030年，美国将有大约一半的人口出现临床肥胖症。肥胖可能会致命，因为 糖尿病、非酒精性脂肪性肝炎、中风和心脏病等并发症的发生。最近， 过氧化物酶体增殖物激活受体δ(PPARδ)激动剂在治疗肥胖和高血压方面显示出巨大的前景。 相关的合并症：增加胰岛素敏感性、体重减轻、耐力和脂肪代谢，而 抑制促炎途径、肝纤维化、平滑肌细胞增殖和内皮细胞 功能障碍。PPAR的内源性配体被认为是花生四烯酸，尽管有大量的研究 显示PPAR结合并被脂肪酸、磷脂酰胆碱(PC)及其代谢物激活。这个 PPAR获得这些在胞浆中产生的亲脂配体的机制尚不清楚。 我们实验室的研究发现了一条PPARδ-FABP5-多不饱和脂肪酸(PUFA)途径，其中PUFA FABP通过FABP穿梭到细胞核，进而上调PPARδ活性。然而，FABP5仅绑定一个子集 已报道的PPARδ配体。为了寻找其他候选脂质运输蛋白(LTPS)，我们进行了一项 LTPS和PPAR之间的蛋白质互补分析(PCA)。我们发现了一种新的相互作用 PPARδ和磷脂酰胆碱转运蛋白(PC-TP)。初步数据显示，这种相互作用与 规范的PPARδ信令。这项提案的总体目标是在生化和功能上表征 PPARδ与PC-TP相互作用的调控。我假设某些PC分子物种 驱动PC-TP易位以抑制PPAR基因的δ反式激活。在目标1中，我将在细胞中使用蛋白质-蛋白质 相互作用试验测试PPARδ与配体结合缺陷的WT或突变体PCTP的结合。在……里面 接下来，我将测试已知的改变PC-TP/PPARδ功能的化学探针在这种相互作用中的作用。这 分析将得到脂质组学的称赞，特别是询问与PC-TP绑定的PC，利用 已知的有利于复杂形成的条件。通过质谱仪检测到与PC-TP结合的脂质将 然后测试它们增强PPARδ结合和抑制的能力。某些扰动可能允许PC- TP与PPARδ相互作用，但可能导致惰性复合体。为了探索这种可能性，我将执行荧光素酶 报道分析和qPCR微阵列专门询问PPARδ控制的基因。在目标2中，我将定义 结合信息确定抑制性全长PPARδ和PC-TP复合体的地形位置 氢-氚交换与质谱学和交联质谱联用得到 实验。这一分析将通过测定络合物的化学计量比和动力学来补充 队形。结合起来，这些方法将在功能和生化方面表征PC-TP如何 通过直接相互作用调节PPARδ，希望确定一个如何异常的分子框架 与肥胖相关的血脂水平可能会影响脂类平衡。

SUMMARY: In 2030 it is predicted that about half of the USA population will be clinically obese. Obesity can be lethal due to development of co-morbidities such as diabetes, nonalcoholic steatohepatitis, stroke, and heart attack. Recently, peroxisome proliferator-activated receptor δ (PPARδ) agonists have shown great promise in treating obesity and associated comorbidities by: increasing insulin sensitivity, weight loss, endurance, and lipid metabolism, while suppressing proinflammatory pathways, liver fibrosis, smooth muscle cell proliferation, and endothelial cell dysfunction. The endogenous ligand for PPARs is thought to be arachidonic acid, although plenty of studies show PPARs bind and are activated by fatty acids, phosphatidylcholines (PCs), and their metabolites. The mechanism by which PPARs gain access to these lipophilic ligands generated in the cytosol remains unknown. Studies in our lab identified a PPARδ-FABP5-polyunsaturated fatty acids (PUFA) pathway, in which PUFAs are shuttled to the nucleus by FABPs which in turn upregulate PPARδ activity. However, FABP5 only binds a subset of reported PPARδ ligands. To find other candidate lipid transport proteins (LTPs), we performed a protein complementation assay (PCA) between LTPs and PPARs. We uncovered a novel interaction between PPARδ and phosphatidylcholine transfer protein (PC-TP). Preliminary data show that this interaction opposes canonical PPARδ signaling. The overall goal of this proposal is to biochemically and functionally characterize the regulation of PPARδ through its interaction with PC-TP. I hypothesize that certain PC molecular species drive PC-TP translocation to inhibit PPARδ transactivation of genes. In Aim 1, I will use in cell protein-protein interaction assays to test PPARδ association with either WT or mutant PCTP, defective in ligand binding. In tandem, I will test the role of chemical probes known to alter PC-TP/ PPARδ function on this interaction. This analysis will be complimented by lipidomics, specifically interrogating PCs bound to PC-TP taking advantage of conditions known to facilitate complex formation. Lipids bound to PC-TP detected via mass spectrometry will then be tested for their ability to enhance PPARδ binding and suppression. Certain perturbations may allow PC- TP to interact with PPARδ but may lead to an inert complex. To probe this possibility, I will perform luciferase reporter assays and qPCR micro-arrays specifically interrogating PPARδ-controlled genes. In Aim 2, I will define the topographic position of the repressive full length PPARδ and PC-TP complex by combining information obtained from hydrogen deuterium exchange coupled to mass spectrometry and crosslinking mass spectrometry experiments. This analysis will be complimented by determining the stoichiometry, and kinetics of complex formation. Combined, these approaches will functionally and biochemically characterize how PC-TP regulates PPARδ through direct interaction in the hopes of determining a molecular framework of how aberrant lipid levels associated with obesity could affect lipid homeostasis.