Phosphatidylcholine transfer protein interacts with PPARd to modulate activity

磷脂酰胆碱转移蛋白与 PPARd 相互作用以调节活性

• 批准号: 10259706
• 负责人: Samuel Adam Druzak
• 金额: $ 4.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-02 至 2022-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10259706
• 关键词: Acute; Adult; Affect; Agonist; Americas; Arachidonic Acids; Automobile Driving; Binding; Biochemical; Biological Assay; Body Weight decreased; Carrier Proteins; Cell Nucleus; Cell Proliferation; Cells; Chemicals; Child; Clinical; Complement; Complex; Coupled; Cytosol; DNA Binding; Data; Dependence; Deuterium; Development; Diabetes Mellitus; Dyslipidemias; Endoplasmic Reticulum; Endothelial Cells; FABP5 gene; Family; Fatty Acids; Functional disorder; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Homeostasis; Hydrogen; Hydrophobicity; Hypertension; In Vitro; Inflammation; Kinetics; Knock-out; Lead; Lecithin; Length; Ligand Binding; Ligand Binding Domain; Ligands; Lipid Binding; Lipids; Liver Fibrosis; Luciferases; Mass Spectrum Analysis; Mediating; Membrane; Metabolism; Mitochondria; Molecular; Mus; Mutation; Myocardial Infarction; Nuclear Receptors; Obesity; PPAR alpha; PPAR delta; Pathway interactions; Peroxisome Proliferator-Activated Receptors; Plasma; Play; Polyunsaturated Fatty Acids; Population; Positioning Attribute; Protein translocation; Proteins; RXR; Regulation; Reporter; Reporting; Role; Signal Transduction; Smooth Muscle Myocytes; Stroke; Structure; Testing; Transactivation; Transcript; Transcriptional Regulation; comorbidity; crosslink; experimental study; fatty acid-binding proteins; insight; insulin sensitivity; knock-down; lipid metabolism; lipid transport; lipidomics; lipophilicity; member; mutant; new therapeutic target; nonalcoholic steatohepatitis; novel; obesity management; phosphatidylcholine transfer protein; protein complex; protein protein interaction; response; stoichiometry; therapeutic target

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.

概括： 在2030年，预测美国大约一半的人口将在临床上肥胖。肥胖可能是致命的 糖尿病，非酒精性脂肪性肝炎，中风和心脏病发作等合并症的发展。最近， 过氧化物组增殖物激活的受体δ（PPARδ）激动剂在治疗肥胖和 相关的合并症：提高胰岛素敏感性，体重减轻，耐力和脂质代谢，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。尽管大量研究 显示PPARS结合并被脂肪酸，磷脂酰胆碱（PC）及其代谢产物激活。这 PPAR能够访问细胞质中产生的这些亲脂性配体的机制仍然未知。 我们实验室的研究确定了PPARδ-FABP5-饱和脂肪酸（PUFA）途径，其中PUFA为 Fabps将其穿梭到核上，这反过来呈上调PPARδ活性。但是，FabP5仅绑定子集 报告的PPARδ配体。为了找到其他候选脂质转运蛋白（LTP），我们进行了 LTP和PPAR之间的蛋白质完成评估（PCA）。我们发现了之间的新颖互动 PPARδ和磷脂酰胆碱转移蛋白（PC-TP）。初步数据表明，这种相互作用相反 规范PPARδ信号传导。该建议的总体目标是在生化和功能上表征 PPARδ通过与PC-TP的相互作用来调节。我假设某些PC分子物种 驱动PC-TP易位以抑制基因的PPARδ反式激活。在AIM 1中，我将用于细胞蛋白蛋白质 与WT或突变体PCTP测试PPARδ相关的相互作用测定，在配体结合中有缺陷。在 串联，我将测试已知在这种相互作用中改变PC-TP/PPARδ功能的化学问题的作用。 分析将由脂质组学称赞，特别是询问与PC-TP约束的PC 已知的促进复杂形成的条件。通过质谱检测到与PC-TP结合的脂质将会 然后测试其增强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物。为了探究这种可能性，我将执行荧光素酶 记者测定和QPCR微阵列专门询问PPARδ控制基因。在AIM 2中，我将定义 通过组合信息，反射全长PPARδ和PC-TP复合物的地形位置 从与质谱和交联质谱法相连的氢氘交换中获得 实验。通过确定化学计量学和复杂的动力学，将对这种分析表示赞赏 形成。这些方法合并在功能和生化上会表征PC-TP的方式 通过直接相互作用调节PPARδ，以期确定一个分子框架的异常 与肥胖相关的脂质水平可能影响脂质稳态。