Effects of lipidomic diversity on GPCR

脂质组多样性对 GPCR 的影响

• 批准号: 9310992
• 负责人: Edward Ray Lyman
• 金额: $ 33.55万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9310992
• 关键词: ADORA2A gene; Affect; Biochemical; Biological Assay; Biological Models; Biophysics; Blood flow; Cardiac Myocytes; Cell Culture Techniques; Cell Membrane Proteins; Cell membrane; Cells; Cellular biology; Characteristics; Cholesterol; Complex; Complex Mixtures; Computer Simulation; Controlled Environment; Coupling; Cultured Cells; Data; Dependence; Diet; Dietary Fats; Environment; Epithelial Cells; Fatty Acids; Follow-Up Studies; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Genetic; Goals; Heart; Individual; Integral Membrane Protein; Kinetics; Knowledge; Leukocytes; Ligand Binding; Ligands; Lipid Bilayers; Lipids; Lovastatin; Measurement; Measures; Membrane; Membrane Proteins; Molecular; Molecular Conformation; Mutagenesis; Neurons; Parkinson Disease; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Physiological; Plasma Cells; Property; Proteins; Purinergic P1 Receptors; Receptor Signaling; Records; Research; Signal Transduction; Site; Specificity; Sterols; Stress; Testing; Therapeutic; Thermodynamics; Tissues; Variant; Viscosity; base; cell type; cellular imaging; feeding; inhibitor/antagonist; membrane model; methyl-beta-cyclodextrin; personalized medicine; physical property; protein function; proteoliposomes; prototype; receptor; receptor function; reconstitution; response; simulation; targeted treatment; therapeutic target; unilamellar vesicle

Integral membrane protein (IMP) function depends on the membrane environment, both through the activity of specific lipids as allosteric modulators and via bulk membrane properties like viscosity, rigidity, and order. Recent lipidomic analyses have established the compositional details of membrane complexity, and provided clear evidence that disparate cell types — and even the same cell type in different individuals — present unique, grossly different membrane environments. Our preliminary observations suggest that these distinct membrane environments influence the conformation and activity of G protein-coupled receptors (GPCRs), and likely modulate their response to targeted therapeutics. Moreover, our recent observations reveal that membrane phenotypes are remarkably susceptible to exogenous perturbations, suggesting that extrinsic factors like diet or lipid synthesis inhibitors could synergize with GPCR-targeted therapeutics. It is therefore critical to define the spectrum of regulatory mechanisms imparted on proteins by lipid bilayers, with this knowledge especially impactful for high value targets, such as GPCRs. Our long-term goal is to exploit tissue-specific differences in membrane environments to predict and tune the efficacy and specificity of therapeutics targeting GPCRs. The objective of this proposal is to leverage advances in lipidomics and GPCR reconstitution to bridge the membrane complexity of live cells with the controlled environments provided by model systems. By combining genetics, cell biology, lipidomics, GPCR reconstitution into proteoliposomes, and molecular simulations we aim to determine the mechanisms by which membrane composition determines ligand binding and signaling characteristics for the adenosine A2A receptor (A2AR). Our central hypothesis is that ligand off-rates and G protein coupling are affected by local membrane order, which is a function of both cholesterol concentration and overall lipid composition. Across model systems, our preliminary observations suggest that cholesterol concentration is a key modulator of GPCR activity, with effects on both ligand binding thermodynamics and signaling kinetics. We will decipher whether the effects of cholesterol are due to specific interactions with the receptor or effects on membrane physical properties by independently varying and measuring these properties in simulations, model membranes, and live cells. Lipidomic analysis of A2AR-relevant target cell types — neurons, leukocytes, cardiomyocytes, and epithelial cells — will inform molecular simulations of complex mixtures, and reconstitution of A2AR into membranes obtained from these same cells will determine the extent of signaling variation across cell types. Finally, we will test whether the effects of lipidomic perturbations — including cholesterol depletion / loading, fatty acid feeding, and drugs that modulate fatty acid and cholesterol synthesis — recapitulate A2AR functional dependence observed in model systems. This final goal will test a prototype therapy based on lipidomic perturbation, to locally and specifically enhance A2AR therapeutics..

整合膜蛋白（IMP）的功能取决于膜环境，两者都通过 特定脂质作为变构调节剂并通过体膜特性（如粘度、刚性和秩序）。最近的 脂质组学分析已经确定了膜复杂性的组成细节，并提供了清晰的信息 有证据表明不同的细胞类型——甚至不同个体的相同细胞类型——呈现出独特的、 截然不同的膜环境。我们的初步观察表明这些不同的膜 环境影响 G 蛋白偶联受体 (GPCR) 的构象和活性，并且可能 调节他们对靶向治疗的反应。此外，我们最近的观察表明，膜 表型非常容易受到外源扰动的影响，这表明饮食或饮食等外在因素 脂质合成抑制剂可以与 GPCR 靶向治疗产生协同作用。因此，定义 脂质双层对蛋白质施加的一系列调节机制，尤其是这一知识 对于 GPCR 等高价值目标具有影响力。 我们的长期目标是利用膜环境中组织特异性的差异来预测和调整 针对 GPCR 的治疗的功效和特异性。该提案的目的是利用 脂质组学和 GPCR 重建的进展将活细胞的膜复杂性与 模型系统提供的受控环境。通过结合遗传学、细胞生物学、脂质组学、GPCR 重组为蛋白脂质体，并进行分子模拟，我们的目的是确定其机制 膜组成决定腺苷 A2A 受体的配体结合和信号传导特征 （A2AR）。我们的中心假设是配体解离速率和 G 蛋白偶联受到局部 膜顺序，是胆固醇浓度和总体脂质组成的函数。 在模型系统中，我们的初步观察表明胆固醇浓度是关键 GPCR 活性调节剂，对配体结合热力学和信号动力学都有影响。我们将 破译胆固醇的影响是否是由于与受体的特定相互作用或对 通过在模拟、模型中独立改变和测量膜的物理特性 膜和活细胞。 A2AR 相关靶细胞类型（神经元、白细胞、 心肌细胞和上皮细胞——将为复杂混合物的分子模拟和重建提供信息 A2AR 进入从这些相同细胞获得的膜中将决定跨细胞的信号传导变异程度 细胞类型。最后，我们将测试脂质组学扰动（包括胆固醇消耗）的影响是否/ 负荷、脂肪酸饲喂以及调节脂肪酸和胆固醇合成的药物 — 概括 A2AR 在模型系统中观察到的功能依赖性。这个最终目标将测试基于脂质组学的原型疗法 扰动，以局部和特异性地增强 A2AR 治疗。