Administrative Supplement to Molecular Drivers of FABP-mediated Endocannabinoid Signaling for Appetite Regulation

FABP 介导的内源性大麻素信号分子驱动食欲调节的行政补充

• 批准号: 10797598
• 负责人: RUTH E. STARK
• 金额: $ 9.88万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797598
• 关键词: Abbreviations; Address; Administrative Supplement; Affinity; Amides; Animal Model; Animals; Appetite Regulation; Applications Grants; Articulation; Autoimmune Diseases; Binding; Binding Proteins; Binding Sites; Biological Assay; Biophysics; Cell Nucleus; Chemicals; Chimera organism; Complex; Desire for food; Diffusion; Dissociation; Drug Design; Drug Modulation; Endocannabinoids; Enterocytes; Enzymes; Esters; Exhibits; Fatty Acids; Fluorescence; Food Intake Regulation; Future; Genetic Transcription; Glycerol; Health; Heart Diseases; Human; Hydrophobicity; In Vitro; Inflammation; Intestines; Knock-out; Knockout Mice; Ligand Binding; Ligand Binding Domain; Ligands; Lipase; Liver; Measures; Mediating; Metabolic; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Structure; Monitor; Mus; NMR Spectroscopy; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Receptors; Obesity; Outcome; Pain; Pattern; Peroxisome Proliferator-Activated Receptors; Peroxisome Proliferators; Phenotype; Physiological; Protein Binding Domain; Proteins; Regulation; Relaxation; Research; Risk; Role; Satiation; Signal Transduction; Signaling Molecule; Site; Surface Plasmon Resonance; Testing; Thermodynamics; Thinness; Titrations; Underrepresented Populations; United States National Institutes of Health; Vertebral column; Work; career; design; endocannabinoid signaling; experimental study; fatty acid amide hydrolase; fatty acid-binding proteins; heart disease risk; in vitro testing; member; neuroprotection; preference; pressure; programs; protein complex; protein protein interaction; receptor binding; recruit; thyroid hormone receptor associated protein 220

Stark, Ruth E. Molecular Drivers of FABP-mediated Endocannabinoid Signaling for Appetite Regulation Metabolic signaling by endogenous cannabinoids (ECs) is essential to the regulation of human appetite, pain, and neuroprotection. Fatty acid-binding proteins (FABPs) can either sequester the hydrophobic ECs or transport them to hydrolytic enzymes; ECs routed to the nucleus also activate the peroxisome proliferator-activated receptors (PPARs). EC levels have been correlated with obesity in knockout mice for liver (L) FABP that is co-expressed with intestinal (I) FABP in enterocytes and also for PPARa knockouts, underscoring the regulatory roles of these proteins. We will probe poorly understood EC-FABP, EC-PPAR, and FABP-EC-PPAR complexes in vitro at near-physiological concentrations, determining affinities, metabolic fates, molecular binding interfaces, and conformational changes to test mechanistic hypotheses regarding EC signaling. Trainees at multiple career stages, including those recruited from underrepresented groups in STEM, will be integrally involved in this research program. Specific questions to be addressed are as follows: (1) How do ECs choose between FABP chaperones to produce obese or lean outcomes? The possibility that ECs are delivered by LFABP to hydrolytic enzymes for metabolic breakdown rather than sequestered by IFABP in the enterocyte will be tested enzymatically, whereas the rationale for LFABP’s diminished affinity will be explored using high- pressure solution-state NMR to identify energetically favored candidate sites for binding. (2) Could transcriptional activity be driven by EC binding preferences for LFABP vs. PPARa? The PPARa ligand-binding domain (PPARa_LBD) will be purified, rigorously delipidated, and tested in vitro for EC- modulated transcriptional activity. EC binding affinities will be compared for PPARa_LBD and FABPs. (3) Could transcriptional activity be driven by EC-modulated FABP-PPAR collisions that cause conformational changes? To determine the interactions involved in the proposed FABP-mediated EC activation of PPARa transcriptional function, we will first use surface plasmon resonance to measure the binding affinity of LFABP-PPARa_LBD protein complexes, on their own and in the presence of ligands with a range of known activation efficacies. The site-specific collision-associated impact on the LFABP partner will be probed by solution-state NMR spectroscopy of the [U-15N]-enriched protein, using chemical shift perturbations of each backbone NH resonance to define the binding interface with PPARa_LBD, any allosteric structural changes that occur upon complex formation, and their modulation by EC ligands. Taken together, these experiments will advance our understanding of (macro)molecular networks that function to achieve metabolic signaling by ECs involved in appetite and pain regulation, thereby advancing our understanding of health risks related to obesity and inflammation. This understanding can guide the design of drugs that modulate these biomedically important ligand-protein and protein-protein interactions. NIH SuRE Grant Proposal

作者：Ruth E. FABP介导的内源性大麻素信号调节食欲的分子驱动因素 内源性大麻素（ECs）的代谢信号传导对于调节人类的代谢是必不可少的。 食欲、疼痛和神经保护。脂肪酸结合蛋白（FABP）可以隔离 疏水EC或运输它们水解酶; EC路由到核也激活 过氧化物酶体增殖物激活受体（PPARs）。EC水平与肥胖相关， 敲除小鼠的肝（L）FABP与肠（I）FABP在肠细胞中共表达， PPARa敲除，强调了这些蛋白质的调节作用。我们将探索鲜为人知的 体外接近生理浓度的EC-FABP、EC-PPAR和FABP-EC-PPAR复合物， 确定亲和力，代谢命运，分子结合界面和构象变化，以测试 关于EC信号传导的机制假说。处于多个职业阶段的受训人员，包括 从STEM中代表性不足的群体中招募，将整体参与这项研究计划。 具体问题如下：（1）EC如何在FABP之间进行选择 产生肥胖或瘦的结果？EC由LFABP递送至 肠上皮细胞中用于代谢分解的水解酶，而不是被IFABP螯合， 酶法测试，而LFABP的亲和力降低的基本原理将使用高- 压力溶液状态NMR，以确定积极有利的候选网站的结合。(2)可以 转录活性驱动EC结合LFABP与PPARa的偏好？PPARa 将纯化配体结合结构域（PPARa_LBD），严格脱脂，并在体外测试EC- 调节转录活性。将比较PPARa_LBD和FABP的EC结合亲和力。 (3)转录活性是否由EC调节的FABP-PPAR碰撞驱动， 构象变化确定拟议的FABP介导的EC中涉及的相互作用 激活PPARa转录功能，我们将首先使用表面等离子体共振来测量 LFABP-PPARa_LBD蛋白复合物自身和在配体存在下的结合亲和力 具有一系列已知的激活功效。特定地点碰撞对低层大气反作用平台的影响 伴侣将通过[U-15 N]富集蛋白质的溶液状态NMR光谱进行探测，使用化学方法， 移动每个骨架NH共振的扰动以限定与PPARa_LBD的结合界面，任何 在复合物形成时发生的变构结构变化，以及它们通过EC配体的调节。采取 总之，这些实验将促进我们对（宏观）分子网络的理解，这些网络的功能是 通过参与食欲和疼痛调节的EC实现代谢信号传导，从而促进我们的 了解与肥胖和炎症有关的健康风险。这种理解可以指导设计 调节这些生物医学上重要的配体-蛋白质和蛋白质-蛋白质相互作用的药物。 NIH SuRE资助提案