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

基本信息

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
项目类别：
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

项目摘要

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

Stark, Ruth E. FABP 介导的内源性大麻素信号通路食欲调节的分子驱动因素内源性大麻素（EC）的代谢信号对于人类的调节至关重要脂肪酸结合蛋白（FABP）可以隔离食欲、疼痛和神经保护。疏水性 EC 或将其转运至水解酶；EC 也可激活过氧化物酶体增殖物激活受体（PPAR）水平与肥胖相关。敲除小鼠肝脏 (L) FABP（与肠细胞 (I) FABP 共表达）以及 PPARa 敲除，强调了这些蛋白质的调节作用，我们将探讨人们知之甚少的问题。 EC-FABP、EC-PPAR 和 FABP-EC-PPAR 复合物在体外接近生理浓度，确定亲和力、代谢命运、分子结合界面和构象变化以进行测试有关 EC 信号传导的机制假设，包括处于多个职业阶段的受训者。从 STEM 中代表性不足的群体中招募的人员将全面参与该研究计划。具体需要解决的问题如下：（1）EC如何在FABP之间进行选择 LFABP 是否有可能产生肥胖或瘦的结果？代谢分解的水解酶而不是被肠上皮细胞中的 IFABP 隔离通过酶法进行测试，而 LFABP 亲和力降低的基本原理将使用高压力溶液状态 NMR 来识别积极有利的候选结合位点 (2) 可以。转录活性是由 EC 对 LFABP 与 PPARa 的结合偏好驱动的吗？配体结合结构域 (PPARa_LBD) 将被纯化、严格脱脂并进行体外 EC-测试将比较 PPARa_LBD 和 FABP 的调节转录活性。 (3) 转录活性是否可以由 EC 调节的 FABP-PPAR 碰撞驱动，从而导致构象变化？确定所提出的 FABP 介导的 EC 中涉及的相互作用为了激活PPARa转录功能，我们首先使用表面等离子共振来测量 LFABP-PPARa_LBD 蛋白复合物本身以及配体存在下的结合亲和力具有一系列已知的激活功效对 LFABP 的特定位点碰撞相关影响。将使用化学方法通过 [U-15N] 富集蛋白质的溶液态 NMR 光谱来探测合作伙伴每个主链 NH 共振的平移扰动来定义与 PPARa_LBD 的结合界面，任何复合物形成时发生的变构结构变化及其通过 EC 配体的调节。总之，这些实验将增进我们对（宏观）分子网络的理解，这些网络的作用是通过涉及食欲和疼痛调节的内皮细胞实现代谢信号传导，从而促进我们的了解与肥胖和炎症相关的健康风险这种了解可以指导设计。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 拨款提案