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的分子驱动器，以调节食欲内源性大麻素（EC）的代谢信号传导对于人类的调节至关重要食欲，疼痛和神经保护作用。脂肪酸结合蛋白（Fabps）可以隔离疏水性EC或将其运输到水解酶；路由到核的EC也激活了过氧化物体增殖物激活受体（PPARS）。 EC水平与肥胖有关肝（L）FABP的敲除小鼠，该肝脏与肠道（I）Fabp共表达肠肠细胞，也适用于肠道（I） PPARA敲除，强调了这些蛋白质的调节作用。我们将探究不理解 EC-FABP，EC-PPAR和FABP-EC-PPAR络合物在近生物学浓度下体外，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信号传导的机械假设。在多个职业阶段的学员，包括从STEM中代表性不足的群体中招募的招募将集成到该研究计划中。要解决的具体问题如下：（1）EC在FABP之间的选择伴侣产生肥胖或瘦肉结果？ LFABP将EC的可能性交付给用于代谢分解的水解酶，而不是由肠肠细胞中的IFABP隔离通过酶法测试，而LFABP的亲和力降低的基本原理将使用高压力溶液状态NMR可有效识别有利于候选位点进行结合。（2）可以转录活性由LFABP与PPARA的EC结合偏好驱动？ PPARA 配体结合结构域（PPARA_LBD）将被纯化，严格分离，并在体外测试EC- 调制转录活性。将比较PPARA_LBD和FABPS的EC结合亲和力。（3）转录活动可能是由EC调制的FABP-PPAR碰撞驱动的构象变化？确定所提出的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蛋白复合物的结合亲和力本身并存在配体的存在具有一系列已知的激活效率。特定地点相关的对LFABP的影响伴侣将通过使用化学的[U-15N]富集蛋白的溶液状态NMR光谱探测每个骨干NH共振的偏移扰动，以定义使用PPARA_LBD的绑定界面，任何复杂形成后发生的变构结构变化及其对EC配体的调节。拍摄这些实验将共同提高我们对（宏）分子网络的理解通过涉及食欲和疼痛调节的EC实现代谢信号传导，从而提高了我们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重要的配体 - 蛋白质和蛋白质蛋白质相互作用的药物。 NIH确定的赠款提案