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

FABP介导的内源性大麻素调节食欲信号的分子驱动 内源性大麻素(ECs)的代谢信号对人体的调节至关重要 食欲、疼痛和神经保护。脂肪酸结合蛋白(FABP)可以隔离 疏水性内皮细胞或将其运输到水解酶；路由到细胞核的内皮细胞也激活 过氧化物酶体增殖物激活受体(PPAR)。EC水平与肥胖有关 肝脏基因敲除小鼠(L)与肠道共表达的FABP(I)FABP在肠细胞中的表达 PPARa基因敲除，强调了这些蛋白质的调节作用。我们将调查了解不多的人 EC-FABP、EC-PPAR和FABP-EC-PPAR复合体在体外近生理浓度下， 确定亲和力、代谢命运、分子结合界面和构象变化进行测试 关于EC信号的机械论假说。多个职业阶段的实习生，包括 从STEM代表性不足的群体中招募，将完整地参与这一研究计划。 需要解决的具体问题如下：(1)欧洲共同体如何在FABP之间做出选择 伴侣会产生肥胖或瘦身的结果吗？LFABP将ECs交付到 分解代谢的水解酶，而不是被IFABP隔离在肠细胞中的水解酶 进行了酶学检测，而LFABP亲和力降低的原因将通过使用高亲和力的 施压溶液状态核磁共振，以确定能量有利的候选结合位点。(2)可以 转录活性是由LFABP与PPARa的EC结合偏好驱动的？PPARA 配体结合区(PPARa_LBD)将被提纯，严格脱脂，并在体外进行EC- 调节转录活性。将比较PPARa_LBD和FABP的EC结合亲和力。 (3)转录活性能否由EC调节的FABP-PPAR碰撞驱动，从而导致 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的转录功能，我们将首先使用表面等离子体共振来测量 LFABP-PPARA_LBD蛋白复合体在自身和配体存在下的结合亲和力 具有一系列已知的激活效率。特定地点碰撞对LFABP的影响 合作伙伴将通过[U-15N]浓缩的蛋白质的溶液状态核磁共振波谱进行探测，使用化学物质 每个主干NH共振的位移扰动，以定义与PPARA_LBD、Any 络合物形成时发生的变构结构变化，以及EC配体对它们的调节。已被占用 总之，这些实验将促进我们对(宏观)分子网络的理解，这些分子网络的功能是 通过参与食欲和疼痛调节的内皮细胞实现代谢信号，从而促进我们的 了解与肥胖和炎症相关的健康风险。这种认识对设计具有指导意义 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 Grant提案

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