FABPs Mediate Activation of PPAR Alpha Receptors by N-Acylethanolamines

描述（由申请人提供）：核受体调节多种生理过程，并代表治疗应用的有吸引力的靶点。过氧化物酶体增殖物激活受体α（PPAR 1）是由脂质的N-酰基乙醇胺（NAE）家族的成员激活的核受体，其包括油酰乙醇胺（OEA）和棕榈酰乙醇胺（PEA），以及内源性大麻素花生四烯酸。PPAR 1受体介导OEA和PEA的抗炎和促炎作用，因此可能代表用于治疗炎症和疼痛的有吸引力的治疗靶点。由于它们的疏水性，NAE不能在没有辅助的情况下穿过含水胞质溶胶。目前尚不清楚OEA或PEA如何通过细胞质到达核PPAR 1受体。最近，我们确定了脂肪酸结合蛋白（FABP）作为内源性大麻素anandamide的细胞内载体。FABP是细胞溶质脂肪酸运输蛋白，其结合位点适应广泛的亲脂性配体，并且同样可以结合OEA和PEA。它们的小尺寸和进入细胞核的能力使得FABP成为NAE至PPAR 1受体的可能载体。我们推测，FABP作为OEA和PEA转运蛋白，并通过影响配体的可用性，可能会调节PPAR 1的活性。本申请的第一个目的是确定FABP是否将NAE转运至核PPAR 1受体。我们将采用化学和遗传方法来抑制FABP功能，并描绘FABP对PPAR 1活性的贡献。这项研究将确定第一个核内NAE携带者，并将深入了解NAE信号的调节。本申请的另一个主要目标涉及鉴定内源性大麻素/大麻素转运抑制剂的分子靶标。由于其亲脂性，NAE被认为是被动扩散通过细胞膜。然而，载体介导的摄取NAEs通过推定的内源性大麻素膜转运蛋白也已提出。尽管缺乏分子证据来证实其存在，但已经合成了数百种针对这种假定转运蛋白的抑制剂，并继续在内源性大麻素研究界积极使用。缺乏真正的细胞转运抑制剂的目标提出了关于这些化合物作为研究工具的特异性和有效性的问题。我们最近发现FABP抑制剂减少花生四烯酸乙醇胺的细胞内转运，转运抑制剂模拟了这种作用。这一目标的目的是提供证据表明，胞质FABP是内源性大麻素转运抑制剂的目标。我们将采用过表达，敲低和直接结合的方法来证明这些化合物与FABP相互作用。由于转运抑制剂具有镇痛特性，确定这些化合物的细胞靶点可能会揭示新的治疗靶点，并将澄清与其在药物滥用研究中的使用相关的模糊性。这项研究的总体结果将大大提高我们对NAE信号传导的理解，并将内源性大麻素生物学中的新功能归因于FABPs。

DESCRIPTION (provided by applicant): Nuclear receptors regulate a diverse set of physiological processes and represent attractive targets for therapeutic applications. Peroxisome proliferator-activated receptor alpha (PPAR1) are nuclear receptors that are activated by members of the N-acylethanolamine (NAE) family of lipids, which includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and the endocannabinoid anandamide. PPAR1 receptors mediate the anti-inflammatory and anorexigenic effects of OEA and PEA and may therefore represent attractive therapeutic targets for the treatment of inflammation and pain. Due to their hydrophobicity, NAEs are unable to traverse the aqueous cytosol unassisted. It is currently not known how OEA or PEA navigate the cellular cytoplasm to reach nuclear PPAR1 receptors. Recently, we identified fatty acid binding proteins (FABPs) as intracellular carriers for the endocannabinoid anandamide. FABPs are cytosolic fatty acid trafficking proteins whose binding sites accommodate a broad range of lipophilic ligands and may likewise bind OEA and PEA. Their small size and ability to enter the nucleus renders FABPs as likely carriers for NAEs to PPAR1 receptors. We hypothesize that FABPs act as OEA and PEA transporters, and by affecting ligand availability, may regulate PPAR1 activity. The first aim of the current application is to determine whether FABPs transport NAEs to nuclear PPAR1 receptors. We will employ chemical and genetic approaches to inhibit FABP function and delineate the contribution of FABPs towards PPAR1 activity. This study will identify the first intranuclear NAE carriers and will shed insights into the regulation of NAE signaling. The other major goal of this application involves identifying the molecular target(s) of endocannabinoid/anandamide transport inhibitors. Owing to their lipophilic nature, NAEs have been proposed to passively diffuse through cellular membranes. However, carrier-mediated uptake of NAEs via a putative endocannabinoid membrane transporter has also been proposed. Despite lacking molecular evidence to substantiate its existence, hundreds of inhibitors targeting this putative transporter have been synthesized and continue to be actively used in the endocannabinoid research community. The lack of a bona fide cellular target for transport inhibitors raises questions about the specificity and validity of these compounds as research tools. We have recently shown that FABP inhibitors reduce the intracellular transport of anandamide, effects that are mimicked by transport inhibitors. The goal of this aim is to provide evidence that cytosolic FABPs are targets of endocannabinoid transport inhibitors. We will employ overexpression, knockdown, and direct binding approaches to demonstrate that these compounds interact with FABPs. Because transport inhibitors possess analgesic properties, identifying cellular targets for these compounds may unmask novel therapeutic targets and will clarify ambiguities associated with their use in drug abuse research. The overall outcome of this study will greatly enhance our understanding of NAE signaling and will ascribe novel functions to FABPs in endocannabinoid biology.