FABPs Mediate Activation of PPAR Alpha Receptors by N-Acylethanolamines

描述(由申请人提供)：核受体调节一系列不同的生理过程，是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(PPAR1)是由N-酰基乙醇胺(NAE)家族成员激活的核受体，包括油酰乙醇胺(OEA)和棕榈酰乙醇胺(PEA)，以及内源性大麻酰胺。PPAR1受体介导了OEA和PEA的抗炎和厌氧作用，因此可能是治疗炎症和疼痛的有吸引力的治疗靶点。由于NAE的疏水性，它们不能在没有辅助的情况下穿过水胞浆。目前尚不清楚OEA或PEA如何通过细胞质到达核PPAR1受体。最近，我们发现脂肪酸结合蛋白(FABP)是内源性大麻素脱氧核糖核酸的胞内载体。FABP是一种胞质脂肪酸转运蛋白，其结合部位可容纳广泛的亲脂配体，也可同样与OEA和PEA结合。它们的小尺寸和进入细胞核的能力使得FABP很可能是NAE到PPAR1受体的载体。我们假设FABP作为OEA和PEA转运体，通过影响配体的可用性，可能调节PPAR1的活性。目前应用的第一个目标是确定FABP是否将NAE转运到核PPAR1受体。我们将使用化学和遗传方法来抑制FABP的功能，并描述FABP对PPAR1活性的贡献。这项研究将确定第一个核内NAE携带者，并将对NAE信号的调控提供见解。本申请的另一个主要目标是确定内源性大麻素/花生胺转运抑制剂的分子靶标(S)。由于其亲脂性，NAE被认为是被动地通过细胞膜扩散的。然而，载体介导的摄取NAE通过假定的内源性大麻素膜转运体也被提出。尽管缺乏分子证据来证实它的存在，但针对这个假定的转运蛋白的数百种抑制剂已经被合成，并继续被积极地用于内源性大麻素的研究领域。缺乏真正的转运抑制剂的细胞靶点，使人们对这些化合物作为研究工具的特异性和有效性提出了质疑。我们最近表明，FABP抑制剂减少了ANANDAME的细胞内转运，这一效应与转运抑制剂类似。这一目标的目的是提供证据，证明胞浆FABP是内源性大麻素转运抑制剂的靶标。我们将使用过度表达、敲除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止痛特性，识别这些化合物的细胞靶点可能会揭示新的治疗靶点，并将澄清与它们在药物滥用研究中使用相关的模棱两可的问题。这项研究的总体结果将极大地提高我们对NAE信号的理解，并将归因于FABP在内源性大麻素生物学中的新功能。 公共卫生相关性：N-乙酰乙醇胺信号的调节为治疗各种疾病提供了有希望的治疗途径。这项建议的重点是确定调节细胞内N-乙醇胺信号的蛋白质。这项研究的发现可能会导致未来治疗疼痛和炎症的方法。

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. PUBLIC HEALTH RELEVANCE: Modulation of N-acylethanolamine signaling offers promising therapeutic avenues for the treatment of various disorders. This proposal focuses upon characterizing proteins that regulate N-acylethanolamine signaling within the cell. The findings of this study may lead to future therapies for the treatment of pain and inflammation.