Chemical probes to assess endocannabinoid localization, storage, and transport

描述(申请人提供)：内源性大麻素(EC)系统中主要的内源性神经递质是花生胺(AEA)和2-花生四烯基甘油(2-AG)。与研究最充分的神经递质不同，这些化学信使是脂类，因此通过不同的生化过程进行调节。在过去的十年里，人们一直在激烈争论，负责EC合成和降解的代谢途径是否主要负责EC信号的启动和终止。最近有证据表明，存在特定的EC结合蛋白，这可能有助于神经元对EC的摄取，这对这一概念提出了挑战。目前的2-AG信号模型也受到了质疑，因为几项新的研究表明，2-AG是在释放之前存储的，而不是按需合成的。缺乏特定EC载体蛋白和2-AG储存的直接证据，使得这些有争议的假说在很大程度上悬而未决。为了解决这些悬而未决的问题，我们将尝试用多方面的化学-生物学方法来完善对EC在哺乳动物大脑中的运输、储存和释放的理解。首先，我们建议通过实施基于活性的蛋白质图谱和多维蛋白质识别技术(ABPP-MudPIT)来全面定位神经系统中的EC结合蛋白质。EC结合蛋白将在无标签光亲和探针的帮助下得到丰富，并通过多维LC-MS/MS鉴定，具有高特异性和亲和力的蛋白质靶标将在功能分析中进一步研究，以阐明它们在EC信号转导中的作用。其次，我们建议开发新的方法来确定2-AG在哺乳动物脑中的细胞和亚细胞定位。我们将首先尝试开发免疫荧光显微镜的2-AG抗体，方法是合成一种能够与免疫原性载体蛋白偶联的末端功能化的2-AG类似物。我们还将尝试开发生物偶联反应，允许对2-AG的内源池进行荧光标记。这两种2-AG显像池的策略都将得益于2-AG相对于其他不饱和单甘油脂的天然高丰度，以及MAGL基因敲除小鼠2-AG水平的选择性增加。作为一个整体，该项目有可能阐明EC的调节机制，并为EC相关疾病的治疗提供新的治疗策略。 与公共健康相关：内源性大麻素是一类神经递质，影响许多正常的生理功能。这个项目的目标是研究内源性大麻素是如何使用化学探针在神经系统中进行调节的。这项工作有可能阐明研究生物系统的新化学策略，并发现内源性大麻素系统中的新治疗靶点。

DESCRIPTION (provided by applicant): Anandamide (AEA) and 2-arachidonylglycerol (2-AG) are the primary endogenous neurotransmitters in the endocannabinoid (EC) system. These chemical messengers, unlike the most well studied neurotransmitters, are lipids and, therefore, regulated through distinct biochemical processes. For the past decade it has been intensely debated whether the metabolic pathways responsible for EC synthesis and degradation are chiefly responsible for EC signal initiation and termination. Challenging this notion, evidence has recently emerged for the existence of specific EC-binding proteins that may facilitate EC uptake in neurons. The current model of 2- AG signaling has also been called into question based on several new studies suggesting that 2-AG is stored prior to release rather than being synthesized 'on-demand'. Direct evidence for specific EC-carrier proteins and for 2-AG storage is lacking, leaving these controversial hypotheses largely unsettled. To address these unresolved questions, we will attempt to refine the understanding of EC transport, storage, and release in the mammalian brain employing a multifaceted chemo-biological approach. First, we propose to comprehensively map EC-binding proteins in the nervous system by implementing activity-based protein profiling and multidimensional protein identification technologies (ABPP-MudPIT). EC-binding proteins will be enriched with the aid of tag-free photoaffinity probes and identified by multidimensional LC-MS/MS. Protein targets exhibiting high specificity and affinity will be further investigated in functional assays to elucidate their role in the EC signaling. Second, we propose to develop new methods to determine the cellular and subcellular localization of 2-AG in the mammalian brain. We will first attempt to develop 2-AG antibodies for immunofluorescence microscopy by synthesizing a terminally functionalized 2-AG analog enabling conjugation to an immunogenic carrier protein. We will also attempt to develop bioconjugation reactions that will allow fluorescent tagging of endogenous pools of 2-AG. Both strategies for imaging pools of 2-AG will be aided by the naturally high abundance of 2-AG relative to other unsaturated monoacylglycerol lipids and the selective increase of 2-AG levels in MAGL knockout mice. As a whole, this project has the potential to illuminate the mechanisms EC regulation and provide new therapeutic strategies for the treatment of EC-related disorders. PUBLIC HEALTH RELEVANCE: The endocannabinoids are a class of neurotransmitters that affect many normal physiological functions. The goal of this project is to investigate how endocannabinoids are regulated in the nervous system using chemical probes. This work has the potential to elucidate novel chemical strategies for investigating biological systems and to uncover novel therapeutic targets in the endocannabinoid system.