Chemical probes to assess endocannabinoid localization, storage, and transport

描述（由申请人提供）：大麻素（AEA）和2-花生四烯酸甘油（2-AG）是内源性大麻素（EC）系统中的主要内源性神经递质。这些化学信使，不像研究最充分的神经递质，是脂质，因此，通过不同的生化过程进行调节。在过去的十年里，一直在激烈的争论是否负责EC的合成和降解的代谢途径是主要负责EC信号的启动和终止。支持这一观点，最近出现的证据表明，存在特定的EC结合蛋白，可能有助于EC在神经元中的摄取。目前的2- AG信号模型也受到质疑，因为几项新的研究表明，2-AG在释放之前被储存，而不是“按需”合成。具体的EC载体蛋白和2-AG存储的直接证据是缺乏的，留下这些有争议的假设在很大程度上悬而未决。为了解决这些悬而未决的问题，我们将试图完善的理解EC运输，存储和释放在哺乳动物大脑采用多方面的化学生物学方法。首先，我们建议通过实施基于活性的蛋白质分析和多维蛋白质鉴定技术（ABPP-MudPIT）来全面绘制神经系统中的EC结合蛋白。EC结合蛋白将富集与无标签的光亲和探针的帮助下，并确定多维LC-MS/MS。蛋白质目标表现出高特异性和亲和力将进一步研究功能测定，以阐明其在EC信号转导的作用。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。我们将首先尝试通过合成能够与免疫原性载体蛋白缀合的末端官能化的2-AG类似物来开发用于免疫荧光显微术的2-AG抗体。我们还将尝试开发生物缀合反应，这将允许荧光标记的内源性池的2-AG。两种用于成像2-AG池的策略将通过2-AG相对于其他不饱和单酰基甘油脂质的天然高丰度和MAGL敲除小鼠中2-AG水平的选择性增加来辅助。总的来说，该项目有可能阐明EC调节机制，并为治疗EC相关疾病提供新的治疗策略。 公共卫生相关性：内源性大麻素是一类影响许多正常生理功能的神经递质。该项目的目标是研究内源性大麻素如何在神经系统中使用化学探针进行调节。这项工作有可能阐明新的化学策略，用于研究生物系统，并发现内源性大麻素系统中的新的治疗靶点。

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.