Chemical probes to assess endocannabinoid localization, storage, and transport

描述（由申请人提供）：Anandamide （AEA）和2-arachidonylglycerol （2-AG）是endocannabinoid （EC）系统中的主要内源性神经递质。这些化学信使，不像研究得最充分的神经递质，是脂质，因此，通过不同的生化过程调节。在过去的十年里，人们一直在激烈地争论是否负责EC合成和降解的代谢途径主要负责EC信号的启动和终止。挑战这一概念，证据最近出现的存在特异性EC结合蛋白，可能促进EC摄取神经元。目前的2-AG信号传导模型也受到了一些新的研究的质疑，这些研究表明2-AG在释放之前是储存的，而不是“按需”合成的。特异性ec载体蛋白和2-AG储存的直接证据缺乏，使得这些有争议的假设在很大程度上不确定。为了解决这些尚未解决的问题，我们将尝试采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谱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结合蛋白将通过无标签光亲和探针进行富集，并通过多维LC-MS/MS进行鉴定。具有高特异性和亲和力的蛋白靶点将在功能分析中进一步研究，以阐明它们在EC信号传导中的作用。其次，我们建议开发新的方法来确定2-AG在哺乳动物大脑中的细胞和亚细胞定位。我们将首先尝试通过合成一种末端功能化的2-AG类似物来结合免疫原性载体蛋白，从而开发用于免疫荧光显微镜的2-AG抗体。我们还将尝试开发生物偶联反应，使内源性2-AG池的荧光标记成为可能。与其他不饱和单酰基甘油脂相比，2-AG的天然高丰度和MAGL敲除小鼠中2-AG水平的选择性增加将有助于2-AG成像池的两种策略。总的来说，本项目有可能阐明EC的调控机制，并为EC相关疾病的治疗提供新的治疗策略。

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.