FUNCTIONAL STUDIES OF FAAH

FAAH 的功能研究

• 批准号: 6741099
• 负责人: BENJAMIN F CRAVATT
• 金额: $ 26.83万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6741099
• 关键词: active sites; amides; amidohydrolases; anandamide; biological signal transduction; cannabinoid receptor; catalyst; enzyme activity; enzyme mechanism; enzyme substrate; fatty acid metabolism; genetically modified animals; hydrolysis; intermolecular interaction; laboratory mouse; laboratory rabbit; membrane activity; membrane channels; protein binding; protein localization; protein protein interaction; protein structure function

The objective of Project I is to understand the molecular and cellular mechanisms of action of fatty acid amide hydrolase (FAAH), a mammalian membrane-bound enzyme responsible for the catabolism of the fatty acid amide (FAA) family of endogenous signaling lipids. FAAs represent an emerging class of chemical messengers that influence a variety of behavioral processes, including, pain sensation, anxiety, sleep, and feeding. Recent genetic and pharmacological studies have demonstrated that FAAH is a principal regulator of FAA-based signaling events in vivo, suggesting that this enzyme may represent an attractive target for the treatment of neurological disorders like pain and anxiety. Nonetheless, many questions remain regarding the function of FAAH and its suitability as a therapeutic target. For example, among the numerous hydrolases present in mammalian proteomes, how does FAAH exert such extraordinary control over the levels and activity of FAAs in vivo? Might FAAH recruit these hydrophobic substrates directly from cellular membranes, thereby giving the enzyme privileged access to these lipid messengers? Likewise, does FAAH operate alone in neurons, or might this enzyme participate in higher-order protein complexes that regulate its localization and/or activity? Our recent determination of the FAAH structure by x-ray crystallography has inspired several new hypotheses to explain this enzyme's mode of action. In this project, we aim to test these hypotheses using a multidisciplinary approach, employing synthetic chemistry, enzymology, cell biology, and proteomic techniques towards the goals of: 1) determining the roles that FAAH channels play in catalysis, 2) characterizing the molecular interactions between FAAH and cell membranes, 3) identifying FAAH-associated proteins in brain and liver, and 4) comparing the cellular stability of WT-FAAH and its natural P129T variant associated with problem drug use. We anticipate that these studies will not only enhance our understanding of the molecular and cellular mechanism of action of FAAH, but also uncover novel ways to target this enzyme with inhibitors for research and therapeutic purposes.

项目 I 的目标是了解脂肪酸酰胺水解酶 (FAAH) 的分子和细胞作用机制，脂肪酸酰胺水解酶是一种哺乳动物膜结合酶，负责内源信号脂质的脂肪酸酰胺 (FAA) 家族的分解代谢。 FAA 代表了一类新兴的化学信使，它影响各种行为过程，包括疼痛感、焦虑、睡眠和进食。最近的遗传和药理学研究表明，FAAH 是体内基于 FAA 的信号传导事件的主要调节因子，这表明这种酶可能是治疗疼痛和焦虑等神经系统疾病的一个有吸引力的靶点。尽管如此，关于 FAAH 的功能及其作为治疗靶点的适用性仍然存在许多问题。例如，在哺乳动物蛋白质组中存在的众多水解酶中，FAAH 如何对体内 FAA 的水平和活性发挥如此非凡的控制作用？ FAAH 是否可以直接从细胞膜招募这些疏水性底物，从而使酶能够优先接触这些脂质信使？同样，FAAH 是否在神经元中单独运作，或者这种酶是否可能参与调节其定位和/或活性的高级蛋白质复合物？我们最近通过 X 射线晶体学确定的 FAAH 结构激发了一些新的研究 解释这种酶的作用方式的假设。在这个项目中，我们的目标是使用多学科方法测试这些假设，采用合成化学、酶学、细胞生物学和蛋白质组学技术，以实现以下目标：1) 确定 FAAH 通道在催化中发挥的作用，2) 表征分子相互作用FAAH 和细胞膜之间的差异，3) 识别大脑和肝脏中的 FAAH 相关蛋白，4) 比较 WT-FAAH 及其与 FAAH 相关的天然 P129T 变体的细胞稳定性问题药物使用。我们预计这些研究不仅将增强我们对 FAAH 分子和细胞作用机制的理解，还将揭示利用抑制剂靶向这种酶以用于研究和治疗目的的新方法。