Structure-Function Studies of Endocannabinoid Biosynthetic Enzymes

内源性大麻素生物合成酶的结构功能研究

• 批准号: 8857387
• 负责人: Jose Olucha - Alvarez
• 金额: $ 5.6万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8857387
• 关键词: 1,2-diacylglycerol; 2-arachidonylglycerol; Affinity Chromatography; Agonist; Anabolism; Architecture; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Blood Pressure; C-terminal; CNR1 gene; Calcium; Cannabinoids; Catalytic Domain; Cells; Chemicals; Chemistry; Cleaved cell; Collaborations; Detergents; Diglycerides; Disease; Drug or chemical Tissue Distribution; Endocannabinoids; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Stability; Enzymes; Esters; Exclusion; Fatty Acids; Fluorescence; G-Protein-Coupled Receptors; Glutathione; Goals; Health; Heart Rate; Human; Hunger; Hydrolysis; Immune; Immune response; Kinetics; Knowledge; Learning; Length; Lipids; Literature; Mammalian Cell; Mammals; Membrane; Membrane Proteins; Metabolic; Methods; Modeling; Molecular Sieve Chromatography; Mus; Neuraxis; Neurotransmitters; Peripheral; Phase; Physiological; Physiological Processes; Play; Positioning Attribute; Property; Proteins; Protocols documentation; Recombinants; Reducing Agents; Reproductive system; Resolution; Role; Serine Hydrolase; Signal Transduction; Solutions; Structure; Structure-Activity Relationship; System; Testing; Thinking; Tissues; Transmembrane Domain; Variant; Work; cannabinoid receptor; crosslink; design; endogenous cannabinoid system; improved; inhibitor/antagonist; light scattering; lipoprotein lipase; novel therapeutics; overexpression; therapeutic target; three dimensional structure; tool; treatment strategy

DESCRIPTION (provided by applicant): The endocannabinoid system (ECS) is an endogenous modulatory system involved in a wide variety of physiological processes, including learning, thinking, immune, metabolic and reproductive systems. 2- arachidonoylglycerol (2-AG) is a lipid messenger of the ECS, full agonist at Cannabinoid Receptors type 1 (CB1) and type 2 (CB2). 2-AG biosynthesis is catalyzed by diacylglycerol lipase (DAGL), a serine hydrolase that cleaves the ester bond at the sn1 position of 2-arachidonoyldiacylglycerol, releasing 2-AG and a fatty acid. There are two known DAG lipases in humans, DAG lipases � and �. Very little is known about the structure and biochemistry of these key ECS enzymes, the only known four helical pass transmembrane serine hydrolases in mammals. Determining the biochemical properties and three dimensional structures of DAG lipases will help us understand their tertiary and quaternary structures, catalytic mechanisms, and substrate binding pocket chemistry and architecture. We propose to use the biochemical and crystallographic tools at our disposal to investigate the structure and biochemistry of DAG lipase �. First, we have developed and will continue to optimize, an overexpression, solubilization, and purification protocol for recombinant DAGLB� using a mammalian cell expression system. Second, we propose to investigate the oligomeric state of DAGL�, both in solution and in the membrane, using size exclusion chromatography, light scattering, and crosslinking methods. We will also investigate the biochemical properties of DAGL�, determining kinetic parameters using fluorescence and LC-MS assays available to us, testing a panel of candidate substrates, inhibitors, and activators proposed in the literature. Finally, we propose to solve the first crystal structure of a full lengh DAG lipase, DAGL�, using in meso lipidic cubic phase methods, in close collaboration with Dr. Ray Stevens' group. The knowledge on DAG lipases obtained through the work proposed in this project has the potential to become an important tool for the rational design of inhibitors against these key therapeutic targets.

描述（由适用提供）：内源性大麻素系统（ECS）是一种涉及各种物理过程的内源调节系统，包括学习，思维，免疫，代谢和生殖系统。 2-蛛网膜烯丙基甘油（2-AG）是ECS的脂质信使，在1型（CB1）和2型（CB2）的大麻素受体中的全部激动剂（CB2）。 2-AG生物合成是由二酰基甘油脂肪酶（DAGL）催化的，这是一种丝氨酸水解酶，可在2-芳基二烯丙基二酰基甘油的SN1位置切割酯键，释放2ag和脂肪酸。人类中有两个已知的DAG脂肪酶，DAG脂肪酶。关于这些关键ECS酶的结构和生物化学知之甚少，这是哺乳动物中唯一已知的四个螺旋跨膜丝氨酸丝氨酸水解酶。确定DAG脂肪酶的生化特性和三维结构将有助于我们了解其第三纪和第四纪结构，催化机制以及底物结合的口袋化学和结构。我们建议使用可以使用的生化和晶体学工具来研究DAG脂肪酶的结构和生化。首先，我们已经开发并将继续优化使用哺乳动物细胞表达系统重组DAGLB的过表达，解决方案和纯化方案。其次，我们建议使用尺寸排除色谱，光散射和交联方法研究溶液和膜中DAGL的低聚状态。我们还将研究DAGL的生化特性，使用可用的荧光和LC-MS分析来确定动力学参数，测试文献中提出的一组候选底物，抑制剂和激活剂。最后，我们建议在与Ray Stevens博士的组密切合作中使用中脂脂立方相方法中的全长DAG脂肪酶DAGL的第一个晶体结构。通过本项目提出的工作获得的关于DAG脂肪酶的知识有可能成为理性设计抑制剂反对的重要工具 这些关键的治疗靶标。