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（由申请人提供）：内源性大麻素系统（ECS）是一种内源性调节系统，涉及多种生理过程，包括学习、思维、免疫、代谢和生殖系统。2-花生四烯酰甘油（2-AG）是一种脂质。 ECS 的信使，1 型大麻素受体 (CB1) 和 2 型大麻素受体 (CB2) 的完全激动剂。二酰基甘油脂肪酶 (DAGL) 是一种丝氨酸水解酶，可裂解 2-花生四烯酰二酰基甘油的 sn1 位置处的酯键，释放 2-AG 和脂肪酸。 人类中有两种已知的 DAG 脂肪酶：DAG 脂肪酶 � 和 � 很少。已知这些关键 ECS 酶的结构和生物化学，这是唯一已知的四螺旋跨膜丝氨酸确定 DAG 脂肪酶的生化特性和三维结构将有助于我们了解其三级和四级结构、催化机制以及底物结合袋化学和结构。 DAG 脂肪酶的结构和生物化学 � 首先，我们已经开发并将继续优化重组的过表达、溶解和纯化方案。其次，我们建议使用尺寸排阻色谱、光散射和交联方法研究 DAGL® 在溶液和膜中的寡聚状态。 DAGL�，使用我们可用的荧光和 LC-MS 测定确定动力学参数，测试文献中提出的一组候选底物、抑制剂和激活剂最后，我们建议解决完整的晶体结构。与 Ray Stevens 博士小组密切合作，在内消旋脂质立方相方法中使用了长度 DAG 脂肪酶，DAGL� 通过本项目中提出的工作获得的有关 DAG 脂肪酶的知识有可能成为理性分析的重要工具。抑制剂的设计 这些关键的治疗靶点。