STRUCTURE AND FUNCTION OF PHOSPHOETHANOLAMINE METHYLTRANSFERASES: NEW ANTI-PARAS

磷酸乙醇胺甲基转移酶的结构和功能：新的 Anti-Paras

• 批准号: 8303674
• 负责人: Joseph Martin Jez
• 金额: $ 32.8万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8303674
• 关键词: Active Sites; Address; Agriculture; Animals; Biochemical; Biochemical Pathway; Biological; Biological Models; Caenorhabditis elegans; Calorimetry; Catalysis; Cessation of life; Chemicals; Complement; Crystallography; Development; Developmental Biology; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Eukaryota; Evolution; Genetic; Goals; Growth; Growth and Development function; Health; Human; In Vitro; Kinetics; Knock-out; Lecithin; Life; Ligand Binding; Livestock; Mammals; Medical; Metabolic; Metabolic Pathway; Metabolism; Methylation; Molecular; Nematoda; Organism; Parasites; Parasitic nematode; Pathway interactions; Pattern; Pharmaceutical Preparations; Phenotype; Phospholipid Metabolism; Phospholipids; Phosphorylcholine; Physiological; Plants; Play; Proteins; Public Health; RNA Interference; Research; Roentgen Rays; Role; Route; Site-Directed Mutagenesis; Structure; Substrate Specificity; Testing; Thermodynamics; Titrations; base; enzyme structure; in vivo; inhibitor/antagonist; insight; phosphoethanolamine; phosphoethanolamine methyltransferase; protein function; research study; small molecule; three dimensional structure

DESCRIPTION (provided by applicant): Caenorhabditis elegans is a powerful model system for genetics and developmental biology, and for identifying biochemical targets to develop drugs active against parasitic nematodes. Eukaryotes, including nematodes and humans, share many similar metabolic pathways, which makes specific targeting challenging. Our recent studies suggest that C. elegans and other nematodes, unlike other animals, may use a plant-like pathway as the primary biosynthetic route to the major phospholipid component phosphatidylcholine. In this metabolic pathway, a pair of phosphoethanolamine methyltransferases (PMT) catalyze the sequential methylation of phosphoethanolamine to phosphocholine, which can then be incorporated into phosphatidylcholine. Importantly, both of the PMT are required for normal growth and development of C. elegans, as RNAi knockout of their expression leads to severe developmental phenotypes including arrested growth and death. This suggests that inhibition of PMT activity may be a possible strategy for controlling nematode growth. Because the PMT are not found in mammals and are highly conserved across multiple parasitic nematodes and protozoans, these enzymes are promising targets for inhibitor development; however, very little is known about how the PMT function at the molecular level. This proposal aims to explore and characterize this new metabolic pathway in C. elegans as a possible target for the development of anti-parasitic compounds. The experiments outlined in this proposal aim to address two basic and unanswered questions - what is the molecular and chemical basis for phosphobase methylation and what is the biological role of this critical metabolic pathway in C. elegans? The planned research sets out to understand the molecular basis for how the PMT function by determining the X-ray crystal structures of these enzymes and by analyzing the kinetic and thermodynamic features that define catalysis and phosphobase substrate specificity (Aims 1 and 2). To complement in vitro studies of the PMT, we will also test the metabolic contribution of these proteins to phosphatidylcholine synthesis and determine their expression patterns (Aim 3). These in vivo experiments will expand our understanding of the essential function these proteins play in the growth and development of nematodes. Because the PMT are highly conserved across nematode parasites of humans, animals, and plants, as well as in protozoan parasites, understanding how these enzymes function and identifying inhibitors targeting them will contribute to developing new anti-parasitic compounds of potential medical, veterinary, and agricultural value. PUBLIC HEALTH RELEVANCE: Relevance to public health. Parasitic nematodes are a major cause of human health problems with an estimated 3 billion people infected worldwide by these organisms. Identifying biochemical targets that differ between the parasite and host species is essential for finding effective new anti-parasitic drugs. Using the free-living nematode Caenorhabditis elegans as a model system, my group has identified a new biochemical pathway essential for worm growth and development that is not found in mammals. This proposal aims to understand the molecular function of the enzymes in the phosphobase methylation pathway and to aid in the development of inhibitors as potential anti-parasitic compounds.

描述（由申请人提供）：秀丽隐杆线虫是一个强大的遗传学和发育生物学模型系统，用于识别生化靶点以开发抗寄生线虫药物。真核生物，包括线虫和人类，有许多相似的代谢途径，这使得特异性靶向具有挑战性。我们最近的研究表明，秀丽隐杆线虫和其他线虫与其他动物不同，它们可能使用类似植物的途径作为主要磷脂成分磷脂酰胆碱的主要生物合成途径。在这一代谢途径中，一对磷酸乙醇胺甲基转移酶（PMT）催化磷酸乙醇胺依次甲基化为磷酸胆碱，然后将其纳入磷脂酰胆碱。重要的是，这两种PMT对于秀丽隐杆线虫的正常生长和发育都是必需的，因为RNAi敲除它们的表达会导致严重的发育表型，包括生长受阻和死亡。这表明抑制PMT活性可能是控制线虫生长的一种可能策略。由于PMT在哺乳动物中未发现，并且在多种寄生线虫和原生动物中高度保守，因此这些酶是开发抑制剂的有希望的靶点；然而，人们对PMT在分子水平上的功能知之甚少。本研究旨在探索和表征秀丽隐杆线虫的这一新的代谢途径，作为开发抗寄生虫化合物的可能靶点。本提案中概述的实验旨在解决两个基本的和未解决的问题-磷酸基甲基化的分子和化学基础是什么？这一关键代谢途径在秀丽隐杆线虫中的生物学作用是什么？计划中的研究通过确定这些酶的x射线晶体结构，并通过分析定义催化和磷酸基底物特异性的动力学和热力学特征（目标1和2），着手了解PMT如何起作用的分子基础。为了补充PMT的体外研究，我们还将测试这些蛋白质对磷脂酰胆碱合成的代谢贡献，并确定它们的表达模式（目的3）。这些体内实验将扩大我们对这些蛋白质在线虫生长和发育中发挥的基本功能的理解。由于PMT在人类、动物和植物的线虫寄生虫以及原生动物寄生虫中高度保守，因此了解这些酶的功能并确定针对它们的抑制剂将有助于开发具有潜在医学、兽医和农业价值的新型抗寄生虫化合物。