STRUCTURE AND FUNCTION OF PHOSPHOETHANOLAMINE METHYLTRANSFERASES: NEW ANTI-PARAS

描述（由申请人提供）：秀丽隐杆线虫是用于遗传学和发育生物学以及用于鉴定生物化学靶标以开发抗寄生线虫活性药物的强大模型系统。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C.与其它动物不同，线虫和其它线虫可使用植物样途径作为主要磷脂组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）催化磷酸乙醇胺连续甲基化为磷酸胆碱，然后磷酸胆碱可以掺入磷脂酰胆碱。重要的是，这两个PMT是C.在线虫中，由于RNAi敲除它们的表达导致严重的发育表型，包括生长停滞和死亡。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，因此这些酶是抑制剂开发的有希望的靶点;然而，关于PMT在分子水平上如何发挥作用知之甚少。 本研究的目的是探索和表征这一新的代谢途径。elegans作为开发抗寄生虫化合物的可能目标。本提案中概述的实验旨在解决两个基本的和未回答的问题-磷酸碱基甲基化的分子和化学基础是什么，以及这一关键代谢途径在C. elegans？计划中的研究旨在通过确定这些酶的X射线晶体结构并通过分析定义催化和磷酸碱基底物特异性的动力学和热力学特征来了解PMT如何发挥作用的分子基础（目标1和2）。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，并确定其表达模式（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所起的重要作用的理解。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疗，兽医和农业价值的新的抗寄生虫化合物。 公共卫生相关性：与公共卫生相关。寄生线虫是人类健康问题的主要原因，估计全世界有30亿人感染这些生物体。确定寄生虫和宿主物种之间不同的生化靶标对于寻找有效的新抗寄生虫药物至关重要。使用自由生活的线虫秀丽隐杆线虫作为模型系统，我的小组已经确定了一个新的生化途径，这是没有发现在哺乳动物蠕虫的生长和发育。该提案旨在了解磷酸碱基甲基化途径中酶的分子功能，并帮助开发抑制剂作为潜在的抗寄生虫化合物。

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.