Mechanistic regulation of ammonia metabolism in Aedes aegypti mosquitoes

项目总结 埃及伊蚊雌性已经进化出有效的代谢途径来管理高氨 在血餐消化过程中释放的浓度。需要葡萄糖中的碳(C)原子 对于通过多个路径的相互作用清除氨和过量氮(N)的处置， 包括糖酵解、氨固定、同化和排泄途径。目前尚不清楚的是 这些相互交错的代谢途径是如何被调控的。长期目标是确定生化和 Ae.氮碳代谢调控的分子基础。埃及伊蚊，所以这种新陈代谢- 可以制定以蚊子控制为基础的战略，以此作为改善公共健康和生活质量的一种方式。 这一应用的总体目标是确定多胺的调节机制。 和葡萄糖/氨代谢。中心假设是，氮废物的适当处理是 由尿酸和多胺通量以及参与糖酵解最后一步的蛋白质控制。这个 拟议研究的理由是，确定监管机制将提供新的 为随后确定目标以设计防治蚊子的创新战略提供机会 控制力。在强劲的初步数据的指导下，核心假设将通过追求两个具体目标来检验： 1)测定多胺在食血蚊体内的代谢通量及其调节机制； 2)确定蚊子体内葡萄糖和氨代谢的调节机制。在第一个下 目的，稳定的同位素标记化合物和先进的LC/MS方法，蛋白质印迹，qRT-PCR，化学 将使用抑制剂和反向遗传学技术。在第二个目标中，基因标准技术 克隆、蛋白质纯化、动力学表征、结晶和结构测定 已执行。此外，免疫沉淀、免疫荧光、RNA干扰和代谢组学分析将 被执行。这种方法是创新的，因为它结合了经典和最先进的技术(I) 在原子水平监测代谢物通量，无需样品衍生化；和(Ii)确定调节机制 多胺、葡萄糖/氨在不同水平的代谢，包括转录和翻译后 级别。这项拟议的研究具有重要意义，因为它有望填补目前对如何 雌性蚊子维持氮和碳的代谢，并调节氮废物在血液中的适当处置 如果没有这种食物，氨的浓度可能会达到致命的浓度。因此，预计这些结果将 发现高氨解毒要求下蚊子特有的调控机制。因此，一个 对氮和碳的生物化学和分子基础的基本了解有了很大的提高 蚊子体内的新陈代谢是可以预见的。也期待在Ae中能学到什么。埃及伊蚊 蚊子通过传统和先进的技术将广泛适用于识别监管 在其他节肢动物传播疾病的媒介以及其他生物系统中的机制。

PROJECT SUMMARY Aedes aegypti females have evolved efficient metabolic pathways for managing the high ammonia concentrations that are released during a blood meal's digestion. Carbon (C) atoms from glucose are required for clearance of ammonia and excess nitrogen (N) disposal through the interplay of multiple pathways, including glycolysis, and ammonia fixation, assimilation and excretion pathways. What remains unknown is how these intersecting metabolic pathways are regulated. The long-term goal is to identify the biochemical and molecular bases underlying the regulation of N and C metabolism in Ae. aegypti, so that novel metabolism- based strategies for mosquito control can be developed as a way to improve public health and quality of life. The overall objective for this application is to identify the mechanisms involved in the regulation of polyamines and glucose/ammonia metabolism. The central hypothesis is that the proper disposition of N waste is controlled by uric acid and polyamine fluxes, and by proteins involved in the last step of glycolysis. The rationale for the proposed research is that the identification of regulatory mechanisms will provide new opportunities for the subsequent identification of targets for the design of innovative strategies to mosquito control. Guided by strong preliminary data, the central hypothesis will be tested by pursuing two specific aims: 1) Determine the metabolic flux of polyamines and the mechanisms of its regulation in blood-fed mosquitoes; 2) Identify mechanisms of regulation of both glucose and ammonia metabolism in mosquitoes. Under the first aim, stable isotopically labeled compounds and advanced LC/MS methods, western blots, qRT-PCR, chemical inhibitors and reverse genetics techniques will be used. In the second aim, standard techniques for gene cloning, protein purification, kinetic characterization, crystallization and structure determination will be performed. Further, immunoprecipitation, inmunofluorence, RNA interference, and metabolomics analysis will be performed. This approach is innovative because it combines classical and state-of-the-art techniques (i) to monitor metabolite flux at atomic level without sample derivatization and (ii) to identify regulatory mechanisms of polyamine, glucose/ammonia metabolism at different levels including transcriptional and post-translational levels. The proposed research is significant because it is expected to fill gaps in current understanding of how female mosquitoes maintain N and C metabolism and regulate the proper disposition of N waste upon a blood meal without which ammonia levels could reach lethal concentrations. Thus, these results are expected to uncover mosquito-specific regulatory mechanisms under high demands of ammonia detoxification. As such, a much-improved fundamental understanding of the biochemical and molecular bases underlying the N and C metabolism in mosquitoes can be anticipated. It is also expected that what can be learned in Ae. aegypti mosquitoes through traditional and advanced technologies will be broadly applicable to identify regulatory mechanisms in other arthropod vectors of diseases, as well as in other biological systems.