Mechanistic regulation of ammonia metabolism in Aedes aegypti mosquitoes

埃及伊蚊氨代谢的机制调控

• 批准号: 10159210
• 负责人: Patricia Yolanda Scaraffia
• 金额: $ 38.03万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-06 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10159210
• 关键词: Aedes; Alanine Transaminase; Amino Acids; Ammonia; Arthropod Vectors; Assimilations; Biochemical; Blood; Carbon; Cessation of life; Chemicals; Crystallization; Culicidae; Data; Deacetylase; Deamination; Development; Diet; Digestion; Disease Vectors; Drug Metabolic Detoxication; Enzymes; Excretory function; Fat Body; Female; Genes; Genetic Techniques; Genetic Transcription; Glucose; Glutamine; Glycolysis; Goals; Heme; Immunoprecipitation; Infection; Isotopes; Kinetics; Knowledge; Leisures; Life; Link; Mediating; Metabolic; Metabolic Pathway; Metabolism; Methods; Mission; Molecular; Monitor; Mosquito Control; Nitrogen; Nutrient; Nutritional status; Ornithine Decarboxylase; Outcome; Oxides; Pathway interactions; Phenotype; Polyamines; Production; Proteins; Public Health; Pyruvate Kinase; Quality of life; Quantitative Reverse Transcriptase PCR; RNA Interference; Recombinants; Refuse Disposal; Regulation; Reproduction; Research; Research Proposals; Sampling; Sirtuins; Stable Isotope Labeling; Structure; Techniques; Technology; Testing; Toxic effect; United States National Institutes of Health; Uric Acid; Western Blotting; XDH gene; base; biological systems; design; egg; feeding; flexibility; gene cloning; glucose metabolism; improved; inhibitor/antagonist; innovation; liquid chromatography mass spectrometry; metabolomics; novel; prolyl-serine; protein purification; public health relevance; reverse genetics; sample fixation; sucking; sugar; urea cycle; wasting

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.

项目摘要 埃及伊蚊雌性已经进化出有效的代谢途径来管理高氨 血液消化过程中释放的浓度。需要来自葡萄糖的碳（C）原子 为了通过多种途径的相互作用清除氨和过量的氮（N）， 包括糖酵解、氨固定、同化和排泄途径。目前尚不清楚的是 这些交叉的代谢途径是如何被调节的。长期目标是确定生物化学和 Ae. N和C代谢调控的分子基础。埃及人，所以新的新陈代谢- 可以制定基于蚊子控制的策略，作为改善公共健康和生活质量的一种方式。 本申请的总体目标是确定参与调节多胺的机制 和葡萄糖/氨代谢。核心假设是，氮废物的适当处置是 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。的 拟议研究的理由是，确定监管机制将提供新的 随后确定目标的机会，以便设计防治蚊子的创新战略 控制在强有力的初步数据的指导下，将通过追求两个具体目标来检验中心假设： 1)测定吸血蚊体内多胺代谢通量及其调控机制; 2)确定蚊子葡萄糖和氨代谢的调节机制。根据第一项 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， 克隆、蛋白纯化、动力学表征、结晶和结构测定将是 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）， 在原子水平上监测代谢物通量，而无需样品衍生化，以及（ii）确定调节机制 多胺，葡萄糖/氨代谢在不同水平，包括转录和翻译后 程度.这项拟议中的研究意义重大，因为它有望填补目前对 雌蚊维持氮和碳的代谢，并调节血液中氮废物的适当处置， 没有这些食物，氨的含量就会达到致命的浓度。因此，这些结果预计将 在氨解毒的高要求下，揭示蚊子特有的调节机制。因此，一 大大提高了对N和C的生化和分子基础的基本理解 蚊子的新陈代谢是可以预料的。它也预计，什么可以学到在Ae。aegypti 蚊子通过传统和先进的技术将广泛适用于确定监管 在其他节肢动物疾病的媒介，以及在其他生物系统的机制。