Mechanistic regulation of ammonia metabolism in Aedes aegypti mosquitoes

埃及伊蚊氨代谢的机制调控

基本信息

批准号：
10394913
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
项目类别：
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/10394913
关键词：
Aedes Alanine Transaminase Amino Acids Ammonia Arthropod Vectors Biochemical Blood Carbon Cessation of life Chemicals Crystallization Culicidae Data Deacetylase Deacetylation Deamination Derivation procedure 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 Learning Leisures Life Link Mediating Metabolic Metabolic Pathway Metabolism Methods Mission Molecular Monitor Mosquito Control Nitrogen Nutrient Nutritional status Ornithine Decarboxylase Outcome 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 innovation liquid chromatography mass spectrometry metabolomics novel protein purification public health relevance reverse genetics sample fixation sucking sugar transmission process urea cycle wasting

项目摘要

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.

项目摘要埃及伊蚊的女性已经发展了有效的代谢途径来管理高氨在血液粉消化过程中释放的浓度。需要来自葡萄糖的碳（C）原子通过多种途径的相互作用清除氨和过量的氮（N）处置包括糖酵解和氨固定，同化和排泄途径。仍然未知的是这些相交的代谢途径如何受到调节。长期目标是确定生化和 AE中N和C代谢调节的基础分子碱基。埃及，所以新陈代谢 - 可以开发基于蚊子控制的策略，以改善公共健康和生活质量。该应用的总体目标是确定多胺调节所涉及的机制和葡萄糖/氨代谢。中心假设是n废物的适当处理是由尿酸和多胺通量控制，以及参与糖酵解最后一步的蛋白质。这拟议研究的理由是，监管机制的识别将提供新的随后确定目标蚊子创新策略的目标的机会控制。在强大的初步数据的指导下，将通过实现两个具体目标来检验中心假设： 1）确定多胺的代谢通量及其在血液喂养蚊子中调节的机制； 2）确定蚊子中葡萄糖和氨代谢的调节机制。在第一个目标，稳定的同位素标记化合物和高级LC/MS方法，蛋白质印迹，QRT-PCR，化学将使用抑制剂和反向遗传技术。在第二个目标中，基因的标准技术克隆，蛋白质纯化，动力学表征，结晶和结构确定将是执行。此外，免疫沉淀，非氟氟，RNA干扰和代谢组学分析将被执行。这种方法具有创新性，因为它将古典和最新技术（i）结合到监测原子水平的代谢物通量，而无需样品衍生化和（ii）确定调节机制多胺，葡萄糖/氨代谢的不同水平，包括转录和翻译后水平。拟议的研究很重要，因为预计它将在当前了解如何雌性蚊子维持N和C代谢，并调节N废物在血液上的适当处理餐点可以达到致命的浓度。因此，这些结果应在高需求氨解毒的需求下，发现蚊子特异性的调节机制。因此，对N和C的生化基础和分子碱基的基本理解已得到深思熟虑可以预料到蚊子中的代谢。还可以预期可以在AE中学到的知识。埃及通过传统技术和先进技术将广泛适用于确定监管的蚊子其他节肢动物媒介以及其他生物系统中的机制。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Distinctive regulatory properties of pyruvate kinase 1 from Aedes aegypti mosquitoes.

DOI：
10.1016/j.ibmb.2018.12.010
发表时间：
2019-01
期刊：
Insect biochemistry and molecular biology
影响因子：
3.8
作者：
N. Petchampai;Claribel Murillo-Solano;J. Isoe;J. Pizarro;P. Y. Scaraffia
通讯作者：
N. Petchampai;Claribel Murillo-Solano;J. Isoe;J. Pizarro;P. Y. Scaraffia

Mass spectrometry-based stable-isotope tracing uncovers metabolic alterations in pyruvate kinase-deficient Aedes aegypti mosquitoes.

DOI：
10.1016/j.ibmb.2020.103366
发表时间：
2020-04
期刊：
Insect biochemistry and molecular biology
影响因子：
3.8
作者：
N. Petchampai;J. Isoe;Thomas D. Horvath;S. Dagan;L. Tan;P. Lorenzi;D. Hawke;P. Y. Scaraffia
通讯作者：
N. Petchampai;J. Isoe;Thomas D. Horvath;S. Dagan;L. Tan;P. Lorenzi;D. Hawke;P. Y. Scaraffia

Unraveling mosquito metabolism with mass spectrometry-based metabolomics.

DOI：
10.1016/j.pt.2021.03.010
发表时间：
2021-08
期刊：
Trends in parasitology
影响因子：
9.6
作者：
Horvath TD;Dagan S;Scaraffia PY
通讯作者：
Scaraffia PY

Pyruvate kinase is post-translationally regulated by sirtuin 2 in Aedes aegypti mosquitoes.

DOI：
10.1016/j.ibmb.2023.104015
发表时间：
2023-11
期刊：
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY
影响因子：
3.8
作者：
Petchampai, Natthida;Isoe, Jun;Balaraman, Prashanth;Oscherwitz, Max;Carter, Brendan H.;Sanchez, Cecilia G.;Scaraffia, Patricia Y.
通讯作者：
Scaraffia, Patricia Y.

Ornithine decarboxylase deficiency critically impairs nitrogen metabolism and survival in Aedes aegypti mosquitoes.

DOI：
10.1096/fj.202200008r
发表时间：
2022-05
期刊：
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
影响因子：
0
作者：
Isoe J;Petchampai N;Joseph V;Scaraffia PY
通讯作者：
Scaraffia PY