Understanding the Functional Roles of Newly Identified Serine ‘Orphan’ Proteases and Two Chymotrypsins in the Aedes aegypti Midgut

了解新鉴定的丝氨酸“孤儿”蛋白酶和两种糜蛋白酶在埃及伊蚊中肠中的功能作用

• 批准号: 10377536
• 负责人: ALBERTO A RASCON
• 金额: $ 10.99万
• 依托单位: SAN JOSE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-22 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377536
• 关键词: Active Sites; Aedes; Affinity; Amino Acid Sequence; Animal Model; Antibodies; Binding; Biochemical; Biological Assay; Biology; Blood; Chromogenic Substrates; Chymase; Crystallography; Culicidae; Development; Digestion; Double-Stranded RNA; Enzymes; Escherichia coli; Family; Female; Fertility; Fluorogenic Substrate; Genetic Transcription; Goals; Growth; Hour; Human; In Vitro; Individual; Insect Vectors; Interruption; Ion Exchange; Kinetics; Lead; Measures; Messenger RNA; Midgut; Mutation; Nutrient; Orphan; Oryctolagus cuniculus; Peptide Hydrolases; Phase; Physiological; Play; Population; Process; Proteins; RNA Interference; Recombinant Proteins; Recombinants; Role; Serine; Serine Protease; Site; Source; Specificity; Structure; Substrate Specificity; Technology; Time; Transcript; Translations; Ursidae Family; Work; X-Ray Crystallography; chymotrypsin; chymotrypsin-like enzyme; egg; expression vector; feeding; in vivo; inhibitor; knock-down; mRNA Expression; novel; pathogen; pathogenic virus; polyclonal antibody; protein expression; protein purification; transmission process; vector; vector control; vector mosquito

Understanding the Functional Roles of Newly Identified Serine `Orphan' Proteases and Two Chymotrypsins in the Aedes aegypti Midgut Project Summary/Abstract The female Aedes aegypti mosquito completely relies on human blood as its main source of nutrients. A single female Ae. aegypti mosquito can lay up to 200 eggs from a single blood meal, and the digestion process is highly dependent on midgut proteolytic enzymes. In vivo studies of four abundant midgut proteases have revealed the importance of only three on maximal fecundity. However, RNAi knockdown studies of all three at once did not have an overall synergistic effect indicating the involvement of other midgut proteases. Indeed, mRNA transcripts of five new serine 'orphan' proteases have been identified, but their physiological roles in this process have not been studied in vivo or in vitro making it unknown how much they contribute to overall fecundity. In addition, the exact role in digestion and overall fecundity of two chymotrypsin-like proteases remains unclear. We hypothesize that the orphan proteolytic and the chymotrypsin-like enzymes play important functional roles in the blood meal digestion process. Because the functions of these proteases are novel in mosquitoes, they will bear broadly on the study of a crucial model organism in vector biology. This proposal will focus on elucidating the physiological roles of these serine proteases using RNAi technology, in vivo and in vitro midgut proteolytic activity assays, fecundity studies (Aim 1), recombinant protein expression and purification for in vitro kinetic and substrate specificity assays, as well as mutational and structural determination using crystallography (Aim 2). Overall, the work will lead to complete understanding of the role and specificity of all known mosquito midgut proteases in the blood meal digestion process and may lead to the development of a potential novel mosquito (vector) control strategy. This is crucial because the mosquito is able to transmit several viral pathogens during blood feeding, and if blood meal digestion is interrupted, the mosquito will not lay eggs minimizing the mosquito population, and in turn minimize pathogen transmission. Furthermore, the information gained from this work may be applied to other vector mosquito species and other blood feeding insect vectors that heavily rely on midgut proteolytic enzymes for digestion.

了解新鉴定的丝氨酸“孤儿”蛋白酶和两种蛋白酶的功能作用 埃及伊蚊中肠中的胰凝乳蛋白酶 项目概要/摘要 雌性埃及伊蚊完全依赖人类血液作为其主要营养来源。单个 女Ae.埃及伊蚊一次吸血可产下多达 200 个卵，其消化过程为 高度依赖中肠蛋白水解酶。对四种丰富的中肠蛋白酶的体内研究 仅揭示了三个对最大繁殖力的重要性。然而，这三者的 RNAi 敲除研究 一旦不具有总体协同效应，表明其他中肠蛋白酶的参与。的确， 五种新的丝氨酸“孤儿”蛋白酶的 mRNA 转录本已被鉴定，但它们在这方面的生理作用 该过程尚未在体内或体外进行研究，因此尚不清楚它们对整体的贡献有多大 繁殖力。此外，两种胰凝乳蛋白酶样蛋白酶在消化和整体繁殖力中的确切作用 仍不清楚。我们假设孤儿蛋白水解酶和胰凝乳蛋白酶样酶发挥重要作用 在血粉消化过程中的功能作用。因为这些蛋白酶的功能在 蚊子，它们将广泛地影响媒介生物学中重要模型生物的研究。该提案将 重点是利用 RNAi 技术在体内和体内阐明这些丝氨酸蛋白酶的生理作用 体外中肠蛋白水解活性测定、繁殖力研究（目标 1）、重组蛋白表达和 用于体外动力学和底物特异性测定以及突变和结构分析的纯化 使用晶体学测定（目标 2）。总的来说，这项工作将导致对角色的完全理解 以及所有已知的蚊子中肠蛋白酶在血粉消化过程中的特异性，并可能导致 开发一种潜在的新型蚊子（媒介）控制策略。这一点至关重要，因为蚊子 能够在吸血期间传播多种病毒病原体，如果血粉消化中断， 蚊子不会产卵，从而减少蚊子数量，从而减少病原体传播。 此外，从这项工作中获得的信息可以应用于其他媒介蚊子物种和其他 严重依赖中肠蛋白水解酶进行消化的吸血昆虫载体。