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: 10598048
• 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/10598048
• 关键词: Active Sites; Aedes; Affinity; Amino Acid Sequence; Antibodies; Binding; Biochemical; Biological Assay; Biology; Blood; Chromogenic Substrates; Chymase; Compensation; 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; 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; Work; X-Ray Crystallography; chymotrypsin; chymotrypsin-like enzyme; egg; expression vector; feeding; in vivo; inhibitor; knock-down; mRNA Expression; model organism; 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.

理解新发现的丝氨酸“孤儿”蛋白和两种蛋白的功能 埃及伊蚊中肠中的凝乳酶 项目摘要/摘要 雌性埃及伊蚊完全依靠人类血液作为其主要营养来源。单人间 雌性Ae.埃及伊蚊一顿血最多可以产卵200粒，消化过程是 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 揭示了只有三个对最大繁殖力的重要性。然而，对这三种基因的RNAi敲除研究都在 一次没有整体的协同效应，表明其他中肠蛋白酶的参与。的确， 已经鉴定了五种新的丝氨酸蛋白酶的mrna转录本，但它们在这一过程中的生理作用 这一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 繁殖力。此外，两种类糜蛋白酶在消化和总体繁殖力中的确切作用 目前仍不清楚。我们推测孤儿蛋白水解酶和类胰凝乳杆菌酶起重要作用。 在血粉消化过程中的功能作用。因为这些蛋白水解酶的功能在 蚊子，它们将广泛地参与媒介生物学中一种重要的模式生物的研究。这项提议将 重点利用RNAi技术在体内和体内阐明这些丝氨酸蛋白酶的生理作用 体外中肠蛋白分解活性分析、繁殖力研究(AIM 1)、重组蛋白表达和 体外动力学和底物特异性分析以及突变和结构分析的纯化 使用结晶学进行测定(目标2)。总体而言，这项工作将导致对这一角色的完全理解 和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 潜在的新型蚊子(媒介)控制策略的开发。这一点至关重要，因为蚊子 在进食过程中能够传播几种病毒病原体，如果血粉消化中断， 蚊子不会产卵，从而将蚊子数量降至最低，进而将病原体传播降至最低。 此外，从这项工作中获得的信息也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食血昆虫媒介。