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”蛋白酶和两个的功能作用 埃德斯埃及埃及中肠里的辣椒蛋白 项目摘要/摘要 雌性埃及埃及蚊子完全依靠人类血液作为其主要营养来源。一个 女性AE。埃及蚊子可以从一顿血餐中产生多达200个鸡蛋，而消化过程是 高度依赖于中肠蛋白水解酶。四种大量中肠蛋白酶的体内研究具有 揭示了只有三个在最大繁殖力上的重要性。但是，RNAi敲低研究的所有三个研究 曾经没有总体协同作用，表明其他中肠蛋白酶的参与。的确， 已经确定了五个新丝氨酸'孤儿蛋白酶的mRNA转录本，但它们在这方面的生理作用 过程尚未在体内或体外研究，因此未知它们对整体贡献了多少 繁殖力。另外，在两种类似胰酪蛋白酶的蛋白酶的消化和整体繁殖力中的确切作用 仍然不清楚。我们假设孤儿蛋白水解和类似辣椒素的酶很重要 在血液粉消化过程中的功能作用。因为这些蛋白酶的功能是新颖的 蚊子，它们将广泛地研究媒介生物学中关键模型生物体的研究。该提议将 专注于使用RNAi技术，体内和IN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（AIM 1），重组蛋白表达和 体外动力学和底物特异性测定的纯化以及突变和结构 使用晶体学测定（AIM 2）。总体而言，这项工作将导致对角色的完整理解 和所有已知蚊子中肠蛋白酶在血液粉消化过程中的特异性，并可能导致 潜在的新型蚊子（向量）控制策略的发展。这是至关重要的，因为蚊子是 能够在血液喂养过程中传播几种病毒病原体，如果中断血液粉消化， 蚊子不会产卵，使蚊子种群最小化，进而将病原体传播最小化。 此外，从这项工作中获得的信息可以应用于其他媒介蚊子和其他 血液喂养的昆虫载体严重依赖中肠蛋白水解酶进行消化。