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”蛋白酶的功能作用， 埃及伊蚊中肠的胰凝乳蛋白酶 项目总结/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 女性Ae。埃及蚊子一次吸血可以产下200个卵， 高度依赖于中肠蛋白水解酶。四种丰富的中肠蛋白酶的体内研究 揭示了只有三个因素对最大繁殖力的重要性。然而，对这三种基因的RNAi敲除研究， 一次不具有总体协同效应，表明其它中肠蛋白酶的参与。的确， 已经鉴定了五种新的丝氨酸“孤儿”蛋白酶的mRNA转录物，但它们在这一过程中的生理作用， 尚未在体内或体外研究这一过程，因此尚不清楚它们对总体 繁殖力此外，两种糜蛋白酶样蛋白酶在消化和总体繁殖力中的确切作用 仍不清楚我们推测孤儿蛋白水解酶和糜蛋白酶样酶起重要作用 在血餐消化过程中的功能作用。因为这些蛋白酶的功能是新的， 蚊子，他们将广泛承担一个关键的模式生物在载体生物学的研究。这项建议会 重点是阐明这些丝氨酸蛋白酶的生理作用，使用RNAi技术，在体内和体内， 体外中肠蛋白水解活性测定、生殖力研究（Aim 1）、重组蛋白表达和 用于体外动力学和底物特异性测定的纯化，以及突变和结构 使用晶体学进行测定（目标2）。总的来说，这项工作将导致完全理解的作用 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， 开发一种潜在的新型蚊子（病媒）控制策略。这一点至关重要，因为蚊子 能够在血液喂养过程中传播几种病毒病原体，如果血液餐消化中断， 蚊子不会产卵，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病媒蚊子物种和其他 严重依赖中肠蛋白水解酶消化的吸血昆虫载体。

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.