The function(s) of serpin-2 in mosquito immunity and physiology

描述（由申请人提供）：疟疾控制和根除策略仍然主要依赖于病媒控制，但病媒群体对杀虫剂的抗药性阻碍了病媒控制。除了抗性管理之外，还需要具有新作用方式的新型杀虫剂，特别是其功效不受现有抗性机制影响的杀虫剂。长期目标是开发一种基于新杀虫剂靶标的疟疾媒介控制方法，即来自非洲疟疾蚊冈比亚按蚊的丝氨酸蛋白酶抑制剂丝氨酸蛋白酶抑制剂（SRPN）2。SRPN2是细胞外蛋白酶级联中的关键负调节因子，可控制酚氧化酶原（proPO）的激活，从而控制黑化——一种强大的节肢动物特异性先天免疫反应。成年雌性蚊子血淋巴中的 SRPN2 耗竭会显着缩短寿命，并且治疗后 9 天每日死亡率会上升。本申请的目的是确定 SRPN2 与其蛋白酶靶标的相互作用，并评估 SRPN2 抑制剂的疟疾控制潜力。拟议研究的基本原理是，有关 SRPN2 生物蛋白酶靶标和作用模式的详细信息将最终允许设计 SRPN2 小分子抑制剂，该抑制剂可以充当控制蚊媒的杀虫剂。根据我们的初步数据，我们将追求以下三个具体目标：（1）确定SRPN2抑制功能的分子靶点； (2)确定SRPN2与其靶蛋白酶之间的分子界面； (3) 模拟 SRPN2 缺失对疾病传播的影响。根据第一个目标，申请人实验室已成功使用的生化和遗传学方法的组合将用于根据其（a）与丝氨酸蛋白酶抑制剂形成共价复合物和（b）恢复丝氨酸蛋白酶抑制剂的耗竭表型的能力来鉴定SRPN2的丝氨酸蛋白酶靶标。第二个目标是通过蛋白质晶体学和突变分析来鉴定 SRPN2 与其靶蛋白酶之一 CLIPB9 之间的分子相互作用。在第三个目标下，将通过数学模型评估 SRPN2 抑制对疟疾传播和针对潜在 SRPN2 抑制剂的耐药性发展的潜在影响。拟议的研究具有创新性，因为它将首次评估蚊子免疫系统作为新型杀虫剂的生理目标。此外，该项目将使用微流体作为一种高度创新的方法来研究蚊子先天免疫，如果成功，将给昆虫生物化学领域带来变革。该项目意义重大，因为它将提供调节蚊子黑色化的丝氨酸蛋白酶级联性质的基础知识。最终，它有潜力推动新一代控制疟疾杀虫剂靶点的开发。

DESCRIPTION (provided by applicant): Malaria control and eradication strategies continue to primarily rely on vector control that is stymied by insecticide resistance in vector populations. In addition to resistance management, novel insecticides with new modes of action - particularly for which efficacy is not affected by existing resistance mechanisms - are required. The long-term goal is to develop a malaria vector control methodology based on a new insecticide target, the serine protease inhibitor serpin (SRPN)2 from the African malaria mosquito, Anopheles gambiae s.s.. SRPN2 is a key negative regulator in the extracellular proteinase cascade that controls activation of prophenoloxidase (proPO) and thus melanization - a powerful, arthropod-specific innate immune response. SRPN2 depletion from the hemolymph of adult female mosquitoes significantly reduces longevity with escalating daily mortality nine days after treatment. The objectives in this application are to identify the interactions of SRPN2 with its proteinase targets and to evaluate the malaria control potential of a SRPN2 inhibitor. The rationale for the proposed research is that detailed information on SRPN2's biological proteinase targets and mode of action will ultimately allow the design of small molecule inhibitors of SRPN2 that could act as an insecticide for mosquito vector control. Guided by our preliminary data, the following three specific aims will be pursued: (1) Determine the molecular targets of SRPN2 inhibitory function; (2) Determine the molecular interface between SRPN2 and its target proteinase(s); and (3) Model the impact of SRPN2 depletion on disease transmission. Under the first aim, a combination of biochemical and genetic approaches that have been used successfully in the applicant's laboratory will be used to identify serine proteinase targets of SRPN2 based on their ability to (a) form covalent complexes with the serpin and (b) revert the serpin's depletion phenotype. Under the second aim, the molecular interactions between SRPN2 and one of its target proteinases, CLIPB9, will be identified by protein crystallography and mutational analysis. Under the third aim, the potential effect of SRPN2 inhibition on malaria transmission and resistance development against a potential SRPN2 inhibitor will be assessed by mathematical modeling. The proposed research is innovative, as it will for the first time evaluate the mosquito immune system as a physiological target for novel insecticides. Additionally, this project will use microfluidics as a highly innovative approach to the study of mosquito innate immunity, which if successful will be transformative to the field of insect biochemistry. This project is significant as it will provide fundamental knowledge of the nature of serine proteinase cascades that regulate melanization in mosquitoes. Ultimately, it has the potential to advance the development of new a generation of insecticide targets for malaria control.