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

描述(申请人提供)：疟疾控制和根除战略继续主要依靠病媒控制，而病媒控制因病媒种群中的杀虫剂耐药性而受阻。除了抗药性管理外，还需要具有新作用模式的新型杀虫剂--特别是其药效不受现有抗药性机制影响的杀虫剂。长期目标是开发一种基于新的杀虫剂靶标的疟疾媒介控制方法，即来自非洲冈比亚按蚊的丝氨酸蛋白酶抑制物丝氨酸蛋白酶抑制物(SRPN)2。SRPN2是细胞外蛋白酶级联反应中的一个关键的负调节因子，它控制着前酚氧化酶(ProPO)的激活，从而控制黑化--一种强大的节肢动物特有的先天性免疫反应。成年雌性蚊子血淋巴中SRPN2的耗尽显著降低了寿命，治疗9天后每日死亡率上升。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。这项拟议研究的基本原理是，有关SRPn2的S生物蛋白酶靶标和作用模式的详细信息最终将使SRPn2的小分子抑制剂得以设计，可以用作蚊媒控制的杀虫剂。在我们的初步数据的指导下，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与其靶蛋白蛋白酶(S)之间的分子界面；(3)模拟SRPN2缺失对疾病传播的影响。在第一个目标下，将使用在申请人的实验室中成功使用的生化和遗传方法的组合来识别SRPN2的丝氨酸蛋白酶靶标，基于它们(A)与丝氨酸形成共价复合体和(B)逆转丝氨酸的耗尽表型的能力。在第二个目标下，将通过蛋白质结晶学和突变分析来鉴定SRPN2与其靶蛋白之一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抑制对疟疾传播的潜在影响和对潜在SRPN2抑制剂的抗药性发展。这项拟议的研究是创新的，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的天然免疫，如果成功，这将对昆虫生物化学领域产生变革。这个项目意义重大，因为它将提供调节蚊子黑化的丝氨酸蛋白酶级联的性质的基本知识。最终，它有可能推动疟疾控制的新一代杀虫剂目标的开发。

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.