Function and regulation of two key serine proteases in insect immunity

项目摘要 细胞外丝氨酸蛋白酶及其非催化同系物协调关键防御机制 在昆虫中。这包括蚊子对病原体和寄生虫的反应， 严重的人类疾病。蛋白水解裂解产生活性酚氧化酶（PO）、硫酯酶 蛋白质（TEPs）、Spätzle和其他细胞因子。PO催化反应性化学品的产生， 隔离并杀死入侵的生物。TEPs调理寄生虫，将它们作为破坏目标。 Spätzle和其他细胞因子触发细胞内信号传导途径，诱导 抗微生物肽和其它防御蛋白。在人类疾病的昆虫载体中，蛋白酶 网络可能会被入侵者的蛋白质逃避或破坏。对系统的知识 来自生物化学模式昆虫如烟草天蛾（Manducasexta）的组分及其相互作用是有用的 为了获得昆虫先天免疫基本方面的详细分子理解， 包括这些蛋白酶级联，并提供了基本知识，可以指导类似的研究 昆虫疾病媒介的系统。本文阐明了M. Sexta蛋白酶网络， 对细菌和真菌的反应激活PO原。在这个网络中，识别蛋白结合到 微生物并以级联模式激活蛋白酶。我们已经注释了193个基因编码 丝氨酸蛋白酶及其同源物，鉴定了幼虫血淋巴中的36种蛋白质， 数据表明，其中一些在免疫信号转导中起关键作用。基于 分子探针，纯化的蛋白质和工作经验，我们建议调查两个关键 步骤的蛋白酶网络，这是保守的双翅目物种，通过结合血淋巴 分级分离，重组蛋白生产和加工，序列和表达信息， 和最先进的质谱分析该项目的具体目标是：1）描述 通过检查肽聚糖、两种识别蛋白和 在结构域水平上的血淋巴蛋白酶-14前体（proHP 14）; 2）鉴定1−2 proHP 6-活化 蛋白酶，并阐明其激活机制。在这个项目中获得的新知识将 在生物化学水平上提高对昆虫免疫的认识，促进相关研究， 病媒物种。

Project Summary Extracellular serine proteases and their non-catalytic homologs coordinate key defense mechanisms in insects. This includes responses of mosquitoes against pathogens and parasites that cause serious human diseases. Proteolytic cleavage generates active phenoloxidases (POs), thioester proteins (TEPs), Spätzle and other cytokines. PO catalyzes the production of reactive chemicals to sequester and kill the invading organisms. TEPs opsonize parasites, targeting them for destruction. Spätzle and other cytokines trigger intracellular signaling pathways to induce the expression of antimicrobial peptides and other defense proteins. In insect vectors of human diseases, the protease networks may be evaded or disrupted by proteins from the intruders. Knowledge of the system components and their interactions from biochemical model insects such as Manduca sexta is useful for gaining detailed molecular understanding of fundamental aspects of insect innate immunity, including these protease cascades and provides basic knowledge that can guide studies of similar systems in insect disease vectors. We have elucidated a part of the M. sexta protease network that activates proPO in response to bacteria and fungi. In this network, recognition proteins bind to microbes and activate the proteases in a cascade mode. We have annotated 193 genes encoding serine proteases and their homologs, identified 36 of the proteins in larval hemolymph, and acquired data indicating that some of them play critical roles in immune signal transduction. Based on the molecular probes, purified proteins and working experience, we propose to investigate two critical steps of the protease network, which are conserved in dipteran species, by combining hemolymph fractionation, recombinant protein production and processing, sequence and expression information, and state-of-the-art mass spectrometry. The specific aims of this project are to: 1) characterize the system initiation by examining interactions of peptidoglycans, two recognition proteins, and hemolymph protease-14 precursor (proHP14) at the domain level; 2) identify 1−2 proHP6-activating proteases and elucidate their activation mechanisms. New knowledge gained in this project will improve understanding of insect immunity at a biochemical level and stimulate related research in vector species.