Proteinase systems in insect hemolymph

描述（由申请人提供）：至少两种昆虫免疫反应需要血淋巴蛋白酶激活血浆蛋白。一种是细胞因子前体的蛋白水解加工，包括Toll配体Sp ^ tzle。第二种蛋白酶介导的反应是酚氧化酶酶原的激活，它随后将循环中的二酚氧化为醌，醌是入侵生物体表面黑色素沉积的前体。这种先天免疫反应可能会影响昆虫媒介与它们传播的病原体和寄生虫感染的结果。血液中蛋白酶调节的反应不是简单的线性途径，而是通常是“蛋白酶网”，其中每个蛋白酶可能切割多个底物，活性蛋白酶可能受到几种抑制剂的调节。我们实验室和其他人之前的工作已经建立了鳞翅目昆虫，Manduca sexta，作为一个非常适合血淋巴蛋白酶生化研究的模型系统。sexta基因组序列的可用性和蛋白质组学技术的新发展使得利用生化系统方法研究sexta血淋巴中的丝氨酸蛋白酶网成为可能。本研究将旨在了解血淋巴蛋白酶途径，它们潜在的交叉连接，以及它们的调控。蛋白质组学系统方法可能揭示以前未发现的蛋白酶激活的昆虫免疫分子。该项目的具体目标是：1。使用Manduca sexta基因组和转录组序列数据来鉴定和注释编码丝氨酸蛋白酶和蛋白酶抑制剂的基因，并为进一步的实验生产试剂。2. 使用蛋白质组学实验的系统方法来确定途径组分和选定的血淋巴蛋白酶的底物库，然后使用纯化蛋白进行实验来验证和表征相互作用。3. 研究蛇蛋白对血淋巴蛋白酶通路的调节，采用体外、离体和体内实验方法。研究的长期目标是深入了解Manduca介导先天免疫反应的蛋白酶级联的调控，将这些知识应用于人类疾病的昆虫载体，并利用Manduca系统的优势进行丝氨酸蛋白酶活性调控的基础研究。

DESCRIPTION (provided by applicant): At least two types of insect immune responses require activation of plasma proteins by proteases in hemolymph. One is the proteolytic processing of cytokine precursors, including the Toll ligand Sp¿tzle. A second protease-mediated response is the activation of phenoloxidase zymogens, which then oxidize circulating diphenols to quinones, precursors for deposition of melanin on the surface of invading organisms. Such Innate immune responses are likely to affect the outcome of infections of insect vectors with the pathogens and parasites they transmit. Responses modulated by proteases in blood are not simply linear pathways, but instead are often "protease webs," in which each protease may cleave multiple substrates, and active proteases may be regulated by several inhibitors. Previous work from our laboratory and others has established the lepidopteran insect, Manduca sexta, as a model system well suited for biochemical investigation of hemolymph proteases. The coming availability of a M. sexta genome sequence and new developments in proteomics technology make possible the investigation of serine protease webs in M. sexta hemolymph, using biochemical systems approaches. This research will be aimed at understanding hemolymph protease pathways, their potential cross-connections, and their regulation. The proteomics systems approach may reveal previously undiscovered protease-activated immune molecules of insects. The specific aims of the project are to: 1. Use Manduca sexta genome and transcriptome sequence data to identify and annotate genes encoding putative serine proteases and protease inhibitors and to produce reagents for further experimentation. 2. Use a systems approach for proteomics experiments to identify pathway components and the substrate repertoire of selected hemolymph proteases, followed by experiments to validate and characterize interactions using purified proteins. 3. Investigate the regulation of protease pathways in hemolymph by serpins, using in vitro, ex vivo, and in vivo experimental approaches. The long term goals of the research are to gain a thorough understanding of the regulation of protease cascades that mediate innate immune responses in Manduca, to apply this knowledge to insect vectors of human diseases, and to apply advantages of the Manduca system for fundamental studies on the regulation of serine protease activity.