Proteinase systems in insect hemolymph

描述(由申请人提供)：至少有两种类型的昆虫免疫反应需要血淋巴中的蛋白酶激活血浆蛋白。一个是细胞因子前体的蛋白质分解过程，包括Toll配体Sp？tzle。蛋白水解酶介导的第二种反应是酚氧化酶酶原的激活，然后将循环中的二苯酚氧化成苯二酚，这是黑色素沉积在入侵生物表面的前体。这种先天免疫反应可能会影响昆虫媒介感染它们所传播的病原体和寄生虫的结果。血液中的蛋白水解酶调节的反应不是简单的线性途径，而是经常是“蛋白酶网”，其中每一种蛋白水解酶可以裂解多个底物，并且活性的蛋白水解酶可能受到几种抑制物的调节。我们实验室和其他人的工作已经建立了鳞翅目昆虫Manduca sexta，作为一个很好地适合于血淋巴蛋白酶的生化研究的模型系统。随着六分枝杆菌基因组序列的出现和蛋白质组学技术的新发展，利用生化系统的方法对六分枝杆菌血淋巴中的丝氨酸蛋白酶网络进行研究成为可能。这项研究旨在了解血淋巴蛋白水解酶途径，它们潜在的交叉连接，以及它们的调节。蛋白质组学系统的方法可能揭示以前未发现的昆虫的蛋白酶激活的免疫分子。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识别和注释编码可能的丝氨酸蛋白酶和蛋白酶抑制剂的基因，并生产用于进一步实验的试剂。2.使用系统的方法进行蛋白质组学实验，以确定选定的血淋巴蛋白水解酶的途径成分和底物库，然后使用纯化的蛋白质进行实验以验证和表征相互作用。3.采用体外、体外和体内实验方法，研究蛇类对血淋巴中蛋白水解酶途径的调节。这项研究的长期目标是彻底了解Manduca中介导先天免疫反应的蛋白酶级联的调节，将这一知识应用于人类疾病的昆虫媒介，并将Manduca系统的优势应用于丝氨酸蛋白酶活性调节的基础研究。

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.