Initiation and Regulation of Immune Protease Pathways

描述(申请人提供)：胞外丝氨酸蛋白酶网络协调昆虫抵御微生物感染的关键机制。蛋白水解性激活原酚氧化酶(PPO)产生酚氧化酶，催化产生活性化合物以隔离和杀死入侵的病原体和寄生虫。SPTZLE前体的蛋白降解过程触发Toll途径，诱导抗菌肽和其他防御蛋白的合成。在人类疾病的昆虫媒介中，这些蛋白酶系统的启动和调节可能会被入侵者的蛋白质扰乱。了解这种生物化学模式昆虫的蛋白质相互作用将有助于理解和操纵节肢动物媒介中的类似系统，以干扰疾病传播。我们已经发现了六分枝杆菌蛋白酶网络的一个分支，它可以检测革兰氏阳性细菌和真菌，并介导PPO的激活。在这个网络中，病原体识别受体与微生物结合，启动蛋白酶途径激活PPO和Toll。我们已经鉴定出一种新的Sushi结构域的蛋白水解酶，它可能参与了响应革兰氏阴性细菌感染的途径的一个分支。蛋白水解酶系统中的正反馈环路控制着防御反应的位置和效力。我们计划通过检测免疫蛋白水解酶途径的启动和调节来扩展我们对PPO激活反应的研究。本项目的具体目标是：1)表征革兰氏阴性菌启动的途径；2)阐明蛋白水解酶系统中的正向调节机制；3)研究六分枝杆菌PPO的结构、功能和激活。这项研究将继续阐明丝氨酸蛋白酶网络在先天性免疫反应中的功能和调节，该网络产生酚氧化酶和抗菌肽，以捕获和杀死导致疟疾和丝虫病的寄生虫。所获得的知识将有助于阻断人类疾病在媒介物种中的传播。

DESCRIPTION (provided by applicant): An extracellular serine protease network coordinates key mechanisms of insect defense against microbial infection. Proteolytic activation of prophenoloxidase (PPO) generates phenoloxidase that catalyzes the production of reactive compounds to sequester and kill invading pathogens and parasites. Proteolytic processing of sp¿tzle precursor triggers the Toll pathway, which induces the synthesis of antimicrobial peptides and other defense proteins. In insect vectors of human diseases, initiation and regulation of these protease systems may be disrupted by proteins from the intruders. Knowledge of such protein interactions from biochemical model insects such as Manduca sexta will be useful for understanding and manipulating similar systems in arthropod vectors to interfere with disease transmission. We have discovered a branch of the M. sexta protease network that detects Gram-positive bacteria and fungi and mediates PPO activation. In this network, pathogen recognition receptors bind to microbes and initiate protease pathways for PPO and Toll activation. We have identified a new Sushi-domain protease that may participate in a branch of the pathway responsive to Gram-negative bacterial infection. A positive feedback loop in the protease system controls locality and potency of the defense responses. We plan to extend our research on the PPO activation reaction by examining the initiation and regulation of immune protease pathways. The specific aims of this project are: 1) Characterization of the pathway initiation by Gram-negative bacteria; 2) Elucidation of the positive regulatory mechanisms in the protease system; 3) Investigation of the structure, function, and activation of M. sexta PPO. PUBLIC HEALTH RELEVANCE This research will continue to elucidate the function and regulation of a serine protease network during innate immune responses, which produces phenoloxidase and antimicrobial peptides to entrap and kill parasites that cause malaria and filariasis. The acquired knowledge will be useful for disrupting the transmission of human diseases in vector species.