Initiation and Regulation of Immune Protease Pathways

描述（申请人提供）：细胞外丝氨酸蛋白酶网络协调昆虫防御微生物感染的关键机制。酚氧化酶原（PPO）的蛋白水解激活产生酚氧化酶，酚氧化酶催化活性化合物的产生，以隔离和杀死入侵的病原体和寄生虫。蛋白酶前体的蛋白水解过程触发Toll通路，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被来自入侵者的蛋白质破坏。从生物化学模式昆虫（如Manduca sexta）中了解这种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m.s sexta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激活。在这个网络中，病原体识别受体与微生物结合并启动PPO和Toll激活的蛋白酶途径。我们已经确定了一个新的寿司结构域蛋白酶，可能参与响应革兰氏阴性细菌感染的途径分支。蛋白酶系统中的正反馈回路控制着防御反应的位置和效力。我们计划通过检查免疫蛋白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)革兰氏阴性菌启动途径的表征；2)阐明蛋白酶系统的正调控机制；3) m.s sexta PPO的结构、功能和活化研究。本研究将继续阐明先天免疫反应中丝氨酸蛋白酶网络的功能和调控，该网络产生酚氧化酶和抗菌肽，以诱捕和杀死引起疟疾和丝虫病的寄生虫。获得的知识将有助于破坏人类疾病在病媒物种中的传播。

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.