Initiation and Regulation of Prophenoloxidase Activation

描述(由申请人提供)：昆虫中的酚氧化酶原激活途径是宿主免疫系统抵御微生物感染的不可或缺的组成部分。酚氧化酶(PO)催化苯二酚的产生，是角质层硬化、伤口愈合和入侵微生物黑色素包裹的前体。由于苯二酚的高反应性和细胞毒性，酚氧化酶原(PPO)的蛋白水解性激活作为一种针对非我的强烈的局部反应必须受到严格的调控。在人类疾病的昆虫媒介中，这一过程的启动和控制可能会受到病原体或寄生虫的蛋白质的干扰。了解生物化学模型昆虫(如六分金龟子)的激活过程将有助于理解和操纵传播人类疾病的食血昆虫的类似系统。从六倍子中分离到的三种PPO激活蛋白(PAPs)需要裂解丝氨酸蛋白酶同源酶(SPHs)来激活PPO。分子克隆和序列比较表明，PAPs和SPHs都含有调控的Clip结构域。我们计划扩大我们目前对PAPs的研究，以深入了解触发和调节Msexta中PPO激活的分子机制。本项目的具体目标是：1)鉴定PPO激活级联的起始酶HP14：2)分离和克隆ProSPH-1和proSPH-2的活化酶； 3)PAP-2中CLIP结构域的功能分析。

DESCRIPTION (provided by applicant): The prophenoloxidase activation pathway in insects is an integral component of the host immune system against microbial infection. Phenoloxidase (PO) catalyzes the production of quinones that are precursors for cuticle sclerotization, wound healing, and melanotic encapsulation of invading microorganisms. Due to high reactivity and cytotoxicity of quinones, proteolytic activation of prophenoloxidase (PPO) has to be tightly regulated as a potent, local reaction against nonself. In insect vectors of human diseases, initiation and control of this process may be interfered by proteins from pathogens or parasites. Knowledge of the activation process from biochemical model insects such as Manduca sexta will be useful for understanding and manipulating a similar system in blood-feeding insects that transmit human diseases. Three PPO-activating proteinases (PAPs) isolated from M. sexta require cleaved serine proteinase homologs (SPHs) for PPO activation. Molecular cloning and sequence comparison indicate that the PAPs and SPHs all contain regulatory clip domains. We plan to expand our current research on PAPs to acquire in-depth understandings of the molecular mechanisms that trigger and regulate the PPO activation in M. sexta. The specific aims of this project are: 1) Characterization of HP14, an initiation proteinase of the PPO activation cascade; 2) Isolation and cDNA cloning of an activating enzyme for proSPH-1 and proSPH-2; 3) Functional analysis of the clip domains in PAP-2.