Initiation and Regulation of Prophenoloxidase Activation

描述（由申请人提供）：昆虫的酚氧化酶原激活途径是宿主免疫系统对抗微生物感染的一个组成部分。酚氧化酶（PO）催化醌的产生，醌是角质层硬化、伤口愈合和入侵微生物的黑素包封的前体。由于醌类化合物的高反应性和细胞毒性，原酚氧化酶（PPO）的蛋白水解活性必须作为一种有效的局部反应受到严格调节。在人类疾病的昆虫媒介中，这一过程的启动和控制可能受到来自病原体或寄生虫的蛋白质的干扰。从生物化学模式昆虫（如Manduca sexta）中获得的激活过程的知识将有助于理解和操纵传播人类疾病的吸血昆虫的类似系统。从sexta中分离到的三种PPO激活蛋白酶（PAPs）需要裂解丝氨酸蛋白酶同源物（SPHs）来激活PPO。分子克隆和序列比较表明，PAPs和SPHs均含有调控片段结构域。我们计划扩大目前对PPO的研究，深入了解m.s sexta中触发和调节PPO激活的分子机制。本项目的具体目标是：1)PPO激活级联的起始蛋白酶HP14的表征；2) proSPH-1和proSPH-2活化酶的分离与cDNA克隆；

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.