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抗微生物感染的组成部分。酚氧化酶（PO）催化醌类的产生，所述醌类是角质层硬化、伤口愈合和入侵微生物的黑色素包封的前体。由于醌类的高反应性和细胞毒性，酚氧化酶原（PPO）的蛋白水解活化必须被严格调节为针对非自身的有效的局部反应。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，如天蛾的激活过程的知识将有助于理解和操纵一个类似的系统在吸血昆虫传播人类疾病。从M. sexta需要裂解的丝氨酸蛋白酶同系物（SPH）来激活PPO。分子克隆和序列比较表明，PAP和SPH都含有调控片段结构域。我们计划扩大我们目前对PAP的研究，以深入了解触发和调节M中PPO活化的分子机制。六。本研究的主要目的是：1）PPO激活级联反应起始蛋白酶HP 14的特性分析：2）分离和克隆PPO激活酶proSPH-1和proSPH-2的cDNA; 3)PAP-2中夹子结构域的功能分析。

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.