Proteinase Systems in Insect Hemolymph

胞外丝氨酸蛋白酶级联调节保护性反应以限制出血和感染 脊椎动物和无脊椎动物，但我们对昆虫中这些途径的了解还处于初级阶段 最好的。该实验室以前的工作已经建立了鳞翅目昆虫Manduca sexta作为模式 适用于血淋巴蛋白水解酶复杂功能的生化表征系统。 该方案中的实验旨在研究血浆丝氨酸蛋白酶及其受体的作用。 昆虫天然免疫反应中的抑制物(蛇毒)，包括酚氧化酶的激活。这项工作将 根据以下模型检验假设。血浆中的模式识别蛋白与微生物结合 表面。然后，这些蛋白质与丝氨酸蛋白酶酶原和其他蛋白质辅因子相互作用，形成 蛋白质复合体，它定位了蛋白酶级联和随后的黑色素沉积到外源 浮出水面。单独的路径(或汇聚路径的单独分支)可以由不同的 微生物的类型，因为它们是由不同的模式识别蛋白质组识别的。抑制 通过与蛇类的特定相互作用产生的蛋白水解酶限制了反应的持续时间和位置。 该项目的具体目标是： 1.研究特定的蛇链对选定的Clip结构域蛋白水解酶的激活和抑制作用，以 确定它们在微生物激活的级联通路中的顺序。 2.确定导致形成蛋白酶激活复合体的蛋白质相互作用，并研究 这类络合物的组装及其在表面定位酚氧化酶活性中的作用。 相关性：先天免疫反应可能会影响感染带有 它们传播的病原体和寄生虫。这项研究的长期目标是获得彻底的 了解介导六分枝杆菌先天免疫反应的蛋白酶级联反应，以应用这一点 对人类疾病的昆虫媒介的认识，并应用六分枝杆菌系统的优势 丝氨酸蛋白酶活性调控的基础研究。

Extracellular serine protease cascades modulate protective responses to limit bleeding and infection in vertebrate and invertebrate animals, but our understanding of these pathways in insects is rudimentary at best. Previous work from this laboratory has established the lepidopteran insect, Manduca sexta, as a model system well suited for biochemical characterization of the complex functions of hemolymph proteases. Experiments in this proposal are designed to investigate the roles of plasma serine proteases and their inhibitors (serpins) in insect innate immune responses, including activation of phenoloxidase. The work will test hypotheses based on the following model. Pattern recognition proteins in plasma bind to microbial surfaces. These proteins then interact with serine protease zymogens and other protein cofactors to form protein complexes, which localize the protease cascade and subsequent melanin deposition to the foreign surface. Separate pathways (or separate branches of converging pathways) may be initiated by different types of microorganisms, as they are recognized by separate sets of pattern recognition proteins. Inhibition of the proteases by specific interactions with serpins limit the duration and location of the response. The specific aims of the projectare: 1. Investigate the activation of selected clip domain proteases and their inhibition by specific serpins, to determine their order in microbe-activated cascade pathways. 2. Identify protein interactions that result in formation of protease activation complexes and investigate the assembly of such complexes and their roles in localizing phenoloxidase activation on surfaces. Relevance: Innate immune responses are likely to affect the outcome of infections of insect vectors with the pathogens and parasites they transmit. The long term goals of the research are to gain a thorough understanding of the protease cascades that mediate innate immune responses in M. sexta, to apply this knowledge to insect vectors of human diseases, and to apply advantages of the M. sexta system for fundamental studies on the regulation of serine protease activity.