Prophenoloxidases of Anopheles gambiae

冈比亚按蚊前酚氧化酶

基本信息

批准号：
8747938
负责人：
HAOBO JIANG
金额：
$ 20.28万
依托单位：
OKLAHOMA STATE UNIVERSITY STILLWATER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-04 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8747938
关键词：
Adult African Anopheles gambiae Bacteria Biochemical Biochemical Reaction Biological Assay Chemicals Complementary DNA Complex Coupled Culicidae Cytolysis Detergents Disease Vectors Double-Stranded RNA Drug or chemical Tissue Distribution Employee Strikes Enzymes Escherichia coli Female Filariasis Foundations Future Genes Genetic Genetic Research Homologous Gene Immune Immune system Immunohistochemistry Individual Insecta Kinetics Knowledge Lead Life Malaria Manduca sexta Mediating Melanins Messenger RNA Methods Modeling Molecular Conformation Molecular Profiling Monoclonal Antibodies Mutate Mycoses Nematoda Parasites Parasitic nematode Peptide Hydrolases Phenotype Physiological Physiological Processes Pigmentation physiologic function Plasmodium Property Proteins Proteomics Reagent Recombinant Proteins Regulation Research Roentgen Rays Serine Protease Site-Directed Mutagenesis Skin tanning Staging Structure Structure-Activity Relationship Substrate Specificity System Testing Tissues Work Wound Healing base defense response disease transmission enzyme structure eumelanin genetic analysis human disease inhibitor/antagonist insight killings knock-down member pathogen polyclonal antibody positional cloning preference pro-phenoloxidase public health relevance resistance mechanism success transmission process vector

项目摘要

DESCRIPTION (provided by applicant): Phenoloxidases take part in key insect physiological processes including pigmentation, cuticle tanning, wound healing, and defense responses. Some mosquito phenoloxidases generate active chemicals and eumelanin to kill and sequester malaria parasites. While reverse genetic analyses have revealed components of this enzyme system, important knowledge gaps still exist due to lack of biochemical analysis. In studies of the Manduca sexta prophenoloxidase (PPO) activation system, we accumulated wealthy knowledge on PPOs, PPO activating proteases, and their regulators. We cloned cDNAs of all nine Anopheles gambiae PPOs, expressed them as functional proteins in E. coli, crystallized three, and solved one PPO structure. Initial kinetic analysis revealed striking differences in substrate preference and catalytic efficiency. We validated the proteomic approach to semi-quantify PPOs and other proteins in the mosquito and detected PPOs in tissues using polyclonal antibodies. By injecting into female adults dsRNA encoding a conserved region in PPOs, we substantially reduced their mRNA and protein levels. Based on these advances, we propose to study the structures, functions, and expression of A. gambiae PPO1-9. Acquired knowledge and reagents will be useful for exploring how parasites evade or suppress the host immune mechanism in the future.

描述（由申请人提供）：酚氧化酶参与关键的昆虫生理过程，包括色素沉着、角质层晒黑、伤口愈合和防御反应。一些蚊子酚氧化酶会产生活性化学物质和真黑素来杀死和隔离疟疾寄生虫。虽然反向遗传分析已经揭示了该酶系统的组成部分，但由于缺乏生化分析，仍然存在重要的知识空白。在Manduca sexta原酚氧化酶（PPO）激活系统的研究中，我们积累了关于PPO、PPO激活蛋白酶及其调节因子的丰富知识。我们克隆了所有九种冈比亚按蚊 PPO 的 cDNA，在大肠杆菌中将它们表达为功能蛋白，结晶了三种，并解析了一种 PPO 结构。初始动力学分析揭示了底物偏好和催化效率的显着差异。我们验证了半定量蚊子中 PPO 和其他蛋白质的蛋白质组学方法，并使用多克隆抗体检测了组织中的 PPO。通过将编码 PPO 保守区域的 dsRNA 注射到雌性成虫体内，我们大大降低了它们的 mRNA 和蛋白质水平。基于这些进展，我们建议研究 A. gambiae PPO1-9 的结构、功能和表达。获得的知识和试剂将有助于探索未来寄生虫如何逃避或抑制宿主免疫机制。