Determining how the giant Streptococcus Pneumoniae IgA1 protease cleaves its host IgA1 substrate and how this interaction can be blocked

确定巨型肺炎链球菌 IgA1 蛋白酶如何裂解其宿主 IgA1 底物以及如何阻断这种相互作用

• 批准号: 9896366
• 负责人: ELAN Z EISENMESSER
• 金额: $ 19.44万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-19 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896366
• 关键词: Active Sites; Address; Advocate; Architecture; Award; Bacterial Meningitis; Bacterial Pneumonia; Binding; Biochemical; Biophysics; Blocking Antibodies; Catalytic Domain; Child; Cleaved cell; Colorado; Communities; Country; Cryoelectron Microscopy; Development; Electron Microscopy; Enzymes; Europe; Generations; Goals; IgA-specific serine endopeptidase; IgA1; Immune response; Immunologics; Infection; Investigation; Letters; Mass Spectrum Analysis; Metalloproteases; Methods; Molecular; Molecular Structure; Monoclonal Antibodies; Nature; Nuclear Magnetic Resonance; Pacific Northwest; Pathogenicity; Peptide Hydrolases; Phagocytes; Pneumococcal Infections; Pneumonia; Polysaccharides; Production; Proteins; Public Health; Publishing; Recombinants; Research Personnel; Resolution; Serotyping; Streptococcus pneumoniae; Structure; Substrate Interaction; Surface; Techniques; Therapeutic; Time; Titan; United States; Vaccine Design; Vaccine Research; Vaccines; Virulence; Virulence Factors; Work; World Health Organization; base; cryogenics; immunogenic; mucosal vaccine; neutralizing monoclonal antibodies; novel; novel strategies; prevent; programs; protein folding; success; therapeutic target; three dimensional structure; universal vaccine; vaccine candidate; vaccine development

PROJECT SUMMARY Streptococcus pneumoniae (SPN) is the primary causative agent for community-acquired pneumonia in the United States and Europe and remains the leading cause of bacterial pneumonia and meningitis in children worldwide, making it a “major global public health problem” according to the World Health Organization. While current vaccines target surface polysaccharides that comprise only a subset of the known serotypes, strategies aimed against a more widely expressed protein virulence factor have been advocated, such as the SPN IgA1 Protease (IgA1P) under investigation here. However, the relatively large size of SPN IgA1P and its IgA1 substrate that it cleaves to thwart the initial host immune response has previously precluded studies aimed at elucidating its molecular structure and interactions. By developing novel strategies through an integrative approach that combines multiple biophysical/biochemical methods including cryo-EM, we have begun elucidating the structure of SPN IgA1P. Remarkably, despite the presence of novel structural folds, the SPN IgA1P active site that is formed between domains is identical to other Zn-metalloproteases and confirms our previously published catalytic mechanism. Considering recent success in utilizing structure-based strategies for the development of broad-based vaccines, our goals here are to provide the first high-resolution structures of IgA1P alone, together with its IgA1 substrate, and with a first-generation monoclonal antibody (mAb) that blocks IgA1P activity. These studies will have a major impact on how we will generate vaccines and potentially therapeutics to block SPN infection through targeting IgA1P. Hypothesis: The large SPN IgA1P comprises multiple independently folded subunits that forms a unique 3- dimensional structure, which includes subunits that join to form the metalloprotease active site to properly orient the IgA1 for hinge cleavage. Furthermore, this active site is occluded by a neutralizing mAb. We will address this hypothesis through the following specific aims: Aim 1) Complete the cryo-EM structure of the novel IgA1P catalytic region to determine how its subunits interact to form the active metalloprotease. Aim 2) Determine how IgA1P interacts with its IgA1 substrate and how this interaction is blocked by a monoclonal antibody.

项目总结 肺炎链球菌(SPN)是引起社区获得性肺炎的主要病原体。 美国和欧洲，仍然是儿童细菌性肺炎和脑膜炎的主要原因 世界范围内，使其成为世界卫生组织所称的“主要的全球公共卫生问题”。而当 目前的疫苗针对的是仅包含已知血清型、策略的子集的表面多糖 针对一种更广泛表达的蛋白，毒力因子已被提倡，如SPN-IgA1 蛋白水解酶(IgA1P)在这里被研究。而SPN IgA1P及其IgA1相对较大 它切割的底物会阻碍最初的宿主免疫反应，此前已经排除了旨在 阐明其分子结构和相互作用。通过综合的 结合多种生物物理/生化方法的方法，包括冷冻EM，我们已经开始 阐明SPN-IgA1P的结构。值得注意的是，尽管存在新的结构性褶皱，SPN 结构域之间形成的IgA1P活性位点与其他锌金属蛋白酶相同，证实了我们的 以前发表的催化机理。考虑到最近在利用基于结构的战略方面取得的成功 对于基础广泛的疫苗的开发，我们的目标是提供第一个高分辨率结构 单独的IgA1P及其IgA1底物，以及第一代单抗(MAb)， 阻断IgA1P活性。这些研究将对我们如何生产疫苗产生重大影响，并有可能 通过靶向IgA1P来阻断SPN感染的治疗 假设：大的SPN IgA1P由多个独立折叠的亚基组成，形成一个独特的3- 空间结构，包括连接形成金属蛋白酶活性部位的亚基，以适当地 为铰链解理调整IgA1的方向。此外，该活性部位被中和单抗阻断。我们会 通过以下具体目标解决这一假设： 目的1)完成新型IgA1P催化区的冷冻EM结构，以确定其 亚基相互作用形成活性金属蛋白酶。 目的2)确定IgA1P如何与其IgA1底物相互作用，以及这种相互作用如何被一种 单抗。