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.

项目摘要 肺炎链球菌（SPN）是社区获得性肺炎的主要病因 美国和欧洲，仍然是儿童肺炎和脑膜炎的主要原因 根据世界卫生组织的说法，在全球范围内，使其成为“主要的全球公共卫生问题”。尽管 当前的疫苗目标表面多糖仅包括已知血清型的一个子集，策略 已经提倡针对更广泛表达的蛋白质病毒因子，例如SPN IGA1 这里正在研究的蛋白酶（IgA1p）。但是，SPN IGA1P及其IGA1的相对大尺寸 它裂解以阻止初始宿主免疫反应的基材先前排除了针对的研究 阐明其分子结构和相互作用。通过通过综合性制定新颖的策略 结合了多种生物物理/生化方法在内的方法，包括冷冻EM，我们已经开始 阐明SPN IGA1P的结构。值得注意的是，SPN是否存在新的结构褶皱 在域之间形成的IgA1p活动位点与其他Zn-MetAllalloteases相同，并确认我们 先前发表的催化机制。考虑到最新利用基于结构的策略的成功 为了开发基于广泛的疫苗，我们的目标是提供第一个高分辨率结构 仅IgA1p，以及其IgA1底物，以及第一代单克隆抗体（MAB） 阻止IgA1p活动。这些研究将对我们将如何生成疫苗以及潜在的可能产生重大影响 通过靶向IgA1p来阻断SPN感染的治疗。 假设：大型SPN IGA1P包括多个独立折叠的亚基，形成独特的3-- 尺寸结构，其中包括连接的亚基，形成金属蛋白酶活性位点以正确 将IgA1定向进行铰链裂解。此外，该活性位点被中和的mAb遮住。我们将 通过以下特定目的解决这一假设： 目标1）完成新型IgA1p催化区域的冷冻EM结构，以确定其如何 亚基相互作用以形成活性金属蛋白酶。 AIM 2）确定IGA1P如何与其IgA1底物相互作用，以及如何通过A阻断这种相互作用 单克隆抗体。