Macromolecular assemblies of transcription factors initiated by pathogen infection

病原体感染引发的转录因子大分子组装

• 批准号: 10000831
• 负责人: Reginald McNulty
• 金额: $ 10.34万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-22 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10000831
• 关键词: 3-Dimensional; Affect; Binding; Biochemical; Biological; C-terminal; CASP1 gene; Cancerous; Caspase; Cell Death; Cells; Cellular biology; Complex; Computer software; Cryoelectron Microscopy; Crystallization; Crystallography; DNA Binding Domain; Data; Development; Disease; Economically Deprived Population; Electron Microscopy; Exposure to; Familial amyloid nephropathy with urticaria and deafness; Filament; Goals; Grain; Helicobacter pylori; Heterogeneity; Human; Image; In Vitro; Incidence; Infection; Inflammasome; Inflammation; Inflammation Mediators; Interleukin-1 beta; Interleukin-18; Lead; Length; Life; Ligands; Location; Macromolecular Complexes; Malignant Neoplasms; Mediating; Mediator of activation protein; Mentors; Methods; Mexican Americans; Mind; Mitochondrial DNA; Modeling; Molecular Conformation; Molecular Structure; Mutation; N-terminal; Organ failure; Pathogenicity; Patients; Pharmaceutical Preparations; Pharmacology; Physiological; Play; Population; Positioning Attribute; Post-Translational Protein Processing; Production; Protein Conformation; Protein p53; Proteins; Regulation; Research; Research Personnel; Research Support; Role; Sampling; Sampling Studies; Shapes; Signal Transduction; Structural Biologist; Structural Protein; Structure; System; TP53 gene; Techniques; Transcriptional Regulation; United States National Institutes of Health; Virus; Virus Diseases; Work; base; cancer therapy; design; experience; experimental study; host-microbe interactions; insight; interest; macromolecular assembly; macrophage; microbial; mouse model; novel therapeutic intervention; pathogen; pathogenic bacteria; pathogenic microbe; prevent; protein structure; three dimensional structure; transcription factor

PROJECT SUMMARY Over the years, I've grown intellectually and demonstrated my potential to become an independent investigator. I have surrounded myself with mentors that have my best professional interests in mind. My work with Prof. Jack Johnson focused on an in vitro system of viruses for studies with primarily by electron microscopy. With Prof. Karin I would grain experience in cell biology studying interactions of transcription factors. I will develop a proficiency in Cryo-EM as I acquire experiences with the nuances associated with imaging transcription factor complexes. This will enable me to establish a single niche I need to pursue fundable research questions as a Structural Biologist, with significant contributions to the field of cell-mediated transcriptional regulation and viral infection. The goal of this project is to increase our understanding of how microbial pathogens lead to aberrant p53 and NLRP3 inflammasome assembly. The tumor suppressor p53 is a transcription factor that prevents cancer by promoting cell death. The NLRP3 inflammasome – which controls the production of bioactive IL-1β and IL-18 via caspase-1, and p53 are key mediators of bacterial-induced inflammation and cancer. Both NLRP3 inflammasome and p53 are subject to substantial post-translational modifications which all influence their structure and function. Although there are crystal structures for isolated domains of p53, and partially for the N- terminal domain of NLRP3, it remains to be determined they form macromolecular complexes within the cell and in what way the functional mechanisms are different in cancer. The p53 N- and C- terminal regions are unstructured, which not only can cause intramolecular heterogeneity, but quaternary structures of the macromolecular complexes may also be heterogeneous. Thus, quaternary structure determination via crystallography would be extremely challenging, if not impossible. My long-term goal is to provide a physiological picture of microbial – host interactions inside of human cells. The overall objective of this application, which is the next step toward attainment of this long-term goal, is to use Cryo-EM to characterize the NLRP3 inflammasome, and p53 tetramer macromolecular complexes in the presence of heterogeneity. With this technique, software can be used to "purify" various biologically relevant conformational or stoichiometric populations. I am interested in the native structure of these proteins and conformational changes that occur upon binding microbial infection. Herein we provide preliminary data towards elucidating the structure and domain organization for the full-length native p53 tetramer using electron microscopy. We will also use 3D Cryo-EM to characterize 3D interactions of NLRP3 inflammasome. The rationale for the proposed research is that once initial native structures of p53 and NLRP3 inflammasome proteins are known, pharmacological drugs can be designed to restore physiological functions of p53 and NLRP3.

项目总结 多年来，我在智力上有所增长，并展示了我成为独立人士的潜力 调查员。我周围都是那些考虑到我最大的职业利益的导师。我的作品 杰克·约翰逊教授专注于病毒的体外系统研究，主要是通过电子手段 显微镜。在卡林教授的指导下，我将拥有研究转录相互作用的细胞生物学经验 各种因素。随着我获得与以下各项相关的细微差别的经验，我将精通冷冻-EM 成像转录因子复合体。这将使我能够建立一个我需要追求的单一利基市场 作为结构生物学家的可资助研究问题，对细胞介导的领域做出了重大贡献 转录调控和病毒感染。 这个项目的目标是增加我们对微生物如何导致P53和P53异常的理解 NLRP3炎症体组装。肿瘤抑制因子P53是一种转录因子，通过以下途径预防癌症 促进细胞死亡。NLRP3炎症体--控制生物活性IL-1β和IL-18的产生 通过caspase-1和p53是细菌诱导的炎症和癌症的关键介质。两个NLRP3 炎症体和P53受到翻译后的大量修饰，这些修饰都会影响它们的 结构和功能。虽然P53的分离结构域有晶体结构，部分N- NLRP3的末端结构域，还有待确定它们在细胞内形成大分子复合体 以及癌症的作用机制在哪些方面有所不同。P53的N-末端和C-末端区域是 非结构化，这不仅会导致分子内的异质性，而且还会导致分子的四级结构 大分子络合物也可能是多相的。因此，四元结构的确定通过 结晶学将极具挑战性，如果不是不可能的话。我的长期目标是提供一个 人体细胞内微生物与宿主相互作用的生理学图景。这样做的总体目标是 应用，这是实现这一长期目标的下一步，是使用Cryo-EM来表征 NLRP3炎症体与P53四聚体大分子复合体中存在异质性。 有了这项技术，软件就可以用来“提纯”各种生物相关的构象或 化学计量人口。我对这些蛋白质的天然结构和构象变化很感兴趣 在结合微生物感染时发生。在这里，我们提供了初步数据，以澄清 电子显微镜下全长天然P53四聚体的结构和结构域组织。我们会 用3DCryo-EM表征NLRP3炎性小体的3D相互作用。建议的理由是 研究表明，一旦知道了P53和NLRP3炎症体蛋白的初始天然结构， 可以设计药理药物来恢复P53和NLRP3的生理功能。