Macromolecular assemblies of transcription factors initiated by pathogen infection

项目摘要 这些年来，我在智力上得到了成长，并展示了我成为一名独立人士的潜力。 调查员我身边的导师都是我最感兴趣的专业人士。我的工作 与Jack约翰逊教授一起专注于体外病毒系统的研究，主要通过电子 显微镜与Karin教授一起，我将获得细胞生物学研究转录相互作用的经验。 因素我将熟练掌握冷冻EM，因为我获得了与以下方面相关的细微差别的经验： 成像转录因子复合物。这将使我能够建立一个单一的利基我需要追求 作为一个结构生物学家，基金的研究问题，与细胞介导的领域的重大贡献 转录调控和病毒感染。 该项目的目标是增加我们对微生物病原体如何导致异常p53的理解， NLRP 3炎性体组装。肿瘤抑制因子p53是一种转录因子，通过以下方式预防癌症： 促进细胞死亡。NLRP 3炎性体-控制生物活性IL-1β和IL-18的产生 通过半胱天冬酶-1和p53是细菌诱导的炎症和癌症的关键介质。两个NLRP 3 炎性小体和p53经历大量的翻译后修饰，这些修饰都影响它们的表达。 结构和功能。虽然存在p53的孤立结构域的晶体结构，并且部分存在N- NLRP 3的末端结构域，但它们在细胞内形成大分子复合物仍有待确定 以及癌症中的功能机制有何不同。p53的N-和C-末端区域是 非结构化的，这不仅可以导致分子内的异质性，但四级结构的 大分子复合物也可以是多相的。因此，四级结构测定通过 结晶学将是极具挑战性的，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图像。本报告的总体目标 实现这一长期目标的下一步是使用Cryo-EM来表征 NLRP 3炎性小体和p53四聚体大分子复合物在异质性存在下的变化。 利用该技术，软件可用于“纯化”各种生物学相关构象或构象异构体。 化学计量种群我感兴趣的是这些蛋白质的天然结构和构象变化 在结合微生物感染时发生。在此，我们提供了初步的数据，以阐明 使用电子显微镜观察全长天然p53四聚体的结构和结构域组织。我们将 还使用3D Cryo-EM来表征NLRP 3炎性体的3D相互作用。建议的理由 研究是一旦已知p53和NLRP 3炎性体蛋白的初始天然结构， 可以设计药理学药物来恢复p53和NLRP 3的生理功能。

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.