Mechanistic investigation of ExoY-family enzyme activation by actin

肌动蛋白激活 ExoY 家族酶的机制研究

• 批准号: 10335145
• 负责人: Jordan Michael Mancl
• 金额: $ 6.98万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10335145
• 关键词: Actins; Address; Adenylate Cyclase; Area; Bacillus anthracis; Bacteria; Bacterial Infections; Bacterial Toxins; Bacterial exotoxin; Binding; Biochemical; Biological Assay; Biophysics; Bordetella pertussis; Bundling; Burkholderia; Calmodulin; Catalysis; Cell physiology; Cells; Characteristics; Clinical; Communication; Complex; Coupled; Cryoelectron Microscopy; Crystallization; Cyclic Nucleotides; Cytoskeletal Proteins; Cytoskeleton; Cytotoxin; Data; Deuterium; Development; Dimerization; Drosophila genus; Eels; Electron Microscopy; Electrons; Enzyme Activation; Enzymes; Equilibrium; Exotoxins; F-Actin; Family; Fellowship; Fostering; Foundations; G Actin; Goals; Heterogeneity; Homologous Gene; Hydrogen; In Vitro; Infection; Intoxication; Investigation; Knowledge; Length; Link; Mass Spectrum Analysis; Mediating; Mentorship; Methods; Microbiology; Microfilaments; Modeling; Molecular; Molecular Sieve Chromatography; Mutagenesis; Nucleotides; Orthologous Gene; Paper; Play; Polymers; Population Heterogeneity; Positioning Attribute; Principal Investigator; Process; Protein Family; Proteins; Pseudomonas; Pseudomonas aeruginosa; Publishing; Research; Resolution; Roentgen Rays; Role; Sampling; Scientist; Second Messenger Systems; Signal Pathway; Signal Transduction; Structure; System; Techniques; Thick; Toxin; Vibrio; Vibrio vulnificus; Virulence Factors; Virulent; Work; X-Ray Crystallography; actin 2; analytical ultracentrifugation; anthrax edema factor; base; beta Actin; biophysical techniques; cell motility; combat; design; edema factor; effectiveness evaluation; emerging human pathogen; genetic regulatory protein; human pathogen; inhibitor; interest; mutant; nanobodies; novel; novel therapeutic intervention; particle; pathogen; post-doctoral training; research and development; skills; small molecule inhibitor; structural biology; therapeutic development; tool; tripolyphosphate

Project Summary: The ExoY family of cytotoxins are prevalent virulent factors expressed by human pathogens of emerging clinical interest. ExoY family toxins function as nucleotidyl cyclases, enzymes that catalyze the conversion of nucleotide triphosphates into cyclic nucleotide monophosphates. Studies have shown that the enzymatic activity of ExoY family enzymes is stimulated by differing bioforms of actin, a prevalent eukaryotic cytoskeletal protein that exists in two distinct forms: monomeric, globular actin (G-actin), and polymeric actin filaments (F-actin). I have recently investigated the relationship between ExoY from Pseudomonas aeruginosa (PaExoY) and its activator, F-actin. I discovered that PaExoY can reorganize actin filaments into thick cable-like bundles. This actin bundling activity is commonly observed among eukaryotic regulatory proteins and my data suggests that it may be coupled to PaExoY catalysis. Actin bundles play key roles in numerous cellular processes, yet they are largely understudied, as they produce highly heterogeneous populations in vitro ill-suited to conventional structural analysis. I have developed an approach, outlined in this proposal, focused on reducing complexity to investigate the structural organization of the PaExoY-actin bundle. This approach integrates cutting edge structural techniques with biochemical and biophysical assays and can be applied to effectively study actin bundles of diverse origin. The results of this work will greatly expand our understanding of the molecular processes underlying actin bundle formation. I will then expand the scope of my studies to investigate the activation of a related toxin from the human pathogen Vibrio vulnificus (VvExoY), which the data suggest is activated by G-actin, not F-actin. A similar approach will be employed to characterize VvExoY-actin interactions and allow for identification of the key factors responsible for ExoY family activation and actin recognition. Such knowledge will provide a vital foundation in the design of small molecule inhibitors with the goal of generating novel therapeutic strategies against bacterial intoxication. Additionally, I will compare the results of this work with other known bacterial toxins, such as anthrax edema factor, to assess the practicality of using ExoY family toxins as a novel research tool to study actin- associated signaling processes. This work has been specifically designed to build off of my established foundation in Pseudomonas microbiology and X-ray crystallography and expand my structural biology skillset, particularly in the area of high-resolution electron microscopy. I have formed a diverse array of collaborators to assist in the proposed research and serve as an effective mentorship team throughout my postdoctoral training. Through close interactions with these experts, I will foster skills not just as an experimentalist, but as a well- rounded scientist, enhancing my skills in areas such as grantsmanship, scientific communication, and mentorship. At the conclusion of this fellowship, I will expand upon this research in the form of a K99/R00 proposal as I transition from my postdoctoral training to a position as an academic principal investigator.

项目概要： ExoY 细胞毒素家族是新兴临床人类病原体表达的普遍毒力因子 兴趣。 ExoY 家族毒素充当核苷酸环化酶，即催化核苷酸转化的酶 三磷酸酯转化为环核苷酸单磷酸酯。研究表明ExoY的酶活性 家族酶受到肌动蛋白不同生物形式的刺激，肌动蛋白是一种普遍存在的真核细胞骨架蛋白 有两种不同的形式：单体、球状肌动蛋白（G-肌动蛋白）和聚合肌动蛋白丝（F-肌动蛋白）。我最近有 研究了铜绿假单胞菌的 ExoY (PaExoY) 与其激活剂 F-肌动蛋白之间的关系。 我发现 PaExoY 可以将肌动蛋白丝重组成粗的电缆状束。这种肌动蛋白捆绑活动 在真核调节蛋白中常见，我的数据表明它可能与 PaExoY 催化。肌动蛋白束在许多细胞过程中发挥着关键作用，但人们对它们的研究却很少， 因为它们在体外产生高度异质的群体，不适合传统的结构分析。我有 制定了本提案中概述的方法，重点是降低调查结构的复杂性 PaExoY-肌动蛋白束的组织。这种方法将尖端的结构技术与 生物化学和生物物理测定，可用于有效研究不同来源的肌动蛋白束。这 这项工作的结果将极大地扩展我们对肌动蛋白束分子过程的理解 形成。然后我将扩大我的研究范围，以调查相关毒素的激活 人类病原体创伤弧菌 (VvExoY)，数据表明它是由 G-肌动蛋白而不是 F-肌动蛋白激活的。类似的 将采用方法来表征 VvExoY-肌动蛋白相互作用并允许识别关键因素 负责 ExoY 家族激活和肌动蛋白识别。这些知识将为以下方面提供重要的基础： 设计小分子抑制剂，旨在产生针对细菌的新治疗策略 中毒。此外，我会将这项工作的结果与其他已知的细菌毒素（例如炭疽）进行比较 水肿因子，评估使用 ExoY 家族毒素作为研究肌动蛋白的新型研究工具的实用性 相关的信令过程。这项工作是专门为建立我已建立的基础而设计的 假单胞菌微生物学和 X 射线晶体学基础并扩展我的结构生物学技能， 特别是在高分辨率电子显微镜领域。我已经组建了各种各样的合作者 协助拟议的研究，并在整个博士后培训期间作为有效的指导团队。 通过与这些专家的密切互动，我不仅会培养作为实验者的技能，而且会培养作为一名优秀的人的技能。 全面的科学家，提高了我在资助、科学传播等领域的技能 指导。在本次研究金结束时，我将以 K99/R00 的形式扩展这项研究 当我从博士后培训过渡到学术首席研究员的职位时，我提出了这个建议。