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.

项目摘要： 细胞毒素的前毒素家族是由新兴临床的人类病原体表达的普遍毒性因子 兴趣。 Exoy家族毒素充当核苷酸循环酶，可促进核苷酸转化的酶 三磷酸成循环核苷酸单磷酸盐。研究表明，Exoy的酶活性 家族酶是由肌动蛋白不同的生物基质刺激的，肌动蛋白是一种普遍存在的真核细胞骨骼蛋白 以两种不同的形式：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（Paexoy）及其活化剂F-肌动蛋白的Exoy之间的关系。 我发现Paexoy可以将肌动蛋白丝重组成厚的电缆状束。这个肌动蛋白捆绑活动 通常在真核调节蛋白中观察到，我的数据表明它可能与 佩索伊催化。肌动蛋白束在众多细胞过程中起关键作用，但它们在很大程度上被研究了， 由于它们在体外不适合传统的结构分析中产生高度异质种群。我有 在该提案中概述了一种方法，重点是降低复杂性以研究结构 Paexoy-Actin捆绑包的组织。这种方法将最先进的结构技术与 生化和生物物理测定法，可用于有效研究各种起源的肌动蛋白束。这 这项工作的结果将大大扩展我们对肌动蛋白束基础分子过程的理解 形成。然后，我将扩大研究范围，以研究从 数据表明的人类病原体颤音（VVEXOY）被G-肌动蛋白而不是F-肌动蛋白激活。类似 将采用方法来表征vvexoy-actin的相互作用，并允许识别关键因素 负责外生家庭激活和肌动蛋白识别。这样的知识将为 小分子抑制剂的设计，目的是产生针对细菌的新型治疗策略 中毒。此外，我将将这项工作的结果与其他已知的细菌毒素（例如炭疽病）进行比较 水肿因素，以评估使用Exoy家族毒素作为一种新的研究工具的实用性来研究肌动蛋白 相关的信号过程。这项工作是专门设计的，以建立我的既定 假单胞菌微生物学和X射线晶体学的基础，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区域。我已经组建了各种各样的合作者 协助拟议的研究，并在我的博士后培训中成为有效的指导团队。 通过与这些专家的密切互动，我将不仅培养技能，不仅是一个实验者，而且还将作为一个很好的技巧 绕过科学家，提高了我在授予技巧，科学交流和 指导。在此研究金的结论结束时，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担任学术首席研究员的职位时，提案。