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: 10154446
• 负责人: Jordan Michael Mancl
• 金额: $ 6.64万
• 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/10154446
• 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; 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/antagonist; interest; mutant; nanobodies; novel; novel therapeutic intervention; organizational structure; 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.

项目总结： 细胞毒素ExoY家族是由人类临床新发病原体表达的流行毒力因子 利息。ExoY家族毒素的功能是核苷酸环化酶，即催化核苷酸转换的酶 三磷酸转变为环核苷酸单磷酸盐。研究表明，ExoY的酶活性 家族酶受到不同生物形式的肌动蛋白的刺激，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)和聚合肌动蛋白细丝(F-肌动蛋白)。我最近有过 研究了铜绿假单胞菌ExoY(PaExoY)与其激活剂F-肌动蛋白的关系。 我发现PaExoY可以将肌动蛋白细丝重组成粗大的索状束。这种肌动蛋白捆绑活动 在真核生物调节蛋白中被普遍观察到，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扮演着关键角色，但对它们的研究很少， 因为它们在体外产生了高度异质的种群，不适合传统的结构分析。我有过 制定了一种方法，在此提案中概述，重点是降低复杂性，以调查结构 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分析，并可用于有效地研究不同来源的肌动蛋白束。这个 这项工作的结果将极大地扩展我们对肌动蛋白束背后的分子过程的理解。 队形。然后，我将扩大我的研究范围，以调查来自 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。一种类似的 将采用一种方法来表征VvExoY-肌动蛋白的相互作用，并允许确定关键因素 负责ExoY家族的激活和肌动蛋白的识别。这些知识将为 小分子抑制剂的设计，目标是产生新的抗细菌治疗策略 喝醉了。此外，我将把这项工作的结果与其他已知的细菌毒素进行比较，如炭疽 探讨ExoY家族毒素作为研究肌动蛋白的新工具的可行性。 关联的信令进程。这项工作是专门设计的，以建立在我的既定 在假单胞菌微生物学和X射线结晶学方面打下基础，并扩大我的结构生物学技能， 特别是在高分辨率电子显微镜领域。我已经组建了一系列不同的合作者来 在我的博士后培训期间，协助拟议的研究，并作为一个有效的指导团队。 通过与这些专家的密切互动，我不仅将培养作为实验者的技能，而且将作为一名优秀的- 多才多艺的科学家，增强了我在一些领域的技能，例如，勇气，科学交流，以及 师徒关系。在本奖学金结束时，我将以K99/R00的形式对这项研究进行扩展 当我从博士后培训过渡到学术首席研究员的职位时，我提出了一个建议。