Mechanistic investigation of ExoY-family enzyme activation by actin

项目摘要： 细胞毒素的ExoY家族是由新兴临床疾病的人类病原体表达的普遍毒力因子， 兴趣ExoY家族毒素作为核苷酸环化酶起作用，所述核苷酸环化酶是催化核苷酸环化酶的酶。 三磷酸转化为环核苷酸单磷酸。研究表明，ExoY的酶活性 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 研究了铜绿假单胞菌ExoY（PaExoY）与其激活剂F-actin的关系。 我发现PaExoY可以将肌动蛋白丝重组成粗的索状束。这种捆绑活动 在真核调节蛋白中经常观察到，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起着关键作用，但它们在很大程度上研究不足， 因为它们在体外产生高度异质的群体，不适合常规的结构分析。我有 开发了一种方法，在本提案中概述，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测定，并可应用于有效地研究不同来源的肌动蛋白束。的 这项工作的结果将大大扩展我们对肌动蛋白束的分子过程的理解 阵然后，我将扩大我的研究范围，以调查一种相关毒素的激活， 人类病原体创伤弧菌（VvExoY），数据表明其由G-肌动蛋白而不是F-肌动蛋白激活。类似的 方法将用于表征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。这些知识将提供一个重要的基础， 小分子抑制剂的设计，目的是产生新的治疗策略， 中毒此外，我将把这项工作的结果与其他已知的细菌毒素（如炭疽）进行比较， 水肿因子，以评估使用ExoY家族毒素作为研究肌动蛋白的新研究工具的实用性， 相关的信号传递过程。这项工作是专门设计来建立我的既定 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。我组建了一批不同的合作者， 在我的博士后培训过程中，协助所提议的研究，并作为一个有效的导师团队。 通过与这些专家的密切互动，我将培养技能，不仅作为一个实验者，但作为一个很好的- 全面的科学家，提高了我在诸如granecraft，科学交流， 导师制在本奖学金结束时，我将以K99/R 00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，我提出了一个建议。

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.