Actin oligomers as novel toxins targeting key steps of actin dynamics

肌动蛋白寡聚物作为针对肌动蛋白动力学关键步骤的新型毒素

• 批准号: 9134177
• 负责人: Dmitri Kudryashov
• 金额: $ 30.42万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9134177
• 关键词: Actin-Binding Protein; Actins; Address; Affinity; Affinity Chromatography; Amaze; Antigen Presentation; Bacteria; Bacterial Toxins; Binding; Binding Sites; Biochemical; Biological Assay; Cell Culture Techniques; Cell physiology; Cellular Morphology; Communicable Diseases; Complement; Complex; Controlled Study; Cytoskeleton; Data; Defense Mechanisms; Elongation Factor; Enzymes; Epithelial; Evolution; F-Actin; Family; Filament; Fostering; G Actin; Genetic Engineering; Goals; Health; Human; Immunoblotting; In Vitro; Intervention; Intestines; Knowledge; Lead; Learning; Life; Light; Mass Spectrum Analysis; Mediating; Mission; Modeling; Molecular; Oligonucleotides; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Permeability; Phagocytosis; Play; Process; Property; Protein Engineering; Protein Isoforms; Proteins; Pyrenes; Research; Role; Stretching; Structure; Targeted Toxins; Tertiary Protein Structure; Testing; Total Internal Reflection Fluorescent; Toxic effect; Toxin; Vibrio cholerae; Vibrio vulnificus; base; cell motility; cellular targeting; crosslink; genetic regulatory protein; improved; inhibitor/antagonist; innovation; microorganism; monolayer; monomer; novel; pathogen; pathogenic bacteria; polymerization; polyproline; profilin; promoter; tool; trait; vasodilator-stimulated phosphoprotein

 DESCRIPTION (provided by applicant): Actin plays many vital roles in eukaryotic innate defense mechanisms against pathogenic microorganisms. Reciprocally, pathogens have developed various elegant and sophisticated ways to disrupt and/or usurp the actin cytoskeleton. By acting on the actin cytoskeleton, pathogenic toxins disturb cell morphology, cell motility, phagocytosis, epithelial permeability, and antigen presentation. Bacterial toxins not onl represent targets for biomedical interventions, but having been tuned to the host cytoskeleton throughout millions of years of co- evolution, they foster our understanding of the cytoskeleton on molecular and cellular levels. The long-term goals are to learn pathogenic mechanisms employed by actin-specific toxins and to utilize the obtained knowledge to illuminate functions of the actin cytoskeleton in norm and pathology. One such poorly understood disruptive mechanism is implemented by the Actin Crosslinking Domain (ACD) toxin produced as a part of larger toxins by pathogenic strains of V.cholerae, V.vulnificus, A.hydrophila, and several other species of bacteria. ACD is an enzyme that covalently crosslinks monomeric actin into oligomers that cannot polymerize. The current paradigm of ACD pathogenesis suggests that the toxin merely depletes functional actin by causing accumulation of bulk amounts of polymerization-incompetent actin oligomers. Instead, this proposal suggests a radically different concept, according to which ACD initiates a unique toxicity cascade by converting actin monomers into highly toxic oligomers that potently disrupt actin dynamics when present at very low concentrations. The central hypothesis is that a unique combination of properties absent in G- and F-actin confers an exceptionally strong interaction of the oligomers with tandem organized G-actin binding proteins and enables them to potently disrupt several key steps of actin dynamics. Guided by strong preliminary data, this concept will be thoroughly tested by pursuing three specific aims: 1) Evaluate the effects of the ACD- crosslinked actin oligomers on actin dynamics controlled by mammalian formins, Arp2/3 complex, WH2 tandem nucleators, and Ena/VASP in solution and at a single filament level in vitro; 2) Confirm predicted cellular targets of the oligomers, identify novel targets, and study cellular effects of the oligomers using a combination of tandem affinity purification, immunoblotting, mass spectrometry, and functional assays; and 3) Apply the acquired knowledge for producing novel ACD-based and ACD-inspired tools for studying actin dynamics at the molecular and cellular levels. These goals will be achieved via a combination of biochemical, biophysical, cellular, analytical, and protein engineering approaches, all of which have been proven to be feasible in preliminary studies conducted by the applicant and his research team. The proposed study is both significant and innovative as it promises to fill a major gap in our understanding of pathogenic mechanisms employed by several life-threatening pathogens and permit the research team to utilize the acquired knowledge by creating tools for studying the role of tandem-organized actin regulators in actin dynamics.

 描述（由适用提供）：肌动蛋白在针对致病微生物的真核先天防御机制中起许多至关重要的作用。相度地，病原体开发了各种优雅而精致的方法来破坏和/或篡夺肌动蛋白细胞骨架。通过作用于肌动蛋白细胞骨架，致病性毒素干扰细胞形态，细胞运动，吞噬作用，上皮渗透性和抗原表现。细菌毒素不开明代表生物医学干预措施的靶标，但是在数百万年的共同进化过程中，它们都被调整为宿主细胞骨架，它们促进了我们对分子和细胞水平上细胞骨架的理解。长期目标是学习通过肌动蛋白特异性毒素进行的致病机制，并利用所获得的知识来阐明肌动蛋白细胞骨架的功能。一种这样的理解的破坏性机制是由肌动蛋白交联结构域（ACD）毒素实现的，该毒素是通过V.Cholerae，V.Vulnificus，A.Hyrophila和其他几种细菌的致病性菌株作为较大毒素的一部分实现的。 ACD是一种共价交联的酶，将单体肌动蛋白交联成不能聚合的低聚物。当前的ACD发病机理的当前范式表明，毒素仅通过引起大量聚合 - 不足的肌动蛋白低聚物的加速而耗尽功能肌动蛋白。取而代之的是，该提案提出了一个根本不同的概念，根据该概念，ACD通过将肌动蛋白单体转换为剧毒毒物的低聚物来启动独特的毒性级联反应，这些降低的浓度很低时可能会破坏肌动蛋白动力学。中心假设是，在G肌动蛋白和F-肌动蛋白中缺乏特性的独特组合赋予了低聚物与串联有组织的G-肌动蛋白结合蛋白的异常强烈的相互作用，并使它们有可能破坏肌动蛋白动力学的几个关键步骤。在强大的初步数据的指导下，将通过追求三个具体目的来彻底测试这个概念：1）评估ACD-交联的肌动蛋白寡聚物对由ARP2/3复合物，WH2 Tandem tandem Nucleators和溶液中的溶液和ENA/VASP在单一材料中的溶液中控制的肌动蛋白动力学对肌动蛋白动力学的影响； 2）确认低聚物的预测细胞靶标，鉴定新靶标，并使用低聚物的细胞效应 串联亲和力纯化，免疫印迹，质谱和功能测定的组合； 3）将获得的知识应用于生产新型ACD和ACD启发的工具，用于在分子和细胞水平上研究肌动蛋白动力学。这些目标将通过生化，生物物理，细胞，分析和蛋白质工程方法的结合来实现，所有这些方法都被证明在申请人及其研究团队进行的初步研究中是可行的。拟议的研究既重要又具有创新性，因为它有望填补我们对几种威胁生命的病原体进行的致病机制的理解，并允许研究团队通过创建工具来利用获得的知识来研究稳定的肌动蛋白调节剂在肌动蛋白动力学中的作用。