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（由申请人提供）：肌动蛋白在真核生物对病原微生物的先天防御机制中起着许多重要作用。反过来，病原体已经开发出各种优雅和复杂的方式来破坏和/或篡夺肌动蛋白细胞骨架。通过作用于肌动蛋白细胞骨架，致病毒素干扰细胞形态、细胞运动、吞噬作用、上皮通透性和抗原呈递。细菌毒素不仅代表生物医学干预的目标，而且在数百万年的共同进化中已经被调谐到宿主细胞骨架，它们促进了我们在分子和细胞水平上对细胞骨架的理解。长期目标是了解肌动蛋白特异性毒素的致病机制，并利用所获得的知识来阐明肌动蛋白细胞骨架在正常和病理学中的功能。一种这样的知之甚少的破坏性机制是通过肌动蛋白交联结构域（ACD）毒素实现的，所述毒素作为由霍乱弧菌、创伤弧菌、曲霉和几种其他细菌物种的致病性菌株产生的较大毒素的一部分。ACD是一种将单体肌动蛋白共价交联成不能交联的寡聚体的酶。目前的ACD发病机制的范例表明，毒素只是耗尽功能性肌动蛋白引起大量的聚合无能的肌动蛋白寡聚体的积累。相反，这个提议提出了一个完全不同的概念，根据该概念，ACD通过将肌动蛋白单体转化为高毒性低聚物来启动独特的毒性级联，当存在于非常低的浓度时，该低毒性低聚物有效地破坏肌动蛋白动力学。核心假设是，一个独特的组合的属性缺乏G-和F-肌动蛋白赋予一个非常强的相互作用的寡聚体与串联组织的G-肌动蛋白结合蛋白，使他们能够有力地破坏几个关键步骤的肌动蛋白动力学。在强有力的初步数据的指导下，这一概念将通过追求三个具体目标进行彻底测试：1）在溶液中和在体外单丝水平上评估ACD交联的肌动蛋白寡聚体对由哺乳动物formins、Arp 2/3复合物、WH 2串联成核剂和Ena/VASP控制的肌动蛋白动力学的影响; 2）确认寡聚体的预测的细胞靶标，鉴定新靶标，并使用 串联亲和纯化、免疫印迹、质谱和功能测定的组合;和3）应用所获得的知识来产生用于在分子和细胞水平上研究肌动蛋白动力学的基于ACD和ACD启发的新工具。这些目标将通过生物化学，生物物理，细胞，分析和蛋白质工程方法的组合来实现，所有这些方法都已在申请人及其研究团队进行的初步研究中证明是可行的。拟议的研究既有意义又有创新性，因为它有望填补我们对几种危及生命的病原体所采用的致病机制的理解中的一个重大空白，并允许研究团队通过创建用于研究串联组织的肌动蛋白调节剂在肌动蛋白动力学中的作用的工具来利用所获得的知识。