Actin oligomers as novel toxins targeting key steps of actin dynamics

 描述(申请人提供)：肌动蛋白在真核生物对病原微生物的天然防御机制中扮演着许多重要的角色。反过来，病原体已经发展出各种优雅和复杂的方法来破坏和/或篡夺肌动蛋白细胞骨架。通过作用于肌动蛋白细胞骨架，致病毒素扰乱细胞形态、细胞运动、吞噬、上皮通透性和抗原呈递。细菌毒素不仅是生物医学干预的靶标，而且在数百万年的共同进化过程中与宿主细胞骨架相适应，它们在分子和细胞水平上促进了我们对细胞骨架的理解。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阐明肌动蛋白细胞骨架在正常和病理中的功能。一种知之甚少的破坏机制是由肌动蛋白交联域(ACD)毒素实现的，该毒素是霍乱弧菌、创伤弧菌、嗜水弧菌和其他几种细菌的致病菌株产生的较大毒素的一部分。ACD是一种酶，它将单体肌动蛋白共价交联成不能聚合的寡聚体。目前ACD发病机制的范例表明，这种毒素仅仅是通过导致大量不具聚合能力的肌动蛋白寡聚体的积累来耗尽功能性肌动蛋白。相反，这一提议提出了一个截然不同的概念，根据该概念，ACD通过将肌动蛋白单体转化为剧毒低聚物来启动一种独特的毒性级联反应，当存在非常低的浓度时，这些寡聚体会有效地扰乱肌动蛋白的动态。中心假说是G-肌动蛋白和F-肌动蛋白所缺乏的一种独特的性质组合赋予了寡聚体与串联组织的G-肌动蛋白结合蛋白特别强的相互作用，并使它们能够有效地扰乱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，这一概念将通过追求三个具体目标进行彻底测试：1)评估ACD-交联型肌动蛋白寡聚体对哺乳动物Forins、arp2/3复合体、WH2串联核酸体和ena/vasp在溶液中和体外单丝水平控制的肌动蛋白动力学的影响；2)确认寡聚体的预测细胞靶点，识别新的靶点，并研究寡聚体的细胞效应。 串联亲和纯化、免疫印迹、质谱学和功能分析的组合；以及3)应用所获得的知识来生产基于ACD和ACD启发的新型工具，在分子和细胞水平上研究肌动蛋白的动力学。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组合来实现，所有这些方法在申请者及其研究团队进行的初步研究中都已被证明是可行的。这项拟议的研究具有重大意义和创新性，因为它有望填补我们对几种威胁生命的病原体所使用的致病机制的了解的一个主要空白，并允许研究团队通过创建工具来研究串联组织的肌动蛋白调节因子在肌动蛋白动力学中的作用，从而利用所获得的知识。

 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.