Molecular Mechanisms of Modulation of N-WASP activity

描述（由申请人提供）：志贺氏杆菌每年在全球造成5.5亿例腹泻或痢疾，110万人死亡。弗氏梭菌是胞浆内革兰氏阴性菌，通过结肠粘膜侵袭和扩散而致病。弗氏沙门氏菌的生存和传播是通过细菌操纵宿主细胞骨架介导的。在宿主细胞质中，细菌在一个极点启动肌动蛋白丝的成核和聚合，形成一个推进的肌动蛋白尾巴。细菌外膜蛋白IcsA与肌动蛋白成核因子Arp 2/3、肌动蛋白成核促进因子N-WASP和Toca-1等宿主因子共同组装肌动蛋白尾部。在静息细胞中，N-WASP维持在自抑制构象，并被Cdc42和Toca-1联合激活。在flexneri肌动蛋白尾部组装过程中，N-WASP的激活与Cdc42无关，我们最近发现N-WASP的激活需要Toca-1。我们的研究表明，Toca-1在肌动蛋白尾部形成过程中发挥了缓解N-WASP自身抑制的作用。一旦激活，与flexneri相关的N-WASP不需要Toca-1的存在就可以进行肌动蛋白聚合。与细胞裂解液中的N-WASP相比，与S. flexneri相关的N-WASP在SDS-PAGE上的迁移发生了移位，表明S. flexneri诱导了共价修饰。我的研究目标是：1)表征S. flexneri对N-WASP的修饰，并确定这种修饰对N-WASP构象和肌动蛋白聚合的影响；2)确定对N-WASP的修饰对细胞运动和侵袭的影响；3)确定对N-WASP进行这种修饰的细菌因子。了解flexneri调节宿主细胞骨架的机制将有助于深入了解与志贺氏菌发病机制相关的细菌操纵宿主细胞骨架的机制，也可能与其他生物体的发病机制有关。此外，由于N-WASP活性受到细胞的高度调节，因此提出的研究结果可能会导致对细胞调节和控制肌动蛋白聚合的基本机制的深入了解。

DESCRIPTION (provided by applicant): Shigella sp. cause 550 million cases of diarrhea or dysentery and 1.1 million deaths annually worldwide. S. flexneri are intracytoplasmic gram negative bacteria which cause disease by invasion and spread through the colonic mucosa. Survival and spread of S. flexneri are mediated by bacterial manipulation of the host cytoskeleton. In the host cell cytoplasm, bacteria initiate nucleation and polymerization of actin filaments at one pole, forming a propulsive actin tail. The bacterial outer membrane protein IcsA is required for actin tail assembly in conjunction with host factors including actin nucleator Arp 2/3, actin nucleation promoting factor N-WASP, and Toca-1. In resting cells, N-WASP is maintained in an autoinhibited conformation and is activated by Cdc42 in conjunction with Toca-1. Activation of N-WASP during S. flexneri actin tail assembly is independent of Cdc42, and we have recently shown that N-WASP activation requires Toca-1. Our studies indicate that Toca-1 plays a role in relieving autoinhibition of N-WASP during actin tail formation. Once activated, N-WASP associated with S. flexneri does not require the presence of Toca-1 for ongoing actin polymerization. N-WASP associated with S. flexneri shows shifted migration on SDS-PAGE compared to N-WASP from cell lysate, suggesting that a covalent modification has been induced by S. flexneri. The goals of my research are to: I) Characterize the modification made to N-WASP by S. flexneri and determine the effect of this modification on N-WASP conformation and actin polymerization, II) Determine the effect of the modification to N-WASP on cell motility and invasion and III) Identify the bacterial factor responsible for making this modification to N-WASP. Understanding mechanisms by which S. flexneri modulates the host cytoskeleton will generate insights into the mechanisms of bacterial manipulation of the host cytoskeleton relevant to Shigella pathogenesis and perhaps to the pathogenesis of other organisms as well. Moreover, because N-WASP activity is highly regulated by the cell, the results of the proposed studies are likely to lead to insights into the fundamental mechanisms of cellular modulation and control of actin polymerization. PUBLIC HEALTH RELEVANCE: Many bacteria that cause disease infect human cells, sometimes modifying structural proteins that give cells shape. My research proposal aims to identify mechanisms used by the bacterium Shigella flexneri to modify these structural proteins. Understanding processes by which bacteria modify these proteins can elucidate new avenues for developing vaccines and treatment strategies