Molecular Mechanisms of Modulation of N-WASP activity

描述（由申请人提供）：志贺氏菌属每年在全世界造成5.5亿例腹泻或痢疾病例和110万例死亡。S.弗氏菌是胞浆内革兰氏阴性细菌，其通过侵入和通过结肠粘膜传播而引起疾病。S.弗氏杆菌是由宿主细胞骨架的细菌操纵介导的。在宿主细胞质中，细菌在一极启动肌动蛋白丝的成核和聚合，形成推进肌动蛋白尾。细菌外膜蛋白IcsA是肌动蛋白尾部组装所需的，与宿主因子结合，包括肌动蛋白成核剂阿普2/3、肌动蛋白成核促进因子N-WASP和Toca-1。在静息细胞中，N-WASP保持自抑制构象，并被Cdc 42与Toca-1联合激活。在S.弗氏肌动蛋白尾部组装不依赖于Cdc 42，我们最近发现N-WASP激活需要Toca-1。我们的研究表明，Toca-1在肌动蛋白尾形成期间在缓解N-WASP的自抑制方面发挥作用。一旦被激活，N-WASP与S. flexneri不需要Toca-1的存在来进行肌动蛋白聚合。N-WASP与S.与来自细胞裂解物的N-WASP相比，弗氏杆菌在SDS-PAGE上显示出移动的迁移，表明S.弗莱克斯内里。本论文的研究目标是：1）表征S. II）确定对N-WASP的修饰对细胞运动性和侵袭性的影响，和III）鉴定负责对N-WASP进行这种修饰的细菌因子。理解S.福氏杆菌调节宿主细胞骨架的研究将有助于深入了解细菌操纵宿主细胞骨架的机制，这些机制与志贺氏菌的发病机制有关，也可能与其他生物的发病机制有关。此外，由于N-WASP活性是高度调节的细胞，所提出的研究的结果可能会导致深入了解细胞调节和控制肌动蛋白聚合的基本机制。

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.