ThiS介导的FtsA类泛素化修饰调控胞质分裂环稳定性的机制与功能研究

细菌细胞分裂蛋白FtsA参与胞质分裂环（Z环）的形成，是新的药物靶点。我们首次发现大肠杆菌类泛素蛋白酶系统ThiS/ThiF/ClpXP调控FtsA的降解，但是具体机制和功能不清楚。FtsA属于肌动蛋白，参与招募微管蛋白FtsZ形成FtsAZ纤维，此纤维与其他分裂蛋白组成Z环。我们推测类泛素蛋白酶系统通过介导FtsA调控Z环的稳定性。本研究将体外鉴定FtsA的修饰位点，分析FtsA的类泛素化修饰对其稳定性的影响；利用光散射及电镜技术实时观察FtsA的纤维化及膜定位情况，揭示类泛素化修饰是否调控FtsA的膜定位；建立FtsAZ自组装体系，通过共聚焦显微镜动态分析类泛素蛋白酶系统调控FtsAZ纤维的形成；进一步在体内检测FtsAZ的定位、纤维化及Z环的稳定性，体内阐述类泛素化修饰FtsA调控Z环稳定性的机制。本研究对于揭示细菌Z环稳定性的调控机制以及开发新型抗生素具有重要理论意义。

Bacterial cell division protein FtsA, regulating the formation of cytokinesis Z ring, is an important target for the development of novel antibacterial drugs. Escherichia coli is an important model organism that studies bacterial cell division and antibiotic activity. Our previous study found that the ubiquitin-like system ThiS/ThiF/ClpXP could modify FtsA in vivo and in vitro, but the mechanism and function is unknown. FtsA, an actin homolog, recruits tubulin like protein FtsZ to form FtsAZ filaments, which is component of cytokinesis Z ring. Based on this, we speculate that the ubiquitin-like system regulates bacterial division by modifying FtsA. In this study, the site of ThiS/ThiF-modified FtsA will be identified in vitro using western blot, and the effect of ubiquitin-like modification of FtsA on its stability will be analyzed. Sequentially, we will observe the multimerization and localization of FtsA using light scattering and electron microscopy to elucidate effect of ubiquitination on membrane localization of FtsA. Furthermore, we will analyze the effect of ubiquitin-like modification of FtsA on the formation and dissociation of FtsAZ by TIRF microscopy using FtsZ self-assembly system in vitro. Detection of intracellular localization of FtsA and the relationship of Z-Ring formation will elucidate ubiquitin-like modification of FtsA regulating cytokinesis. Therefore, this study will have important theoretical significance for revealing the regulatory mechanism of bacterial cytokinesis and developing novel antibiotics.