Substrate selection by the FtsH protease

大肠杆菌必需的AAA型蛋白酶FtsH具有蛋白质质量控制和调节功能。我们正在研究FtsH在控制热休克反应中的作用，通过降低热休克σ因子RpoH（s32）。多个因子（DnaK、DnaJ、RNA聚合酶）参与该过程。已经使用两种策略来产生rpoH中的随机突变：（i）接头插入诱变，和（ii）在设计用于选择蛋白酶切割位点的细菌双杂交系统中的易错诱变。第一种方法得到的RpoH蛋白的构象变化和蛋白质-蛋白质相互作用将通过生物物理技术进行研究。双杂交筛选的初步结果表明，RpoH的一个重要的周转元件位于氨基酸47附近。该区域中的其他突变将通过定点诱变构建。将在体内和体外分析RpoH变体的降解。将检查它们与参与FtsH介导的蛋白水解的伴侣蛋白的相互作用。为了扩大我们的研究底物选择的FtsH蛋白酶，我们计划利用双杂交技术的其他FtsH底物（1 cII和cIII）。此外，将建立一个遗传筛选，以确定最重要的FtsH底物LpxC（EnvA）中的周转元件。

The essential AAA-type protease FtsH of Escherichia coli has protein quality control and regulatory functions. We are studying the role of FtsH in controlling the heat shock response by degradation of the heat shock sigma factor RpoH (s32). Multiple factors (DnaK, DnaJ, RNA polymerase) are involved in this process. Two strategies have been used to generate random mutations in rpoH: (i) linker insertion mutagenesis, and (ii) error-prone mutagenesis in a bacterial two-hybrid system designed to select for protease cleavage sites. Conformational changes and protein-protein interactions of RpoH proteins derived from the first approach are going to be studied by biophysical techniques. Initial results from the two-hybrid screening suggest that an important turnover element of RpoH is located around amino acid 47. Additional mutations in this region will be constructed by site-directed mutagenesis. Degradation of the RpoH variants will be analyzed in vivo and in vitro. Their interaction with partner proteins involved in FtsH-mediated proteolysis will be examined. To extent our studies on substrate selection by the FtsH protease, we plan to exploit the two-hybrid technology for other FtsH substrates (l cII and cIII). Moreover, a genetic screen to identify the turnover element in the most important FtsH substrate LpxC (EnvA) will be established.