Substrate selection by the FtsH protease

FtsH 蛋白酶的底物选择

• 批准号: 5422941
• 负责人: Professor Dr. Franz Narberhaus
• 金额: --
• 依托单位: Lehrstuhl für Biologie der Mikroorganismen
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2008-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5422941?language=en
• 关键词: Substrate; selection; FtsH; protease

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.

大肠杆菌必需的AAA型蛋白水解酶FtsH具有蛋白质质量控制和调节功能。我们正在研究FtsH通过降解热休克Sigma因子RpoH来控制热休克反应的作用(S32)。这一过程涉及多种因素(DNAK、DNAJ、RNA聚合酶)。已经使用两种策略来产生rpoH的随机突变：(I)连接子插入突变，和(Ii)在细菌双杂交系统中容易出错的突变，该系统旨在选择蛋白酶裂解位点。从第一种方法得到的RpoH蛋白的构象变化和蛋白质-蛋白质相互作用将用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。这个区域的更多突变将通过定点突变来构建。RpoH变异体的降解将在体内和体外进行分析。它们与参与FtsH介导的蛋白分解的伙伴蛋白的相互作用将被检测。为了扩大我们对FtsH蛋白酶底物选择的研究，我们计划将双杂交技术开发到其他FtsH底物(L CII和CIII)。此外，还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。