Mechanisms of protease-substrate interactions in the E. coli cytosol

大肠杆菌胞质中蛋白酶与底物相互作用的机制

• 批准号: 5361995
• 负责人: Professor Dr. Bernd Bukau
• 金额: --
• 依托单位: Zentrum für Molekulare Biologie (ZMBH)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2002
• 资助国家: 德国
• 起止时间: 2001-12-31 至 2008-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5361995?language=en
• 关键词: Mechanisms; protease; substrate; interactions; E.

The folding state of proteins within cells is controlled by an energy-dependent quality control system, consisting of ATP-dependent proteases and molecular chaperones. Misfolded or aggregated proteins can be either refolded to the native structure or degraded. The functional interplay between proteases and chaperones in this process is poorly understood. While substrate specificities and substrate populations of major chaperones of the E. coli cytosol are known, equivalent data for proteases are missing. We plan to identify natural substrates of major cytosolic proteases (Lon, ClpAP, ClpXP, HslUV) and to determine their substrate binding motifs and the kinetic features of the protease-substrate interactions. We will also determine the relationship between proteases and chaperones in their activity on misfolded soluble and aggregated proteins as well as on reporter proteins with engineered protease and/or chaperone binding sequences. Finally, we will have a major focus on the mechanistic analysis of a novel adaptor protein for the ClpAP protease machine, ClpS, which we discovered recently. ClpS changes the substrate specificity of the ClpAP proteolytic machine by inhibiting the degradation of SsrA-tagged substrates, while stimulating the removal of aggregated proteins. The structural basis of the ClpA-ClpS interaction and the resulting consequences on ClpA substrate interaction and the chaperone mechanism of ClpA will be investigated.

蛋白质在细胞内的折叠状态是由一个能量依赖的质量控制系统控制的，该系统由依赖于ATP的蛋白酶和分子伴侣组成。错误折叠或聚集的蛋白质可以重新折叠为天然结构，也可以降解。在这一过程中，蛋白水解酶和伴侣之间的功能相互作用还知之甚少。虽然大肠杆菌胞浆主要伴侣的底物特异性和底物种群是已知的，但有关蛋白酶的同等数据尚不清楚。我们计划鉴定主要胞浆蛋白水解酶(Lon、ClpAP、ClpXP、HslUV)的天然底物，并确定它们的底物结合基序和蛋白酶-底物相互作用的动力学特征。我们还将确定蛋白酶和伴侣在错误折叠的可溶蛋白和聚集蛋白以及具有工程蛋白酶和/或伴侣结合序列的报告蛋白上的活性之间的关系。最后，我们将主要关注我们最近发现的一种新的ClpAP蛋白酶机器的适配蛋白CLPS的机制分析。CLPS通过抑制SsrA标记底物的降解，同时刺激聚集蛋白的去除，改变了ClpAP蛋白水解机的底物特异性。我们将研究ClpA-CLPS相互作用的结构基础及其对ClpA底物相互作用的影响，以及ClpA的伴侣机制。