Analysis of the role of HSP100/Clp and their adaptor proteins in general and regulated proteolysis in Bacillus subtilis

以前我们已经证明，在枯草芽孢杆菌的感受态形成过程中，适配蛋白MecA是转录因子ComK被ClpCP酶调控的蛋白分解所必需的。然而，最近的实验数据表明，MecA也是AAA+ATPase中HSP100亚家族的伴侣蛋白ClpC识别、解聚和复性先前热聚集蛋白所必需的。此外，MecA能够通过ClpCP特异性识别和降解未折叠或聚集的蛋白质。一个Meca Paralogue YpbH显示了同样的活动。因此，接头蛋白对于ClpC在枯草杆菌蛋白质质量控制中的一般功能是必需的。为了了解接合蛋白MecA和类似物YpbH在枯草杆菌蛋白质质量控制系统中的作用，我们想要对接合蛋白的配对进行生化表征和鉴定。因此，我们想要测试MecA和YpbH与HSP 100蛋白ClpC、CLPE和ClpX在有或没有ClpP的情况下的功能相互作用。另一方面，我们想要确定接头蛋白的一般底物特异性，因为底物识别和与HSP100的相互作用是这类新蛋白发挥功能的先决条件。此外，还设计了实验来了解接头蛋白的靶向降解机制。最后，首先介绍了了解这些接头蛋白及其HSP100伙伴蛋白在枯草杆菌蛋白质质量控制网络中的作用的实验。

Previously we could demonstrate that the adaptor protein MecA is necessary for the regulated proteolysis of the transcription factor ComK in competence development of Bacillus subtilis by ClpCP protease. However recent experimental data demonstrate that MecA is also necessary for the recognition, disaggregation and refolding of previously heat aggregated proteins by ClpC a chaperone of the HSP 100 subfamily of AAA+ ATPases. In addition MecA enables the specific recognition and degradation of unfolded or aggregated proteins by ClpCP. A MecA paralogue YpbH shows the same activity. The adaptor proteins are therefore necessary for the general function of ClpC in protein quality control in B. subtilis. To understand the role of the adaptor proteins MecA and the paralogue YpbH in the protein quality control system of B. subtilis we want to biochemically characterise and identify the partners of the adaptor proteins. Therefore we want to test the functional interaction of MecA and YpbH with those possible partner the HSP 100 proteins ClpC, ClpE and ClpX with and without ClpP. On the other hand we want to determine the general substrate specificity of the adaptor proteins, because the connection of substrate recognition and interaction with the HSP 100 is a prerequisite for the function of this new class of proteins. In addition experiments are devised to develop an understanding of the targeting for degradation mechanism of the adaptor proteins. Finally first experiments to understand the in vivo role of these adaptor proteins and their HSP 100 partner proteins in the protein quality control network of B. subtilis are introduced.