Table_11_Proteomic analysis of the regulatory networks of ClpX in a model cyanobacterium Synechocystis sp. PCC 6803.xlsx

Protein homeostasis is tightly regulated by protein quality control systems such as chaperones and proteases. In cyanobacteria, the ClpXP proteolytic complex is regarded as a representative proteolytic system and consists of a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. However, the functions and molecular mechanisms of ClpX in cyanobacteria remain unclear. This study aimed to decipher the unique contributions and regulatory networks of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We showed that the interruption of clpX led to slower growth, decreased high light tolerance, and impaired photosynthetic cyclic electron transfer. A quantitative proteomic strategy was employed to globally identify ClpX-regulated proteins in Synechocystis cells. In total, we identified 172 differentially expressed proteins (DEPs) upon the interruption of clpX. Functional analysis revealed that these DEPs are involved in diverse biological processes, including glycolysis, nitrogen assimilation, photosynthetic electron transport, ATP-binding cassette (ABC) transporters, and two-component signal transduction. The expression of 24 DEPs was confirmed by parallel reaction monitoring (PRM) analysis. In particular, many hypothetical or unknown proteins were found to be regulated by ClpX, providing new candidates for future functional studies on ClpX. Together, our study provides a comprehensive ClpX-regulated protein network, and the results serve as an important resource for understanding protein quality control systems in cyanobacteria.

蛋白质稳态 (Protein homeostasis) 由伴侣蛋白 (chaperones)、蛋白酶 (proteases) 等蛋白质质量控制系统严格调控。在蓝细菌 (cyanobacteria) 中，ClpXP蛋白水解复合物被视作典型的蛋白水解系统，由六聚体ATP酶ClpX与十四聚体肽酶ClpP组成。然而，蓝细菌中ClpX的功能与分子机制仍有待阐明。本研究旨在解析模式蓝细菌集胞藻 (Synechocystis sp. PCC 6803，下文简称集胞藻) 中ClpX的独特作用与调控网络。研究发现，clpX基因的中断会导致生长速率减缓、高光耐受性下降以及光合循环电子传递功能受损。本研究采用定量蛋白质组学策略，对集胞藻细胞中受ClpX调控的蛋白质进行全局鉴定。最终共鉴定得到172个差异表达蛋白 (differentially expressed proteins, DEPs)，其表达水平在clpX中断后发生显著变化。功能富集分析显示，这些DEPs参与了多种生物学过程，包括糖酵解、氮同化、光合电子传递、ATP结合盒 (ATP-binding cassette, ABC) 转运蛋白系统以及双组分信号转导通路。通过平行反应监测 (parallel reaction monitoring, PRM) 技术，验证了24个DEPs的表达变化。尤为值得关注的是，诸多假设蛋白或功能未知蛋白被证实受ClpX调控，为后续ClpX相关功能研究提供了全新的候选靶点。综上，本研究构建了全面的ClpX调控蛋白质网络，其研究结果可为解析蓝细菌的蛋白质质量控制系统提供重要的参考资源。