Balogh Dóra, Eckel Konstantin, Fetzer Christian, Sieber Stephan A
Center for Functional Protein Assemblies (CPA), Department of Chemistry, Chair of Organic Chemistry II, Technische Universität München 85748 Garching Germany
RSC Chem Biol. 2022 May 31;3(7):955-971. doi: 10.1039/d2cb00077f. eCollection 2022 Jul 6.
exhibits two ClpP isoforms (ClpP1/ClpP2) which assemble into a heterooligomeric complex with enhanced proteolytic activity. Herein, we demonstrate that the formation of this complex depends on temperature and reaches a maximum ratio of about 1 : 1 at 30 °C, while almost no complex formation occurred below 4 °C. In order to decipher the role of the two isoforms at elevated temperatures, we constructed ClpP1, ClpP2 and ClpP1/2 knockout strains and analyzed their protein regulation in comparison to the wild type (WT) strain whole proteome mass-spectrometry (MS) at 37 °C and 42 °C. While the Δ strain only altered the expression of very few proteins, the Δ and Δ strains revealed the dysregulation of many proteins at both temperatures. These effects were corroborated by crosslinking co-immunoprecipitation MS analysis. Thus, while ClpP1 serves as a mere enhancer of protein degradation in the heterocomplex, ClpP2 is essential for ClpX binding and functions as a gatekeeper for substrate entry. Applying an integrated proteomic approach combining whole proteome and co-immunoprecipitation datasets, several putative ClpP2 substrates were identified in the context of different temperatures and discussed with regards to their function in cellular pathways such as the SOS response.
表现出两种ClpP同工型（ClpP1/ClpP2），它们组装成具有增强蛋白水解活性的异源寡聚复合物。在此，我们证明这种复合物的形成取决于温度，在30°C时达到约1:1的最大比例，而在4°C以下几乎不发生复合物形成。为了解析这两种同工型在高温下的作用，我们构建了ClpP1、ClpP2和ClpP1/2基因敲除菌株，并在37°C和42°C下通过全蛋白质组质谱（MS）分析与野生型（WT）菌株相比它们的蛋白质调控情况。虽然Δ菌株仅改变了极少数蛋白质的表达，但Δ和Δ菌株在这两个温度下都显示出许多蛋白质的失调。这些效应通过交联共免疫沉淀质谱分析得到了证实。因此，虽然ClpP1在异源复合物中仅作为蛋白质降解的增强剂，但ClpP2对于ClpX结合至关重要，并作为底物进入的守门人发挥作用。应用结合全蛋白质组和共免疫沉淀数据集的综合蛋白质组学方法，在不同温度的背景下鉴定了几种推定的ClpP2底物，并讨论了它们在细胞途径如SOS反应中的功能。
