Department of Molecular Systems Biology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Germany.
Institute of Biochemistry, Leipzig University, Leipzig, Germany.
Front Immunol. 2023 Sep 22;14:1244431. doi: 10.3389/fimmu.2023.1244431. eCollection 2023.
Although macrophages are known to be affected by their redox status, oxidation is not yet a well-recognized post-translational modification (PTM) in regulating macrophages and immune cells in general. While it has been described that the redox status of single cysteines in specific proteins is relevant for macrophage functions, global oxidation information is scarce. Hence, we globally assessed the impact of oxidation on macrophage activation using untargeted proteomics and PTM-omics. We exposed THP-1 macrophages to lipopolysaccharide (LPS) for 4 h and 24 h and applied a sequential iodoTMT labeling approach to get information on overall oxidation as well as reversible oxidation of cysteines. Thus, we identified 10452 oxidation sites, which were integratively analyzed with 5057 proteins and 7148 phosphorylation sites to investigate their co-occurance with other omics layers. Based on this integrative analysis, we found significant upregulation of several immune-related pathways, e.g. toll-like receptor 4 (TLR4) signaling, for which 19 proteins, 7 phosphorylation sites, and 39 oxidation sites were significantly affected, highlighting the relevance of oxidations in TLR4-induced macrophage activation. Co-regulation of oxidation and phosphorylation was observed, as evidenced by multiply modified proteins related to inflammatory pathways. Additionally, we observed time-dependent effects, with differences in the dynamics of oxidation sites compared to proteins and phosphorylation sites. Overall, this study highlights the importance of oxidation in regulating inflammatory processes and provides a method that can be readily applied to study the cellular redoxome globally.
尽管巨噬细胞的氧化还原状态已被证实会受到影响，但氧化作用尚未被广泛认可为调节巨噬细胞和一般免疫细胞的翻译后修饰（PTM）之一。虽然已经描述了特定蛋白质中单个半胱氨酸的氧化还原状态与巨噬细胞功能有关，但关于全局氧化信息却很少。因此，我们使用非靶向蛋白质组学和 PTM 组学，全面评估了氧化对巨噬细胞激活的影响。我们将 THP-1 巨噬细胞暴露于脂多糖（LPS）4 小时和 24 小时，并应用顺序碘代 TMT 标记方法来获取有关整体氧化和半胱氨酸可逆氧化的信息。因此，我们鉴定了 10452 个氧化位点，并与 5057 种蛋白质和 7148 个磷酸化位点进行综合分析，以研究它们与其他组学层的共现关系。基于这种综合分析，我们发现了几个免疫相关途径的显著上调，例如 Toll 样受体 4（TLR4）信号通路，其中 19 种蛋白质、7 个磷酸化位点和 39 个氧化位点受到显著影响，突出了氧化在 TLR4 诱导的巨噬细胞激活中的重要性。氧化和磷酸化的共调控得到了观察，这可以从与炎症途径相关的多重修饰蛋白中得到证明。此外，我们还观察到了时间依赖性效应，与蛋白质和磷酸化位点相比，氧化位点的动态变化存在差异。总的来说，这项研究强调了氧化在调节炎症过程中的重要性，并提供了一种可以方便地用于全面研究细胞氧化还原组的方法。
