Oxidation is an underappreciated post-translational modification in the regulation of immune responses associated with changes in phosphorylation

Abstract

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.

Unveiling the dynamics of acetylation and phosphorylation in SGBS and 3T3-L1 adipogenesis

“…Labelled phosphopeptides and their corresponding background proteome were eluted and measured analogously to Karkossa et al.. 91 In short, phosphopeptides were eluted over a 170 min four-step linear gradient starting at 4% solvent B (solvent A: 0.1% FA in water; solvent B: 80% ACN/0.1% FA in water), via 18% B after 78 min, 30% B after 115 min, 55% B after 145 min reaching 99% B after 150 min followed by flushing of the column for 5 min at 99% B and subsequent equilibration to initial conditions. Settings for the acquisition of positive mode full MS scans were the following: scan range of 350-1550 m/z , AGC target of 3 × 10 6 ions, resolution of 120,000 and a maxIT of 150 ms.…”