Global thiol proteome analysis provides novel insights into the macrophage inflammatory response and its regulation by the thioredoxin system

Oxidative modifications of protein cysteine thiols regulate various physiological processes, including innate immune and inflammatory responses. The thioredoxin system plays a key role in redox regulation and signaling by reversing thiol modifications. Here, we characterized the dynamic thiol proteome of human macrophages upon activation by the prototypical pro‐inflammatory agent, bacterial lipopolysaccharide (LPS), and/or perturbation of the thioredoxin system. To this end, we profiled the cellular and redox proteome of human THP-1-derived macrophages during the early phase of LPS activation and/or inhibition of thioredoxin system activity by auranofin, by employing a peptide-centric, resin-assisted capture, redox proteomic workflow. Among 4200 identified cysteines, the oxidation of nearly 10% was selectively affected by LPS or auranofin treatments. Notably, the proteomic analysis uncovered a subset of ~100 cysteines, mapped to proteins involved in diverse processes, whose oxidation is antagonistically regulated by LPS and thioredoxin. Compared to the redox proteome, the cellular proteome was largely unchanged, highlighting the importance of redox modifications as a mechanism that allows for rapid modulation of macrophage activities in response to a pro-inflammatory or a pro-oxidant insult. Structural-functional analyses provided mechanistic insights into redox regulation of selected proteins, including the glutathione synthesizing enzyme, glutamate cysteine ligase and the autophagy adaptor SQSTM1/p62. Overall, this work expands our knowledge of the macrophage thiol proteome and suggests mechanisms by which macrophages adapt and fine-tune their responses according to a changing inflammatory and redox environment.