Redox Proteomics of the Inflammatory Secretome Identifies a Common Set of Redoxins and Other Glutathionylated Proteins Released in Inflammation, Influenza Virus Infection and Oxidative Stress

Abstract

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

“…Park et al (25,26), who generated adducts by overnight incubation of reduced Prxs with 10 mM GSSG, focused mainly on glutathionylation of Cys-83 in Prx1 (which is not present in Prx2) and reversal by sulfiredoxin. Others have detected glutathionylated Prxs in proteomic screens and observed secretion from cells under oxidative and inflammatory stress (27,28). Reduction of Prx3 by Grx2 has also been observed, but in this case, the disulfide was reduced directly without involving a glutathionylated intermediate (6).…”

“…Glutathionylation of the isolated proteins has been observed but only after extended incubation with nonphysiological GSSG concentrations (25,26), and recycling of Prx1 and Prx2 by GSH and Grx1 was not apparent in initial studies (21). However, glutathione adducts have been detected in cell systems (25,27,28) and could therefore be physiologically relevant. We have characterized the interactions between GSH and Prx2 using mass spectrometry to detect products.…”

Redox Proteomics Applied to the Thiol Secretome

“…Therefore, it is imperative that each protein is validated. In our studies, we normally provide the list of the identified proteins as supplementary data but report only those validated in the main body of the manuscript (23). This makes it possible for others to eventually validate other proteins of interest.…”

“…Typically, a Western blot is required to measure the protein in the supernatant of LPS-stimulated versus unstimulated cells. This should be done with a proper number of replicates and, possibly, followed by densitometry and statistical analysis (23). Of course, if an enzyme-linked immunosorbent assay (ELISA) or any other assay (such as an enzymatic assay in case the protein is an enzyme) are available, that would be preferable as it may be more quantitative than a Western blot.…”

“…Then comparable volumes/dilutions of the cell lysate or the supernatant should be analyzed by Western blot. Using this approach to the study of PRDX1 secretion, for instance, we calculated that at least 40% cell death would be required to account for the observed release of PRDX1 (23).…”