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

PLoS One. 2015 May 18;10(5):e0127086. doi: 10.1371/journal.pone.0127086. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antioxidants / pharmacology
  • Blotting, Western
  • Cell Line
  • Dexamethasone / pharmacology
  • Down-Regulation / drug effects
  • Glutathione / metabolism*
  • Humans
  • Inflammation / complications
  • Inflammation / metabolism*
  • Inflammation / pathology
  • Influenza, Human / complications
  • Influenza, Human / metabolism*
  • Influenza, Human / pathology
  • Lipopolysaccharides / pharmacology
  • Mice
  • Oxidation-Reduction / drug effects
  • Oxidative Stress* / drug effects
  • Peroxiredoxins / metabolism
  • Profilins / metabolism
  • Proteins / metabolism*
  • Proteomics*
  • RAW 264.7 Cells
  • Sulfhydryl Compounds / pharmacology
  • Thioredoxins / metabolism
  • Vimentin / metabolism

Substances

  • Antioxidants
  • Lipopolysaccharides
  • Profilins
  • Proteins
  • Sulfhydryl Compounds
  • Vimentin
  • Thioredoxins
  • Dexamethasone
  • Peroxiredoxins
  • Glutathione

Grant support

This work was supported by the RM Phillips Charitable Trust (to PG), European Development Fund, Interreg Project Peptide Research Network of Excellence, Perene (to PG), the EU COST action EU-ROS (to PG), the Deutsche Forschungsgemeinschaft (DFG SFB593-N01 to CHL), the von Behring-Röntgen Foundation (to CHL), the Italian Ministry of Instruction, University and Research (grant number PON 0101802 to ATP). PC was supported by a fellowship from the Institute Pasteur/Cenci-Bolognetti Foundation, Rome. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.