Thiol regulation of pro-inflammatory cytokines and innate immunity: protein S-thiolation as a novel molecular mechanism.
ABSTRACT Inflammation or inflammatory cytokines and oxidative stress have often been associated, and thiol antioxidants, particularly glutathione, have often been seen as possible anti-inflammatory mediators. However, whereas several cytokine inhibitors have been approved for drug use in chronic inflammatory diseases, this has not happened with antioxidant molecules. We outline the complexity of the role of protein thiol-disulfide oxidoreduction in the regulation of immunity and inflammation, the underlying molecular mechanisms (such as protein glutathionylation) and the key enzyme players such as Trx (thioredoxin) or Grx (glutaredoxin).
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ABSTRACT: Glutathione oxidation and protein glutathionylation are considered hallmarks of oxidative stress in cells because they reflect thiol redox status in proteins. Our aims were to analyze redox status of thiols and to identify mixed disulfides and targets of redox signaling in pancreas in experimental acute pancreatitis as a model of acute inflammation associated with glutathione depletion. Glutathione depletion in pancreas in acute pancreatitis is not associated with any increase in oxidized glutathione levels or protein glutathionylation. Cystine and homocystine levels as well as protein cysteinylation and gamma-glutamyl cysteinylation markedly rose in pancreas after induction of pancreatitis. Protein cysteinylation was non-detectable in pancreas under basal conditions. Targets of disulfide stress were identified by western blotting, diagonal electrophoresis and proteomic methods. Cysteinylated albumin was detected. Redox-sensitive PP2A and tyrosine protein phosphatase activities diminished in pancreatitis and this loss was abrogated by N-acetyl cysteine. According to our findings, disulfide stress may be considered a specific type of oxidative stress in acute inflammation associated with protein cysteinylation and gamma-glutamyl cysteinylation and oxidation of the pair cysteine/cystine, but without glutathione oxidation or changes in protein glutathionylation. Two types of targets of disulfide stress were identified: redox buffers, such as ribonuclease inhibitor or albumin; and redox-signaling thiols that include thioredoxin 1, APE1/Ref1, Keap1, tyrosine and serine/threonine phosphatases, protein disulfide isomerase. These targets exhibit great relevance in DNA repair, cell proliferation, apoptosis, endoplasmic reticulum stress, and inflammatory response. Disulfide stress would be a specific mechanism of redox signaling independent of glutathione redox status involved in inflammation.Free Radical Biology and Medicine 01/2014; · 5.27 Impact Factor
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ABSTRACT: Bottom-up mass spectrometry studies typically employ a reduction and alkylation step that eliminates a class of post-translational modification, S-thiolation. Given that molecular oxygen can mediate S-thiolation from reduced thiols, which are abundant in the reducing intracellular milieu, we investigated the possibility that some S-thiolation modifications are artifacts of protein preparation. SOD1 was chosen for this case study as it has a reactive surface cysteine residue, which is readily cysteinylated in vitro. The ability of oxygen to generate S-thiolation artifacts was tested by comparing purification of SOD1 from post-mortem human cerebral cortex under aerobic and anaerobic conditions. S-thiolation was ∼50% higher in aerobically processed preparations, consistent with oxygen-dependent artifactual S-thiolation. The ability of endogenous small molecule disulfides (e.g. cystine) to participate in artifactual S-thiolation was tested by blocking reactive protein cysteine residues during anaerobic homogenization. A 50-fold reduction in S-thiolation occurred, indicating the majority of S-thiolation observed aerobically was artifact. Tissue-specific artifacts were explored by comparing brain- and blood-derived protein, with remarkably more artifacts observed in brain-derived SOD1. Given the potential for such artifacts, rules-of-thumb for sample preparation are provided. This study demonstrates that without taking extraordinary precaution, artifactual S-thiolation of highly reactive, surface exposed, cysteine residues can result. This article is protected by copyright. All rights reservedProteomics 03/2014; · 3.97 Impact Factor
Dataset: 28.Moreno ML et al FRBM 2014