Search

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, resulting in more than one million deaths each year worldwide. This pathogen generates large amounts of hydrogen peroxide (H 2 O 2 ), which will be converted to hypothiocyanous acid (HOSCN) by lactoperoxidase (LPO) in the human respiratory tract. S. pneumoniae has been shown to be more resistant to HOSCN than some bacteria, and sensitizing S. pneumoniae to HOSCN may be a novel treatment strategy for combating this deadly pathogen. In this study we investigated the role of the low molecular weight thiol glutathione in HOSCN resistance. S. pneumoniae does not synthesize glutathione but imports it from the environment via an ABC transporter. Upon treatment of S. pneumoniae with HOSCN, bacterial glutathione was reversibly oxidized in a time- and dose-dependent manner, and intracellular proteins became glutathionylated. Bacterial death was observed when the reduced glutathione pool dropped below 20%. A S. pneumoniae mutant unable to import glutathione (ΔgshT) was more readily killed by exogenous HOSCN. Furthermore, bacterial growth in the presence of LPO converting bacterial H 2 O 2 to HOSCN was significantly impeded in mutants that were unable to import glutathione, or mutants unable to recycle oxidized glutathione (Δgor). This research highlights the importance of glutathione in protecting S. pneumoniae from HOSCN. Limiting glutathione utilization by S. pneumoniae may be a way to limit colonization and pathogenicity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Excessive production of microbicidal oxidants by neutrophils can damage host tissue. The short-term response of cells to oxidative stress is well understood, but the mechanisms behind long-term consequences require further clarification. Epigenetic pathways mediate cellular adaptation, and are therefore a potential target of oxidative stress. Indeed, there is evidence that many proteins and metabolites involved in epigenetic pathways are redox sensitive. In this review we provide an overview of the epigenetic landscape and discuss the potential for redox regulation. Using this information, we highlight specific examples where neutrophil oxidants react with epigenetic pathway components. We also use published data from redox proteomics to map out known intersections between oxidative stress and epigenetics that may signpost helpful directions for future investigation. Finally, we discuss the role neutrophils play in adaptive pathologies with a focus on tumour initiation and progression. We hope this information will stimulate further discourse on the emerging field of redox epigenomics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The bactericidal activity of the physiological oxidant hypochlorous acid (HOCl) is commonly studied in a variety of laboratory media. Reactive with numerous targets, HOCl will rapidly lose its toxicity via reduction or be converted to chloramines and other less toxic species. The objective of this study was to test the influence of various media, temperature and reaction time on the toxicity of HOCl. After incubating bacteria in media dosed with reagent HOCl, the bactericidal outcome was measured by colony forming ability. In parallel, we determined the HOCl and chloramine content after dosing media alone. Our results showed that more reagent HOCl was required to kill bacteria in culture media than in aqueous buffer, and this corresponded to the lower concentration of reactive chlorine species achieved in the media. RPMI and MOPS minimal medium retained significant oxidising equivalents after HOCl-dosing, but more nutrient-rich media such as MEM, DMEM, LB and TSB, had higher scavenging capacity. Other factors that lowered the bactericidal strength of HOCl were longer lag-times and raised temperature when pre-dosing media, and insufficient incubation time of cells with the HOCl-treated media. In summary, we demonstrate that the choice of media as well as procedural details within experiments crucially impact the cellular toxicity of HOCl. These factors influence the nature and concentration of oxidants generated, and therefore are critical in affecting cellular responses., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Peroxiredoxins (Prxs) are thiol peroxidases with a key role in antioxidant defense and redox signaling. They could be important in neutrophils for handling the large amount of oxidants that these cells produce. We investigated the redox state of Prx1 and Prx2 in HL-60 promyelocytic cells differentiated to neutrophil-like cells (dHL-60) and in human neutrophils. HL-60 cell differentiation with dimethyl sulfoxide caused a large decrease in expression of both Prxs, and all-trans retinoic acid also decreased Prx1 expression. Prx1 was mostly reduced in dHL-60 cells. NADPH oxidase activation by phorbol myristate acetate (PMA) or ingestion of Staphylococcus aureus induced rapid oxidation to disulfide-linked dimers, and eventually hyperoxidation. The NADPH oxidase inhibitor, diphenyleneiodonium, prevented Prx1 dimerization in stimulated dHL-60 cells, and decreased the extent of oxidation under resting conditions. In contrast, Prx1 and Prx2 were present in neutrophils from human blood as disulfides, and PMA or S. aureus caused no further oxidation. They remained oxidized on incubation with diphenyleneiodonium in media. Although this suggests that Prx redox cycling could be deficient in neutrophils, thioredoxin expression and thioredoxin reductase activity were similar in neutrophils and dHL-60 cells. Additionally, neutrophil thioredoxin was initially reduced and underwent oxidation after PMA activation. Thus, although the Prxs respond to oxidant generation in dHL-60 cells, in neutrophils they appear "locked" as disulfides. On this basis we propose that neutrophil Prxs are inefficient antioxidants and contribute little to peroxide removal during the oxidative burst, and speculate that they might be involved in other cell processes., (Copyright © 2019. Published by Elsevier Inc.)

Hydrogen peroxide (H 2 O 2 ) acts as a signaling molecule in cells by oxidising cysteine residues in regulatory proteins such as phosphatases, kinases and transcription factors. It is unclear exactly how many of these proteins are specifically targeted by H 2 O 2 because they appear too unreactive to be directly oxidised. One proposal is that peroxiredoxins (Prxs) initially react with H 2 O 2 and then oxidise adjacent proteins via a thiol relay mechanism. The aim of this study was to identify constitutive interaction partners of Prx2 in Jurkat T-lymphoma cells, in which thiol protein oxidation occurs at low micromolar concentrations of H 2 O 2 . Immunoprecipitation and proximity ligation assays identified a physical interaction between collapsin response mediator protein 2 (CRMP2) and cytoplasmic Prx2. CRMP2 regulates microtubule structure during lymphocyte migration and neuronal development. Exposure of Jurkat cells to low micromolar levels of H 2 O 2 caused rapid and reversible oxidation of CRMP2, in parallel with Prx2 oxidation, despite purified recombinant CRMP2 protein reacting slowly with H 2 O 2 (k~1 M -1 s -1 ). Lowering Prx expression should inhibit oxidation of proteins oxidised by a relay mechanism, however knockout of Prx2 had no effect on CRMP2 oxidation. CRMP2 also interacted with Prx1, suggesting redundancy in single knockout cells. Prx 1 and 2 double knockout Jurkat cells were not viable. An interaction between Prx2 and CRMP2 was also detected in other human and rodent cells, including primary neurons. However, low concentrations of H 2 O 2 did not cause CRMP2 oxidation in these cells. This indicates a cell-type specific mechanism for promoting CRMP2 oxidation in Jurkat cells, with insufficient evidence to attribute oxidation to a Prx-dependent redox relay., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundant in the lungs of patients with cystic fibrosis but fails to prevent their recurrent infections. Calprotectin is a major protein of neutrophils and composed of two monomers, S100A8 and S100A9. We show that the ability of calprotectin to limit growth of Staphylococcus aureus and Pseudomonas aeruginosa is exquisitely sensitive to oxidation by hypochlorous acid. The N-terminal cysteine residue on S100A9 was highly susceptible to oxidation which resulted in cross-linking of the protein monomers. The N-terminal methionine of S100A8 was also readily oxidized by hypochlorous acid, forming both the methionine sulfoxide and the unique product dehydromethionine. Isolated human neutrophils formed these modifications on calprotectin when their myeloperoxidase generated hypochlorous acid. Up to 90% of the N-terminal amine on S100A8 in bronchoalveolar lavage fluid from young children with cystic fibrosis was oxidized. Oxidized calprotectin was higher in children with cystic fibrosis compared to disease controls, and further elevated in those patients with infections. Our data suggest that oxidative stress associated with inflammation in cystic fibrosis will stop metal sequestration by calprotectin. Consequently, strategies aimed at blocking extracellular myeloperoxidase activity should enable calprotectin to provide nutritional immunity within the airways., (Copyright © 2015 Elsevier Inc. All rights reserved.)

There is strong evidence for the involvement of reactive oxygen species in ischemia/reperfusion injury. Although oxidation of individual thiol proteins has been reported, more extensive redox proteomics of hearts subjected to ischemia/reperfusion has not been performed. We have carried out an exploratory study using mass spectrometry with isotope-coded affinity tags (ICAT) aimed at identifying reversible oxidative changes to protein thiols in Langendorff perfused isolated mouse hearts subjected to 20 min ischemia with or without aerobic reperfusion for 5 or 30 min. Reduced thiols were blocked by adding N-ethylmaleimide during protein extraction, then reversibly oxidized thiols in extracts of control perfused and treated hearts were reduced and labeled with the light and heavy ICAT reagents, respectively. Protein extracts were mixed in equal amounts and relative proportions of the isotope-labeled peaks were used to quantify oxidative changes between the control and the treated groups. Approximately 300 peptides with ICAT signatures were reliably identified in each sample, with 181 peptides from 118 proteins common to all treatments. A proportion showed elevated ICAT ratios, consistent with reversible thiol oxidation. This was most evident after early reperfusion, with apparent reversal after longer reperfusion. In comparison, there was gradual accumulation of protein carbonyls and loss of GSH with longer reperfusion. Many of the thiol changes were in mitochondrial proteins, including components of electron transport complexes and enzymes involved in lipid metabolism. The results are consistent with mitochondria being a major site of oxidant generation during early cardiac reperfusion and mitochondrial thiol proteins being targets for oxidation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-alpha-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3.

Exposure of cells to sublethal oxidative stress results in the modulation of various signaling pathways. Oxidants can activate and inactivate transcription factors, membrane channels, and metabolic enzymes, and regulate calcium-dependent and phosphorylation signaling pathways. Oxidation and reduction of thiol proteins are thought to be the major mechanisms by which reactive oxidants integrate into cellular signal transduction pathways. This review focuses on mechanisms for sensing and transmitting redox signals, from the perspective of their chemical reactivity with specific oxidants. We discuss substrate preferences for different oxidants and how the kinetics of these reactions determines how each oxidant will react in a cell. This kinetic approach helps to identify initial oxidant-sensitive targets and elucidate mechanisms involved in transmission of redox signals. It indicates that only those proteins with very high reactivity, such as peroxiredoxins, are likely to be direct targets for hydrogen peroxide. Other more modestly reactive thiol proteins such as protein tyrosine phosphatases are more likely to become oxidized by an indirect mechanism. The review also examines oxidative changes observed during receptor-mediated signaling, the strengths and limitations of detection methods for reactive oxidant production, and the evidence for hydrogen peroxide acting as the second messenger. We discuss areas where observations in cell systems can be rationalized with the reactivity of specific oxidants and where further work is needed to understand the mechanisms involved.

Isothiocyanates are phytochemicals with anti-cancer properties that include the ability to trigger apoptosis. A substantial body of evidence suggests that reaction of the electrophilic isothiocyanate moiety with cysteine residues in cellular proteins and glutathione accounts for their biological activity. In this study we investigated the effect of several different isothiocyanates on the redox states of the cysteine-dependent peroxiredoxins (Prx) in Jurkat T lymphoma cells, and compared this to known effects on the selenoprotein thioredoxin reductase, glutathione reductase and intracellular GSH levels. Interestingly, oxidation of mitochondrial Prx3 could be detected as early as 5 min after exposure of cells to phenethyl isothiocyanate, with complete oxidation occurring at doses that only had small inhibitory effects on total cellular thioredoxin reductase and glutathione reductase activities. Peroxiredoxin oxidation was specific to the mitochondrial isoform with cytoplasmic Prx1 and Prx2 maintained in their reduced forms at all analyzed time points and concentrations of isothiocyanate. Phenethyl isothiocyanate could react with purified Prx3 directly, but it did not oxidize Prx3 or promote its oxidation by hydrogen peroxide. A selection of aromatic and alkyl isothiocyanates were tested and while all lowered cellular GSH levels, only the isothiocyanates that caused Prx3 oxidation were able to trigger cell death. We propose that pro-apoptotic isothiocyanates selectively disrupt mitochondrial redox homeostasis, as indicated by Prx3 oxidation, and that this contributes to their pro-apoptotic activity.

It is hypothesized that activation of death receptors disrupts the redox homeostasis of cells and that this contributes to the induction of apoptosis. The redox status of the peroxiredoxins, which are extremely sensitive to increases in H2O2 and disruption of the thioredoxin system, were monitored in Jurkat T lymphoma cells undergoing Fas-mediated apoptosis. The only detectable change during the early stages of apoptosis was oxidation of mitochondrial peroxiredoxin 3. Increased H2O2 triggers peroxiredoxin overoxidation to a sulphinic acid; however during apoptosis peroxiredoxin 3 was captured as a disulfide, suggesting impairment of the thioredoxin system responsible for maintaining peroxiredoxin 3 in its reduced form. Peroxiredoxin 3 oxidation was an early event, occurring within the same timeframe as increased mitochondrial oxidant production, caspase activation and cytochrome c release. It preceded other major apoptotic events including mitochondrial permeability transition and phosphatidylserine exposure, and glutathione depletion, global thiol protein oxidation and protein carbonylation. Peroxiredoxin 3 oxidation was also observed in U937 cells stimulated with TNF-alpha. We hypothesize that the selective oxidation of peroxiredoxin 3 leads to an increase in mitochondrial H2O2 and that this may influence the progression of apoptosis.

Neutrophil oxidants are hypothesized to damage the gastric mucosa and promote carcinogenesis in people infected with Helicobacter pylori. To investigate this process we used wild-type and chronic granulomatous disease (CGD) mice with a targeted disruption of the gp91(phox) subunit of the NADPH oxidase. The mice were inoculated with a mouse-adapted strain of H. pylori and changes in gastric pathology were examined 12 and 30 weeks after infection. Glandular atrophy, a precursor lesion in the development of intestinal-type gastric carcinoma, and epithelial cell proliferation were both dramatically increased in the gastric body of CGD animals within 12 weeks. This correlated strongly with increased numbers of neutrophils in the mucosa (Pearson coefficient 0.97, P < 0.001). H. pylori is a noninvasive bacterium, and there was no increase in bacterial numbers in the CGD animals. Closer examination of the gastric tissue indicated the presence of degenerate neutrophils associated with atrophy. We hypothesize that the release of granule constituents from these neutrophils contributes to tissue damage. This is exacerbated by the absence of a functional NADPH oxidase, and is consistent with this enzyme complex having an important role in dampening the inflammatory response.