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Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation., Competing Interests: Conflict of interest J. M. M. contributes to the development of necroptosis inhibitors in collaboration with Anaxis Pharma Pty Ltd. G.S.S. consults for Genentech Inc. All other authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Excessive generation of oxidants by immune cells results in acute tissue damage. One mechanism by which oxidant exposure could have long-term effects is modulation of epigenetic pathways. We hypothesized that methylation of newly synthesized DNA in proliferating cells can be altered by oxidants that target DNA methyltransferase activity or deplete its substrate, the methyl donor SAM. To this end, we investigated the effect of two oxidants produced by neutrophils, H 2 O 2 and glycine chloramine, on maintenance DNA methylation in Jurkat T lymphoma cells. Using cell synchronization and MS-based analysis, we measured heavy deoxycytidine isotope incorporation into newly synthesized DNA and observed that a sublethal bolus of glycine chloramine, but not H 2 O 2 , significantly inhibited DNA methylation. Both oxidants inhibited DNA methyltransferase 1 activity, but only chloramine depleted SAM, suggesting that removal of substrate was the most effective means of inhibiting DNA methylation. These results indicate that immune cell-derived oxidants generated during inflammation have the potential to affect the epigenome of neighboring cells., (© 2020 O'Connor et al.)

During aerobic growth, the Gram-positive facultative anaerobe and opportunistic human pathogen Streptococcus pneumoniae generates large amounts of hydrogen peroxide that can accumulate to millimolar concentrations. The mechanism by which this catalase-negative bacterium can withstand endogenous hydrogen peroxide is incompletely understood. The enzyme alkylhydroperoxidase D (AhpD) has been shown to contribute to pneumococcal virulence and oxidative stress responses in vivo We demonstrate here that Sp AhpD exhibits weak thiol-dependent peroxidase activity and, unlike the previously reported Mycobacterium tuberculosis AhpC/D system, Sp AhpD does not mediate electron transfer to Sp AhpC. A 2.3-Å resolution crystal structure revealed several unusual structural features, including a three-cysteine active site architecture that is buried in a deep pocket, in contrast to the two-cysteine active site found in other AhpD enzymes. All single-cysteine Sp AhpD variants remained partially active, and LC-MS/MS analyses revealed that the third cysteine, Cys-163, formed disulfide bonds with either of two cysteines in the canonical Cys-78- X-X -Cys-81 motif. We observed that Sp AhpD formed a dimeric quaternary structure both in the crystal and in solution, and that the highly conserved Asn-76 of the AhpD core motif is important for Sp AhpD folding. In summary, Sp AhpD is a weak peroxidase and does not transfer electrons to AhpC, and therefore does not fit existing models of bacterial AhpD antioxidant defense mechanisms. We propose that it is unlikely that Sp AhpD removes peroxides either directly or via AhpC, and that Sp AhpD cysteine oxidation may act as a redox switch or mediate electron transfer with other thiol proteins., (© 2020 Meng et al.)

Myeloperoxidase is a major neutrophil antimicrobial protein, but its role in immunity is often overlooked because individuals deficient in this enzyme are usually in good health. Within neutrophil phagosomes, myeloperoxidase uses superoxide generated by the NADPH oxidase to oxidize chloride to the potent bactericidal oxidant hypochlorous acid (HOCl). In this study, using phagocytosis assays and LC-MS analyses, we monitored GSH oxidation in Pseudomonas aeruginosa to gauge their exposure to HOCl inside phagosomes. Doses of reagent HOCl that killed most of the bacteria oxidized half the cells' GSH, producing mainly glutathione disulfide (GSSG) and other low-molecular-weight disulfides. Glutathione sulfonamide (GSA), a HOCl-specific product, was also formed. When neutrophils phagocytosed P. aeruginosa , half of the bacterial GSH was lost. Bacterial GSA production indicated that HOCl had reacted with the bacterial cells, oxidized their GSH, and was sufficient to be solely responsible for bacterial killing. Inhibition of NADPH oxidase and myeloperoxidase lowered GSA formation in the bacterial cells, but the bacteria were still killed, presumably by compensatory nonoxidative mechanisms. Of note, bacterial GSA formation in neutrophils from patients with cystic fibrosis (CF) was normal during early phagocytosis, but it was diminished at later time points, which was mirrored by a small decrease in bacterial killing. In conclusion, myeloperoxidase generates sufficient HOCl within neutrophil phagosomes to kill ingested bacteria. The unusual kinetics of phagosomal HOCl production in CF neutrophils confirm a role for the cystic fibrosis transmembrane conductance regulator in maintaining HOCl production in neutrophil phagosomes., (© 2019 Dickerhof et al.)

Regulation of growth factor signaling involves reversible inactivation of protein tyrosine phosphatases (PTPs) through the oxidation and reduction of their active site cysteine. However, there is limited mechanistic understanding of these redox events and their co-ordination in the presence of cellular antioxidant networks. Here we investigated interactions between PTP1B and the peroxiredoxin 2 (Prx2)/thioredoxin 1 (Trx1)/thioredoxin reductase 1 (TrxR1) network. We found that Prx2 becomes oxidized in PDGF-treated fibroblasts, but only when TrxR1 has first been inhibited. Using purified proteins, we also found that PTP1B is relatively insensitive to inactivation by H 2 O 2 but found no evidence for a relay mechanism in which Prx2 or Trx1 facilitates PTP1B oxidation. Instead, these proteins prevented PTP1B inactivation by H 2 O 2 Intriguingly, we discovered that TrxR1/NADPH directly protects PTP1B from inactivation when present during the H 2 O 2 exposure. This protection was dependent on the concentration of TrxR1 and independent of Trx1 and Prx2. The protection was blocked by auranofin and required an intact selenocysteine residue in TrxR1. This activity likely involves reduction of the sulfenic acid intermediate form of PTP1B by TrxR1 and is therefore distinct from the previously described reactivation of end-point oxidized PTP1B, which requires both Trx1 and TrxR1. The ability of TrxR1 to directly reduce an oxidized phosphatase is a novel activity that can help explain previously observed increases in PTP1B oxidation and PDGF receptor phosphorylation in TrxR1 knockout cells. The activity of TrxR1 is therefore of potential relevance for understanding the mechanisms of redox regulation of growth factor signaling pathways., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Typical 2-Cys peroxiredoxins (Prxs) react rapidly with H2O2 to form a sulfenic acid, which then condenses with the resolving cysteine of the adjacent Prx in the homodimer or reacts with another H2O2 to become hyperoxidized. Hyperoxidation inactivates the Prx and is implicated in cell signaling. Prxs vary in susceptibility to hyperoxidation. We determined rate constants for disulfide formation and hyperoxidation for human recombinant Prx2 and Prx3 by analyzing the relative proportions of hyperoxidized and dimeric products using mass spectrometry as a function of H2O2 concentration (in the absence of reductive cycling) and in competition with catalase at a fixed concentration of H2O2. This gave a second order rate constant for hyperoxidation of 12,000 M(-1) s(-1) and a rate constant for disulfide formation of 2 s(-1) for Prx2. A similar hyperoxidation rate constant for Prx3 was measured, but its rate of disulfide formation was ~10-fold higher, making it is more resistant than Prx2 to hyperoxidation. There are two active sites within the homodimer, and at low H2O2 concentrations one site was hyperoxidized and the other present as a disulfide. Prx with two hyperoxidized sites formed progressively at higher H2O2 concentrations. Although the sulfenic acid forms of Prx2 and Prx3 are ~1000-fold less reactive with H2O2 than their active site thiols, they react several orders of magnitude faster than most reduced thiol proteins. This observation has important implications for understanding the mechanism of peroxide sensing in cells.

Peroxiredoxins (Prx) are thiol peroxidases that exhibit exceptionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are ∼3-4 Å or ∼6-7 Å away from the active site peroxidative cysteine (C(p)), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooperative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure suggest strong catalytic effects of H-bond-donating functional groups to the C(p) sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cation in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bonding to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H(2)O(2) and may have wider implications for protein thiol reactivity toward peroxides., (© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.)

Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti-inflammatory activity. However, knowledge of their activity at a molecular level is limited. The objective of this study was to identify biological targets of phenethyl isothiocyanate (PEITC) using an affinity purification approach. An analogue of PEITC was synthesized to enable conjugation to a solid-phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was the major protein captured from cell lysates. Site-directed mutagenesis and mass spectrometry showed that PEITC covalently modified the N-terminal proline residue of MIF. This resulted in complete loss of catalytic tautomerase activity and disruption of protein conformation, as determined by impaired recognition by a monoclonal antibody directed to the region that receptors and interacting proteins bind to MIF. The conformational change was supported by in silico modeling. Monoclonal antibody binding to plasma MIF was disrupted in humans consuming watercress, a major dietary source of PEITC. The isothiocyanates have significant potential for development as MIF inhibitors, and this activity may contribute to the biological properties of these phytochemicals.

Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H(2)O(2). Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 x 10(7) M(-1) s(-1) for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.

Neutrophils kill bacteria by ingesting them into phagosomes where superoxide and cytoplasmic granule constituents, including myeloperoxidase, are released. Myeloperoxidase converts chloride and hydrogen peroxide to hypochlorous acid (HOCl), which is strongly microbicidal. However, the role of oxidants in killing and the species responsible are poorly understood and the subject of current debate. To assess what oxidative mechanisms are likely to operate in the narrow confines of the phagosome, we have used a kinetic model to examine the fate of superoxide and its interactions with myeloperoxidase. Known rate constants for reactions of myeloperoxidase have been used and substrate concentrations estimated from neutrophil morphology. In the model, superoxide is generated at several mm/s. Most react with myeloperoxidase, which is present at millimolar concentrations, and rapidly convert the enzyme to compound III. Compound III turnover by superoxide is essential to maintain enzyme activity. Superoxide stabilizes at approximately 25 microM and hydrogen peroxide in the low micromolar range. HOCl production is efficient if there is adequate chloride supply, but further knowledge on chloride concentrations and transport mechanisms is needed to assess whether this is the case. Low myeloperoxidase concentrations also limit HOCl production by allowing more hydrogen peroxide to escape from the phagosome. In the absence of myeloperoxidase, superoxide increases to >100 microM but hydrogen peroxide to only approximately 30 microM. Most of the HOCl reacts with released granule proteins before reaching the bacterium, and chloramine products may be effectors of its antimicrobial activity. Hydroxyl radicals should form only after all susceptible protein targets are consumed.

Diphenyleneiodonium (DPI) is a broad-spectrum flavoprotein inhibitor commonly used to inhibit oxidant production by the NADPH oxidase of phagocytic and nonphagocytic cells. A previous study has shown that DPI can sensitize T24 bladder carcinoma cells to Fas-mediated apoptosis. We observed DPI to deplete intracellular reduced glutathione (GSH) in T24 cells and a range of other primary and transformed cell types. The effect was immediate, with 50% loss of intracellular GSH within 2 h of treatment with DPI. The glutathione was quantitatively recovered in the extracellular medium, indicating that efflux was occurring. The loss of GSH was blocked with bromosulfophthalein, an inhibitor of the canalicular GSH transporters. We conclude that DPI induces a dramatic efflux of cellular GSH from T24 cells via a specific transport channel. This provides a potential mechanism for its proapoptotic effect, and it also has important implications for the regulation of glutathione homeostasis in cells.

Myeloperoxidase is proposed to play a central role in bacterial killing by generating hypochlorous acid within neutrophil phagosomes. However, it has yet to be demonstrated that these inflammatory cells target hypochlorous acid against bacteria inside phagosomes. In this investigation, we treated Staphylococcus aureus with varying concentrations of reagent hypochlorous acid and found that even at sublethal doses, it converted some tyrosine residues in their proteins to 3-chlorotyrosine and 3,5-dichlorotyrosine. To determine whether or not ingested bacteria were exposed to hypochlorous acid in neutrophil phagosomes, we labeled their proteins with [(13)C(6)]tyrosine and used gas chromatography with mass spectrometry to identify the corresponding chlorinated isotopes after the bacteria had been phagocytosed. Chlorinated tyrosines were detected in bacterial proteins 5 min after phagocytosis and reached levels of approximately 2.5/1000 mol of tyrosine at 60 min. Inhibitor studies revealed that chlorination was dependent on myeloperoxidase. Chlorinated neutrophil proteins were also detected and accounted for 94% of total chlorinated tyrosine residues formed during phagocytosis. We conclude that hypochlorous acid is a major intracellular product of the respiratory burst. Although some reacts with the bacteria, most reacts with neutrophil components.