Redox proteomic analysis of HO -treated Jurkat cells and effects of bicarbonate and knockout of peroxiredoxins 1 and 2.

Oxidation of thiol proteins and redox signaling occur in cells exposed to HO but mechanisms are unclear. We used redox proteomics to seek evidence of oxidation of specific proteins either by a mechanism involving reaction of HO with CO/bicarbonate to give the more reactive peroxymonocarbonate, or via a relay involving peroxiredoxins (Prdxs). Changes in oxidation state of specific Cys-SH residues on treating Jurkat T lymphoma cells with HO were measured by isotopically labeling reduced thiols and analysis by mass spectrometry.

Effect of peroxiredoxin 1 or peroxiredoxin 2 knockout on the thiol proteome of Jurkat cells.

Peroxiredoxins are important regulators of cellular peroxide metabolism. As antioxidants, they restrict oxidation of other cell proteins, but as signaling molecules they can act as sensors and promote thiol protein oxidation via a redox relay mechanism. The presence of peroxiredoxins could therefore influence other thiol proteins, even in cells experiencing endogenous redox activity.

Phenotypic spectrum in a family with a novel p.I21S dominant-activating mutation.

Objectives: Dominant-activating (DA) lesions in have been reported in 18 individuals to date. Some have required haematopoietic stem cell transplantation (HSCT) for their (severe) combined immunodeficiency syndrome phenotype. We aimed to investigate clinical and cellular features of a kindred harbouring a novel variant in p.

Peroxidasin Inhibition by Phloroglucinol and Other Peroxidase Inhibitors.

Human peroxidasin (PXDN) is a ubiquitous peroxidase enzyme expressed in most tissues in the body. PXDN represents an interesting therapeutic target for inhibition, as it plays a role in numerous pathologies, including cardiovascular disease, cancer and fibrosis. Like other peroxidases, PXDN generates hypohalous acids and free radical species, thereby facilitating oxidative modifications of numerous biomolecules.

Carbon dioxide/bicarbonate is required for sensitive inactivation of mammalian glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains an active site Cys and is one of the most sensitive cellular enzymes to oxidative inactivation and redox regulation. Here, we show that inactivation by hydrogen peroxide is strongly enhanced in the presence of carbon dioxide/bicarbonate. Inactivation of isolated mammalian GAPDH by HO increased with increasing bicarbonate concentration and was sevenfold faster in 25 mM (physiological) bicarbonate compared with bicarbonate-free buffer of the same pH.

Superoxide: The enigmatic chemical chameleon in neutrophil biology.

The burst of superoxide produced when neutrophils phagocytose bacteria is the defining biochemical feature of these abundant immune cells. But 50 years since this discovery, the vital role superoxide plays in host defense has yet to be defined. Superoxide is neither bactericidal nor is it just a source of hydrogen peroxide.

Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo.

Multiple roles of reactive oxygen species (ROS) and their consequences for health and disease are emerging throughout biological sciences. This development has led researchers unfamiliar with the complexities of ROS and their reactions to employ commercial kits and probes to measure ROS and oxidative damage inappropriately, treating ROS (a generic abbreviation) as if it were a discrete molecular entity. Unfortunately, the application and interpretation of these measurements are fraught with challenges and limitations.

Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology.

'Reactive oxygen species' (ROS) is a generic term that defines a wide variety of oxidant molecules with vastly different properties and biological functions that range from signalling to causing cell damage. Consequently, the description of oxidants needs to be chemically precise to translate research on their biological effects into therapeutic benefit in redox medicine. This Expert Recommendation article pinpoints key issues associated with identifying the physiological roles of oxidants, focusing on HO and O.

Neutrophil NET Formation with Microbial Stimuli Requires Late Stage NADPH Oxidase Activity.

Neutrophils respond to a range of stimuli by releasing extracellular traps (NETs), a mesh consisting of chromatin plus granule and cytoplasmic proteins. We have investigated NET release in response to phorbol myristate acetate (PMA), (PAO1), and , and the involvement of NADPH oxidase (NOX2) and myeloperoxidase (MPO) activities. An oxidative mechanism was involved with each stimulus, and the NOX2 inhibitor diphenylene iodonium (DPI) gave almost total inhibition.

Hydrogen peroxide signaling via its transformation to a stereospecific alkyl hydroperoxide that escapes reductive inactivation.

During systemic inflammation, indoleamine 2,3-dioxygenase 1 (IDO1) becomes expressed in endothelial cells where it uses hydrogen peroxide (HO) to oxidize L-tryptophan to the tricyclic hydroperoxide, cis-WOOH, that then relaxes arteries via oxidation of protein kinase G 1α. Here we show that arterial glutathione peroxidases and peroxiredoxins that rapidly eliminate HO, have little impact on relaxation of IDO1-expressing arteries, and that purified IDO1 forms cis-WOOH in the presence of peroxiredoxin 2. cis-WOOH oxidizes protein thiols in a selective and stereospecific manner.

Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential.

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein.

The Enigma of 2-Cys Peroxiredoxins: What Are Their Roles?

2-Cys peroxiredoxins are abundant thiol proteins that react efficiently with a wide range of peroxides. Unlike other enzymes, their exceptionally high reactivity does not rely on cofactors. The mechanism of oxidation and reduction of peroxiredoxins places them in a good position to act as antioxidants as well as key players in redox signaling.

Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2.

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with HO and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site.

Induction of the reactive chlorine-responsive transcription factor RclR in Escherichia coli following ingestion by neutrophils.

Neutrophils generate hypochlorous acid (HOCl) and related reactive chlorine species as part of their defence against invading microorganisms. In isolation, bacteria respond to reactive chlorine species by upregulating responses that provide defence against oxidative challenge. Key questions are whether these responses are induced when bacteria are phagocytosed by neutrophils, and whether this provides them with a survival advantage.

A curious case of cysteines in human peroxiredoxin I.

Peroxiredoxins (Prxs) are antioxidant proteins that are involved in cellular defence against reactive oxygen species and reactive nitrogen species. Humans have six peroxiredoxins, hPrxI-VI, out of which hPrxI and hPrxII belongs to the typical 2-Cys class sharing 90% conservation in their amino acid sequence including catalytic residues required to carry out their peroxidase and chaperone activities. Despite the high conservation between hPrxI and hPrxII, hPrxI behaves differently from hPrxII in its peroxidase and chaperone activity.

Evaluating the bactericidal action of hypochlorous acid in culture media.

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.

Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2.

Peroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites.

Peroxidasin mediates bromination of tyrosine residues in the extracellular matrix.

Peroxidasin is a heme peroxidase that oxidizes bromide to hypobromous acid (HOBr), a powerful oxidant that promotes the formation of the sulfilimine crosslink in collagen IV in basement membranes. We investigated whether HOBr released by peroxidasin leads to other oxidative modifications of proteins, particularly bromination of tyrosine residues, in peroxidasin-expressing PFHR9 cells. Using stable isotope dilution LC-MS/MS, we detected the formation of 3-bromotyrosine, a specific biomarker of HOBr-mediated protein modification.

Hydrogen peroxide reactivity and specificity in thiol-based cell signalling.

Reversible oxidation of thiol proteins is an important cell signalling mechanism. In many cases, this involves generation or exposure of the cells to H2O2, and oxidation of proteins that are not particularly H2O2-reactive. There is a conundrum as to how these proteins are oxidised when other highly reactive proteins such as peroxiredoxins are present.

Inhibition of DNA methylation in proliferating human lymphoma cells by immune cell oxidants.

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.

Deciphering the in vivo redox behavior of human peroxiredoxins I and II by expressing in budding yeast.

Peroxiredoxins (Prxs), scavenge cellular peroxides by forming recyclable disulfides but under high oxidative stress, hyperoxidation of their active-site Cys residue results in loss of their peroxidase activity. Saccharomyces cerevisiae deficient in human Prx (hPrx) orthologue TSA1 show growth defects under oxidative stress. They can be complemented with hPRXI but not by hPRXII, but it is not clear how the disulfide and hyperoxidation states of the hPrx vary in yeast under oxidative stress.

Enhanced hyperoxidation of peroxiredoxin 2 and peroxiredoxin 3 in the presence of bicarbonate/CO.

Hydrogen peroxide undergoes an equilibrium reaction with bicarbonate/CO to produce peroxymonocarbonate (HCO). Peroxymonocarbonate is more reactive with thiols than HO but it makes up only a small fraction of the HO in physiological bicarbonate buffers so the increase in rate of oxidation of low molecular weight thiols is modest. However, for some thiol proteins such as protein tyrosine phosphatases, the rate enhancement is very much greater.

Bicarbonate is essential for protein-tyrosine phosphatase 1B (PTP1B) oxidation and cellular signaling through EGF-triggered phosphorylation cascades.

Protein-tyrosine phosphatases (PTPs) counteract protein tyrosine phosphorylation and cooperate with receptor-tyrosine kinases in the regulation of cell signaling. PTPs need to undergo oxidative inhibition for activation of cellular cascades of protein-tyrosine kinase phosphorylation following growth factor stimulation. It has remained enigmatic how such oxidation can occur in the presence of potent cellular reducing systems.

Peroxiredoxin expression and redox status in neutrophils and HL-60 cells.

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.