Formation of Calprotectin-Derived Peptides in the Airways of Children with Cystic Fibrosis.

Calprotectin is released by activated neutrophils along with myeloperoxidase (MPO) and proteases. It plays numerous roles in inflammation and infection, and is used as an inflammatory biomarker. However, calprotectin is readily oxidized by MPO-derived hypohalous acids to form covalent dimers of its S100A8 and S100A9 subunits. The dimers are susceptible to degradation by proteases. We show that detection of human calprotectin by ELISA declines markedly because of its oxidation by hypochlorous acid and subsequent degradation. Also, proteolysis liberates specific peptides from oxidized calprotectin that is present at inflammatory sites. We identified six calprotectin-derived peptides by mass spectrometry and detected them in the bronchoalveolar lavage fluid of children with cystic fibrosis (CF). We assessed the peptides as biomarkers of neutrophilic inflammation and infection. The content of the calprotectin peptide ILVI was related to calprotectin (r = 0.72, p = 0.01, n = 10). Four of the peptides were correlated with the concentration of MPO (r > 0.7, p ≤ 0.01, n = 21), while three were higher (p < 0.05) in neutrophil elastase-positive (n = 14) than -negative samples (n = 7). Also, five of the peptides were higher (p < 0.05) in the bronchoalveolar lavage fluid from children with CF with infections (n = 21) than from non-CF children without infections (n = 6). The specific peptides liberated from calprotectin will signal uncontrolled activity of proteases and MPO during inflammation. They may prove useful in tracking inflammation in respiratory diseases dominated by neutrophils, including coronavirus disease 2019.

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. The level of 3-bromotyrosine in extracellular matrix proteins from normally cultured cells was 1.1 mmol/mol tyrosine and decreased significantly in the presence of the peroxidasin inhibitor, phloroglucinol. A negligible amount of 3-bromotyrosine was detected in peroxidasin-knockout cells. 3-Bromotyrosine formed both during cell growth in culture and in the isolated decellularized extracellular matrix when embedded peroxidasin was supplied with hydrogen peroxide and bromide. The level of 3-bromotyrosine was significantly higher in extracellular matrix than intracellular proteins, although a low amount was detected intracellularly. 3-Bromotyrosine levels increased with higher bromide concentrations and decreased in the presence of physiological concentrations of thiocyanate and urate. However, these peroxidase substrates showed moderate to minimal inhibition of collagen IV crosslinking. Our findings provide evidence that peroxidasin promotes the formation of 3-bromotyrosine in proteins. They show that HOBr produced by peroxidasin is selective for, but not limited to, the crosslinking of collagen IV. Based on our findings, the use of 3-bromotyrosine as a specific biomarker of oxidative damage by HOBr warrants further investigation in clinical conditions linked to high peroxidasin expression.

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin.

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H2O2 and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m(-1) s(-1)) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.

Hyperoxidation of peroxiredoxins 2 and 3: rate constants for the reactions of the sulfenic acid of the peroxidatic cysteine.

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.

Cardiac effects of myoregulin in ischemia-reperfusion.

Myoregulin is a recently discovered micropeptide that controls calcium levels by inhibiting the intracellular calcium pump sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). Keeping calcium levels balanced in the heart is essential for normal heart functioning, thus myoregulin has the potential to be a crucial regulator of cardiac muscle performance by reducing the rate of intracellular Ca2+ uptake. We provide the first report of myoregulin mRNA expression in human heart tissue, absence of expression in human plasma, and the effects of myoregulin on cardiac hemodynamics in an ex vivo Langendorff isolated rat heart model of ischemia/reperfusion. In this preliminary study, myoregulin provided a cardio-protective effect, as assessed by preservation of left ventricular contractility and relaxation, during ischemia/reperfusion. This study provides the foundation for future research in this area.

Inactivation of human myeloperoxidase by hydrogen peroxide.

Human myeloperoxidase (MPO) uses hydrogen peroxide generated by the oxidative burst of neutrophils to produce an array of antimicrobial oxidants. During this process MPO is irreversibly inactivated. This study focused on the unknown role of hydrogen peroxide in this process. When treated with low concentrations of H2O2 in the absence of reducing substrates, there was a rapid loss of up to 35% of its peroxidase activity. Inactivation is proposed to occur via oxidation reactions of Compound I with the prosthetic group or amino acid residues. At higher concentrations hydrogen peroxide acts as a suicide substrate with a rate constant of inactivation of 3.9 × 10(-3) s(-1). Treatment of MPO with high H2O2 concentrations resulted in complete inactivation, Compound III formation, destruction of the heme groups, release of their iron, and detachment of the small polypeptide chain of MPO. Ten of the protein's methionine residues were oxidized and the thermal stability of the protein decreased. Inactivation by high concentrations of H2O2 is proposed to occur via the generation of reactive oxidants when H2O2 reacts with Compound III. These mechanisms of inactivation may occur inside neutrophil phagosomes when reducing substrates for MPO become limiting and could be exploited when designing pharmacological inhibitors.

2-thioxanthines are mechanism-based inactivators of myeloperoxidase that block oxidative stress during inflammation.

Myeloperoxidase (MPO) is a prime candidate for promoting oxidative stress during inflammation. This abundant enzyme of neutrophils uses hydrogen peroxide to oxidize chloride to highly reactive and toxic chlorine bleach. We have identified 2-thioxanthines as potent mechanism-based inactivators of MPO. Mass spectrometry and x-ray crystal structures revealed that these inhibitors become covalently attached to the heme prosthetic groups of the enzyme. We propose a mechanism whereby 2-thioxanthines are oxidized, and their incipient free radicals react with the heme groups of the enzyme before they can exit the active site. 2-Thioxanthines inhibited MPO in plasma and decreased protein chlorination in a mouse model of peritonitis. They slowed but did not prevent neutrophils from killing bacteria and were poor inhibitors of thyroid peroxidase. Our study shows that MPO is susceptible to the free radicals it generates, and this Achilles' heel of the enzyme can be exploited to block oxidative stress during inflammation.

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. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.

Redox potential and peroxide reactivity of human peroxiredoxin 3.

Peroxiredoxins (Prxs) are a ubiquitous family of thiol peroxidases that protect cells from peroxides and have a putative role in redox signaling. In this study, we investigated the redox properties of human Prx 3, a typical 2-Cys Prx that is localized to the mitochondrial matrix. We found that Prx 3 displayed strong reactivity with H(2)O(2), with a competitive kinetic approach generating a second order rate constant of 2 x 10(7) M(-1) s(-1). This is considerably higher than typical thiols and similar to values for other mammalian 2-Cys Prxs. In contrast, Prx 3 reacted very slowly with the thiol alkylating agents iodoacetamide and N-ethylmaleimide. Using dithiothreitol redox buffers, we measured the redox potential of Prx 3 of -290 mV. This is similar to the redox potential of mitochondrial thioredoxin 2 and is consistent with optimal operation of Prx 3 in the mitochondrial matrix.

Interaction of adenanthin with glutathione and thiol enzymes: selectivity for thioredoxin reductase and inhibition of peroxiredoxin recycling.

The diterpenoid, adenanthin, represses tumor growth and prolongs survival in mouse promyelocytic leukemia models (Liu et al., Nat. Chem. Biol. 8, 486, 2012). It was proposed that this was done by inactivating peroxiredoxins (Prxs) 1 and 2 through the formation of an adduct specifically on the resolving Cys residue. We confirmed that adenanthin underwent Michael addition to isolated Prx2, thereby inhibiting oxidation to a disulfide-linked dimer. However, contrary to the original report, both the peroxidatic and the resolving Cys residues could be derivatized. Glutathione also formed an adenanthin adduct, reacting with a second-order rate constant of 25±5 M(-1) s(-1). With 50 µM adenanthin, the peroxidatic and resolving Cys of Prx2 reacted with half-times of 7 and 40 min, respectively, compared with 10 min for GSH. When erythrocytes or Jurkat T cells were treated with adenanthin, we saw no evidence for a reaction with Prxs 1 or 2. Instead, adenanthin caused time- and concentration-dependent loss of GSH followed by dimerization of the Prxs. Prxs undergo continuous oxidation in cells and are normally recycled by thioredoxin reductase and thioredoxin. Our results indicate that Prx reduction was inhibited. We observed rapid inhibition of purified thioredoxin reductase (half-time 5 min with 2 µM adenanthin) and in cells, thioredoxin reductase was much more sensitive than GSH and loss of both preceded accumulation of oxidized Prxs. Thus, adenanthin is not a specific Prx inhibitor, and its reported antitumor and anti-inflammatory effects are more likely to involve more general inhibition of thioredoxin and/or glutathione redox pathways.

Increased thrombin-induced polymerization of fibrinogen associated with high protein carbonyl levels in plasma from patients post myocardial infarction.

Increased levels of protein carbonyls have been measured in plasma of patients following a myocardial infarction (Mocatta et al. J. Am. Coll. Cardiol.49:1993-2000; 2007). In this study, we have examined representative plasma samples from this group of patients. We show that carbonyls are formed preferentially on fibrinogen and that there is a strong correlation between fibrinogen and total plasma protein carbonyls. Functional properties of fibrinogen isolated from post myocardial plasmas were investigated by measuring thrombin-catalyzed polymerization. Fibrinogen from plasma with upper quartile protein carbonyls (mean 0.16 nmol/mg protein) polymerized approximately 1.4 times more rapidly and gave 1.4-fold higher maximum turbidity (12 per group, P<0.001) than fibrinogen from lower quartile carbonyl plasma (mean 0.007 nmol/mg), which behaved similarly to control plasma. Significant differences were also apparent when related to the carbonyl content of the fibrinogen itself. These changes in the high carbonyl plasma reflect a faster rate of lateral aggregation of small oligomers to form fibrin polymers that comprise thicker, more loosely woven fibers. In vivo this could be translated into a tendency to clot faster and form more fragile clots. High protein carbonyls in fibrinogen were not associated with an increased content of nitrotyrosine or chlorotyrosine. Nitrotyrosine levels were significantly lower in fibrinogen than total plasma protein, with no difference between patients and controls. Chlorotyrosine levels in fibrinogen (but not total protein) were significantly higher for the post myocardial patients. Our data suggest that high fibrinogen protein carbonyls in heart disease could be a prothrombotic risk factor.

Investigations into charge heterogeneity of wool intermediate filament proteins.

Genomic studies have shown that there are four abundant type I and type II intermediate filament proteins (IFPs) in wool. When separated using 2D-PAGE, the type I IFPs separated into four clearly defined major rows. The type II IFPs separated into two distinct staggered rows. The large number of spots seen by 2D-PAGE has previously been attributed to charge heterogeneity caused by post-translational modification of the protein. However, analysis of wool IFPs by 2D-PAGE techniques and mass spectrometry suggested an absence of phosphorylation or glycosylation modifications. Investigations with both the type I and type II IFPs showed that when single protein spots from a 2D-PAGE separation are eluted, re-focused and re-electrophoresed, several spots are formed on both the acidic and basic side of the original spot. Amino acid analysis, mass spectrometry and Ellman's assay support the hypothesis that the proteins have the same sequence but vary in isoelectric charge, due to differences in exposure of charged residues on the molecular surface. The cause of IFP charge heterogeneity is thus proposed to be a conformational equilibrium between several different forms of the same protein in the rehydration solution used for the first dimension.

Two-dimensional gel electrophoresis of wool intermediate filament proteins.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods have been used to provide high-resolution separation of wool intermediate filament proteins (IFPs). An improved method of extraction was developed based on a previously published method. The improved method for extraction eliminates the use of dialysis and freeze-drying between the extraction and rehydration steps, allowing the extraction and rehydration for the first dimension gel to be achieved in one day. Improvements to the method for maintaining reducing conditions and chaotrope constitution, combined with low %T polyacrylamide gels, allowed the high-resolution separation of the two keratin IFP families and their individual family members. The IFPs were separated to produce a clearly defined spot pattern of higher intensity, with numerous minor spots not previously observed, and a marked improvement in the vertical resolution. Further work to analyse the composition of each of the protein spots has been made much easier by being able to separate the IFPs into discrete spots.

The differential expression of proteins in the cortical cells of wool and hair fibres.

Three different cell types have been identified in the cortex of wool: orthocortex, mesocortex and paracortex. Fine wool fibres, particularly Merino sheep, are noted for their bilateral distribution of orthocortical and paracortical cells, with the latter following the concave side of the crimp wave. Furthermore, studies have indicated that the paracortex has a higher concentration of cysteine than the orthocortex. This has been supported by in situ hybridization studies in the follicle that have shown that sulphur-rich proteins are initially expressed on the paracortical side of the fibre, with some becoming more uniformly spread, laterally, over the entire fibre as the keratinization process progresses. In contrast, proteins high in glycine and tyrosine tend to be expressed initially on the orthocortical side of the follicle. While these in vitro studies have pointed to where specific proteins are located in the follicle, elucidating the situation for the mature fibre has been less easy. A range of approaches have been used to separate orthocortical and paracortical cells and these have only been able to provide evidence for a higher level of cysteine in the latter. Electrophoretic studies have found a number of differences in protein expression between the two sides but have not specifically identified which proteins. Thus, there appears to be good evidence for the paracortex containing a higher proportion of proteins in the ultra-high sulphur class but there is some uncertainty regarding the exact distribution of proteins high in glycine and tyrosine.

The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents.

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.

The effect of oxidation or alkylation on the separation of wool keratin proteins by two-dimensional gel electrophoresis.

Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new spots appeared at more acidic pIs. Mass spectral studies indicated that cysteine alkylation was relatively fast, with 70-95% of the cysteines in the keratin proteins being alkylated within the first 10 min, while in the case of the HGTPs there was evidence for noncysteine alkylation occurring within this period. Alkylation of proteins for periods of up to 6 h prior to electrofocusing is being promoted as a better alternative to the current 2-DE protocol of the inclusion of a reductant in the immobilized pH gradient rehydration solution. This study has clearly demonstrated that long alkylation times do not suit all protein types or classes.