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Jay W Heinecke - One of the best experts on this subject based on the ideXlab platform.
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High density lipoprotein is targeted for oxidation by myeloperoxidase in rheumatoid arthritis
Annals of the rheumatic diseases, 2013Co-Authors: Anuradha Vivekanandan-giri, Jaeman Byun, Jay W Heinecke, Jessica L. Slocum, Chongren Tang, Robin L. Sands, Brenda W. Gillespie, Rajiv Saran, Mariana J. Kaplan, Subramaniam PennathurAbstract:Objective Phagocyte-derived myeloperoxidase (MPO) and pro-inflammatory high density lipoprotein (HDL) associate with rheumatoid arthritis (RA), but the link between MPO and HDL has not been systematically examined. In this study, we investigated whether MPO can oxidise HDL and determined MPO-specific oxidative signature by apoA-1 by peptide mapping in RA subjects with and without known cardiovascular disease (CVD). Methods Two MPO oxidation products, 3-Chlorotyrosine and 3-nitrotyrosine, were quantified by tandem mass spectrometry (MS/MS) in in vitro model system studies and in plasma and HDL derived from healthy controls and RA subjects. MPO levels and cholesterol efflux were determined. Site-specific nitration and chlorination of apoA-1 peptides were quantified by MS/MS. Results RA subjects demonstrated higher levels of MPO, MPO-oxidised HDL and diminished cholesterol efflux. There was marked increase in MPO-specific 3-Chlorotyrosine and 3-nitrotyrosine content in HDL in RA subjects consistent with specific targeting of HDL, with increased nitration in RA subjects with CVD. Cholesterol efflux capacity was diminished in RA subjects and correlated inversely with HDL 3-Chlorotyrosine suggesting a mechanistic role for MPO. Nitrated HDL was elevated in RACVD subjects compared with RA subjects without CVD. Oxidative peptide mapping revealed site-specific unique oxidation signatures on apoA-1 for RA subjects with and without CVD. Conclusions We report an increase in MPO-mediated HDL oxidation that is regiospecific in RA and accentuated in those with CVD. Decreased cholesterol efflux capacity due to MPO-mediated chlorination is a potential mechanism for atherosclerosis in RA and raises the possibility that oxidant resistant forms of HDL may attenuate this increased risk.
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myeloperoxidase impairs abca1 dependent cholesterol efflux through methionine oxidation and site specific tyrosine chlorination of apolipoprotein a i
Journal of Biological Chemistry, 2006Co-Authors: Baohai Shao, Constanze Bergt, Pattie S Green, Xiaoyun Fu, Nathan Brot, John F Oram, Jay W HeineckeAbstract:Abstract High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-Chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-Chlorotyrosine in vivo, indicating that MPO oxidizes HDL in humans. We previously reported that Tyr-192 is the major site that is chlorinated in apolipoprotein A-I (apoA-I), the chief protein in HDL, and that chlorinated apoA-I loses its ability to promote cholesterol efflux from cells by the ATP-binding cassette transporter A1 (ABCA1) pathway. However, the pathways that promote the chlorination of specific Tyr residues in apoA-I are controversial, and the mechanism for MPO-mediated loss of ABCA1-dependent cholesterol efflux of apoA-I is unclear. Using site-directed mutagenesis, we now demonstrate that lysine residues direct tyrosine chlorination in apoA-I. Importantly, methionine residues inhibit chlorination, indicating that they can act as local, protein-bound antioxidants. Moreover, we observed near normal cholesterol efflux activity when Tyr-192 of apoA-I was mutated to Phe and the oxidized protein was incubated with methionine sulfoxide reductase. Thus, a combination of Tyr-192 chlorination and methionine oxidation is necessary for depriving apoA-I of its ABCA1-dependent cholesterol transport activity. Our observations suggest that biologically significant oxidative damage of apoA-I involves modification of a limited number of specific amino acids, raising the feasibility of producing oxidation-resistant forms of apoA-I that have enhanced anti-atherogenic activity in vivo.
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ablation of the inflammatory enzyme myeloperoxidase mitigates features of parkinson s disease in mice
The Journal of Neuroscience, 2005Co-Authors: Dongkug Choi, Subramaniam Pennathur, Celine Perier, Kim Tieu, Peter Teismann, Du Chu Wu, Vernice Jacksonlewis, Miquel Vila, Jeanpaul G Vonsattel, Jay W HeineckeAbstract:Parkinson's disease (PD) is characterized by a loss of ventral midbrain dopaminergic neurons, which can be modeled by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Inflammatory oxidants have emerged as key contributors to PD- and MPTP-related neurodegeneration. Here, we show that myeloperoxidase (MPO), a key oxidant-producing enzyme during inflammation, is upregulated in the ventral midbrain of human PD and MPTP mice. We also show that ventral midbrain dopaminergic neurons of mutant mice deficient in MPO are more resistant to MPTP-induced cytotoxicity than their wild-type littermates. Supporting the oxidative damaging role of MPO in this PD model are the demonstrations that MPO-specific biomarkers 3-Chlorotyrosine and hypochlorous acid-modified proteins increase in the brains of MPTP-injected mice. This study demonstrates that MPO participates in the MPTP neurotoxic process and suggests that inhibitors of MPO may provide a protective benefit in PD.
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tyrosine 192 in apolipoprotein a i is the major site of nitration and chlorination by myeloperoxidase but only chlorination markedly impairs abca1 dependent cholesterol transport
Journal of Biological Chemistry, 2005Co-Authors: Baohai Shao, Constanze Bergt, Pattie S Green, John F Oram, John C Voss, Michael N Oda, Jay W HeineckeAbstract:High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-Chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-Chlorotyrosine in humans, indicating that MPO oxidizes HDL in vivo. In the current studies, we used tandem mass spectrometry to identify the major sites of tyrosine oxidation when lipid-free apolipoprotein A-I (apoA-I), the major protein of HDL, was exposed to MPO or peroxynitrite (ONOO(-)). Tyrosine 192 was the predominant site of both nitration and chlorination by MPO and was also the major site of nitration by ONOO(-). Electron paramagnetic spin resonance studies of spin-labeled apoA-I revealed that residue 192 was located in an unusually hydrophilic environment. Moreover, the environment of residue 192 became much more hydrophobic when apoA-I was incorporated into discoidal HDL, and Tyr(192) of HDL-associated apoA-I was a poor substrate for nitration by both myeloperoxidase and ONOO(-), suggesting that solvent accessibility accounted in part for the reactivity of Tyr(192). The ability of lipid-free apoA-I to facilitate ATP-binding cassette transporter A1 cholesterol transport was greatly reduced after chlorination by MPO. Loss of activity occurred in concert with chlorination of Tyr(192). Both ONOO(-) and MPO nitrated Tyr(192) in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr(192) is the predominant site of nitration and chlorination when MPO or ONOO(-) oxidizes lipid-free apoA-I but that only chlorination markedly reduces the cholesterol efflux activity of apoA-I. This impaired biological activity of chlorinated apoA-I suggests that MPO-mediated oxidation of HDL might contribute to the link between inflammation and cardiovascular disease.
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human neutrophils use the myeloperoxidase hydrogen peroxide chloride system to chlorinate but not nitrate bacterial proteins during phagocytosis
Journal of Biological Chemistry, 2002Co-Authors: Henry Rosen, Jan R Crowley, Jay W HeineckeAbstract:Abstract The generation of extracellular oxidants by neutrophils has been widely investigated, but knowledge about the chemical reactions that occur in the phagolysosome, the cellular compartment that kills pathogens, is more limited. One important pathway may involve the production of potent halogenating agents such as hypochlorous acid (HOCl) by the myeloperoxidase-hydrogen peroxide-halide system. However, explorations of the oxidation chemistry of phagolysosomes have been hampered by the organelle's inaccessibility. To overcome this limitation, we recoveredEscherichia coli that had been internalized by human neutrophils. We then analyzed the bacterial proteins for 3-Chlorotyrosine, a stable marker of damage by HOCl. Mass spectrometric analysis revealed that levels of 3-Chlorotyrosine in E. coli proteins increased markedly after the bacteria were internalized by human neutrophils. This increase failed to occur inE. coli exposed to neutrophils deficient in NADPH oxidase or myeloperoxidase, implicating H2O2 and myeloperoxidase in the halogenation reaction. The extent of protein chlorination by normal neutrophils paralleled bacterial killing. Our observations support the view that the phagolysosome of human neutrophils uses the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate bacterial proteins. In striking contrast, human neutrophils failed to nitrate bacterial proteins unless the medium was supplemented with 1 mm nitrite, and the level of nitration was low. Protein chlorination associated with bacterial killing was unaffected by the presence of nitrite in the medium. Nitration required NADPH oxidase but appeared to be independent of myeloperoxidase, suggesting that neutrophils can nitrate proteins through a pathway that requires nitrite but is independent of myeloperoxidase.
Anthony J Kettle - One of the best experts on this subject based on the ideXlab platform.
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Myeloperoxidase and Protein Oxidation in the Airways of Young Children with Cystic Fibrosis
2013Co-Authors: Anthony J Kettle, Anna L.p. Chapman, Revathy Senthilmohan, Timothy Chan, Iris Osberg, Tessa J. Mocatta, Jeffrey S. WagenerAbstract:Cystic fibrosis (CF) is characterized by considerable oxidative stress. However, it is not known whether oxidative stress is an important feature early in this disease. We have investigated a group of infants and young children with CF to establish whether oxidants are produced in their airways. Bronchoalveolar lavage fluid (BALF) was assayed for myeloperoxidase as a measure of neutrophilic inflammation, and 3-Chlorotyrosine as a biomarker of the potent oxidant hypochlorous acid, which is formed by myeloperoxidase. Protein carbonyls were also measured as a nonspecific indicator of reactive oxidant production. Myeloperoxidase and 3-Chlorotyrosine levels in BALF from children with CF were 10- and fivefold higher, respectively, than in disease control subjects. There was a strong correlation between myeloperoxidase and 3-Chlorotyrosine. Myeloperoxidase levels were fourfold higher in children with infections in their airways. Median protein carbonyls were elevated by only twofold compared with disease control subjects, but some children had extremely high levels of protein oxidation. We conclude that hypochlorous acid is produced early in CF and that it is a candidate for precipitating the fatal decline in lung function associated with this disease. Also, there must be other sources of oxidants because protein carbonyls were not related to either inflammation or infection
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Measuring chlorine bleach in biology and medicine.
Biochimica et biophysica acta, 2013Co-Authors: Anthony J Kettle, Anna L.p. Chapman, Irada Khalilova, Amelia M. Albrett, Nina Dickerhof, Louisa V. Forbes, Rufus TurnerAbstract:Abstract Background Chlorine bleach, or hypochlorous acid, is the most reactive two-electron oxidant produced in appreciable amounts in our bodies. Neutrophils are the main source of hypochlorous acid. These champions of the innate immune system use it to fight infection but also direct it against host tissue in inflammatory diseases. Neutrophils contain a rich supply of the enzyme myeloperoxidase. It uses hydrogen peroxide to convert chloride to hypochlorous acid. Scope of review We give a critical appraisal of the best methods to measure production of hypochlorous acid by purified peroxidases and isolated neutrophils. Robust ways of detecting it inside neutrophil phagosomes where bacteria are killed are also discussed. Special attention is focused on reaction-based fluorescent probes but their visual charm is tempered by stressing their current limitations. Finally, the strengths and weaknesses of biomarker assays that capture the footprints of chlorine in various pathologies are evaluated. Major conclusions Detection of hypochlorous acid by purified peroxidases and isolated neutrophils is best achieved by measuring accumulation of taurine chloramine. Formation of hypochlorous acid inside neutrophil phagosomes can be tracked using mass spectrometric analysis of 3-Chlorotyrosine and methionine sulfoxide in bacterial proteins, or detection of chlorinated fluorescein on ingestible particles. Reaction-based fluorescent probes can also be used to monitor hypochlorous acid during phagocytosis. Specific biomarkers of its formation during inflammation include 3-Chlorotyrosine, chlorinated products of plasmalogens, and glutathione sulfonamide. General significance These methods should bring new insights into how chlorine bleach is produced by peroxidases, reacts within phagosomes to kill bacteria, and contributes to inflammation. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
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myeloperoxidase and oxidative stress in rheumatoid arthritis
Rheumatology, 2012Co-Authors: Lisa K Stamp, Revathy Senthilmohan, Irada Khalilova, Joanna M Tarr, Rufus Turner, Richard Haigh, Paul G Winyard, Anthony J KettleAbstract:Objective. To determine whether MPO contributes to oxidative stress and disease activity in RA and whether it produces hypochlorous acid in SF. Methods. Plasma and where possible SF were collected from 77 RA patients while 120 healthy controls supplied plasma only. MPO and protein carbonyls were measured by ELISAs. 3-Chlorotyrosine in proteins and allantoin in plasma were measured by mass spectrometry. Results. Plasma MPO concentrations were significantly higher in patients with RA compared with healthy controls [10.8 ng/ml, inter-quartile range (IQR): 7.214.2; P 3.2) and those with low disease activity (LDA; DAS-28 43.2) (HDA 27.9 ng/ml, 20.234.1 vs LDA 22.1 ng/ml, 16.934.9; P > 0.05). There was a significant relationship between plasma MPO and DAS-28 (r = 0.35; P = 0.005). Plasma protein carbonyls and allantoin were significantly higher in patients with RA compared with the healthy controls. MPO protein was significantly higher in SF compared with plasma (median 624.0 ng/ml, IQR 258.42433.0 vs 30.2 ng/ml, IQR 25.150.9; P < 0.0001). The MPO present in SF was mostly active. 3-Chlorotyrosine, a specific biomarker of hypochlorous acid, was present in proteins from SF and related to the concentration of MPO (r = 0.69; P = 0.001). Protein carbonyls in SF were associated with MPO protein concentration (r = 0.40; P = 0.019) and 3-Chlorotyrosine (r = 0.66; P = 0.003).
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Myeloperoxidase and protein oxidation in the airways of young children with cystic fibrosis.
American journal of respiratory and critical care medicine, 2004Co-Authors: Anthony J Kettle, Anna L.p. Chapman, Revathy Senthilmohan, Timothy Chan, Iris Osberg, Tessa J. Mocatta, Jeffrey S. WagenerAbstract:Cystic fibrosis (CF) is characterized by considerable oxidative stress. However, it is not known whether oxidative stress is an important feature early in this disease. We have investigated a group of infants and young children with CF to establish whether oxidants are produced in their airways. Bronchoalveolar lavage fluid (BALF) was assayed for myeloperoxidase as a measure of neutrophilic inflammation, and 3-Chlorotyrosine as a biomarker of the potent oxidant hypochlorous acid, which is formed by myeloperoxidase. Protein carbonyls were also measured as a nonspecific indicator of reactive oxidant production. Myeloperoxidase and 3-Chlorotyrosine levels in BALF from children with CF were 10- and fivefold higher, respectively, than in disease control subjects. There was a strong correlation between myeloperoxidase and 3-Chlorotyrosine. Myeloperoxidase levels were fourfold higher in children with infections in their airways. Median protein carbonyls were elevated by only twofold compared with disease control...
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3 Chlorotyrosine as a marker of protein damage by myeloperoxidase in tracheal aspirates from preterm infants association with adverse respiratory outcome
Pediatric Research, 2003Co-Authors: Hendrikje I Buss, Anthony J Kettle, Revathy Senthilmohan, Brian A Darlow, N Mogridge, Christine C WinterbournAbstract:Oxidative injury is implicated in the development of chronic lung disease in preterm infants with respiratory distress. However, direct evidence of a causal role is limited and the source of reactive oxidants has not been identified. We have previously shown that protein carbonyl levels in tracheal aspirates correlate positively with myeloperoxidase, suggesting that neutrophil oxidants could be the source of this protein injury. We have extended these observations by measuring 3-Chlorotyrosine, a specific biomarker of the neutrophil oxidant, hypochlorous acid, in tracheal aspirate proteins (144 samples) from 69 infants with birth weight <1500 g. 3-Chlorotyrosine levels were higher in these infants than in larger infants without respiratory distress (median 83 compared with 13 μmol/mol tyrosine). They correlated strongly with myeloperoxidase activity (correlation coefficient 0.75, p < 0.0001) and to a lesser extent with protein carbonyls. 3-Chlorotyrosine levels (at 1 wk after birth) correlated negatively with birth weight or gestational age. They were significantly higher in infants who developed chronic lung disease (oxygen requirement at 36 wk postmenstrual age) than in those who did not (median 88 and 49 μmol/mol tyrosine, respectively) and correlated with days of supplemental oxygen. 3-Chlorotyrosine was also significantly higher in infants who had lung infection or were Ureaplasma urealyticum positive. Our results are the first evidence that chlorinated proteins are produced in the lungs of premature infants and that they are higher in infection. The higher 3-Chlorotyrosine levels in infants who develop chronic lung disease suggest that neutrophil oxidants contribute to the pathology of this disease.
Revathy Senthilmohan - One of the best experts on this subject based on the ideXlab platform.
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Myeloperoxidase and Protein Oxidation in the Airways of Young Children with Cystic Fibrosis
2013Co-Authors: Anthony J Kettle, Anna L.p. Chapman, Revathy Senthilmohan, Timothy Chan, Iris Osberg, Tessa J. Mocatta, Jeffrey S. WagenerAbstract:Cystic fibrosis (CF) is characterized by considerable oxidative stress. However, it is not known whether oxidative stress is an important feature early in this disease. We have investigated a group of infants and young children with CF to establish whether oxidants are produced in their airways. Bronchoalveolar lavage fluid (BALF) was assayed for myeloperoxidase as a measure of neutrophilic inflammation, and 3-Chlorotyrosine as a biomarker of the potent oxidant hypochlorous acid, which is formed by myeloperoxidase. Protein carbonyls were also measured as a nonspecific indicator of reactive oxidant production. Myeloperoxidase and 3-Chlorotyrosine levels in BALF from children with CF were 10- and fivefold higher, respectively, than in disease control subjects. There was a strong correlation between myeloperoxidase and 3-Chlorotyrosine. Myeloperoxidase levels were fourfold higher in children with infections in their airways. Median protein carbonyls were elevated by only twofold compared with disease control subjects, but some children had extremely high levels of protein oxidation. We conclude that hypochlorous acid is produced early in CF and that it is a candidate for precipitating the fatal decline in lung function associated with this disease. Also, there must be other sources of oxidants because protein carbonyls were not related to either inflammation or infection
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myeloperoxidase and oxidative stress in rheumatoid arthritis
Rheumatology, 2012Co-Authors: Lisa K Stamp, Revathy Senthilmohan, Irada Khalilova, Joanna M Tarr, Rufus Turner, Richard Haigh, Paul G Winyard, Anthony J KettleAbstract:Objective. To determine whether MPO contributes to oxidative stress and disease activity in RA and whether it produces hypochlorous acid in SF. Methods. Plasma and where possible SF were collected from 77 RA patients while 120 healthy controls supplied plasma only. MPO and protein carbonyls were measured by ELISAs. 3-Chlorotyrosine in proteins and allantoin in plasma were measured by mass spectrometry. Results. Plasma MPO concentrations were significantly higher in patients with RA compared with healthy controls [10.8 ng/ml, inter-quartile range (IQR): 7.214.2; P 3.2) and those with low disease activity (LDA; DAS-28 43.2) (HDA 27.9 ng/ml, 20.234.1 vs LDA 22.1 ng/ml, 16.934.9; P > 0.05). There was a significant relationship between plasma MPO and DAS-28 (r = 0.35; P = 0.005). Plasma protein carbonyls and allantoin were significantly higher in patients with RA compared with the healthy controls. MPO protein was significantly higher in SF compared with plasma (median 624.0 ng/ml, IQR 258.42433.0 vs 30.2 ng/ml, IQR 25.150.9; P < 0.0001). The MPO present in SF was mostly active. 3-Chlorotyrosine, a specific biomarker of hypochlorous acid, was present in proteins from SF and related to the concentration of MPO (r = 0.69; P = 0.001). Protein carbonyls in SF were associated with MPO protein concentration (r = 0.40; P = 0.019) and 3-Chlorotyrosine (r = 0.66; P = 0.003).
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plasma concentrations of myeloperoxidase predict mortality after myocardial infarction
Journal of the American College of Cardiology, 2007Co-Authors: Tessa J. Mocatta, Revathy Senthilmohan, Anna P Pilbrow, Vicky A Cameron, Chris Frampton, Mark A Richards, Christine C WinterbournAbstract:Objectives This study investigated relationships between plasma myeloperoxidase (MPO), protein oxidation markers, and clinical outcome retrospectively in patients after acute myocardial infarction (MI). Background Reactive oxidants are implicated in cardiovascular disease, and elevated plasma MPO is reported to predict adverse outcome in acute coronary syndromes. Methods Detailed demographic information, radionuclide ventriculography, neurohormone measurements, and clinical history were obtained for 512 acute MI patients at hospital admission. Plasma levels of MPO and protein carbonyls were measured in patients and 156 heart-healthy control subjects. 3-Chlorotyrosine was measured in selected patients. Patient mortality was followed for 5 years. Results Plasma MPO and protein carbonyl concentrations were higher in MI patients 24 h to 96 h after admission than in control subjects (medians: MPO 55 ng/ml vs. 39 ng/ml, and protein carbonyls 48 pmol/mg vs. 17 pmol/mg protein, p Conclusions Myeloperoxidase and protein carbonyl levels are elevated in plasma after acute MI, apparently via independent mechanisms. High MPO is a risk factor for long-term mortality and adds prognostic value to LVEF and plasma NT-proBNP measurements.
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Myeloperoxidase and protein oxidation in the airways of young children with cystic fibrosis.
American journal of respiratory and critical care medicine, 2004Co-Authors: Anthony J Kettle, Anna L.p. Chapman, Revathy Senthilmohan, Timothy Chan, Iris Osberg, Tessa J. Mocatta, Jeffrey S. WagenerAbstract:Cystic fibrosis (CF) is characterized by considerable oxidative stress. However, it is not known whether oxidative stress is an important feature early in this disease. We have investigated a group of infants and young children with CF to establish whether oxidants are produced in their airways. Bronchoalveolar lavage fluid (BALF) was assayed for myeloperoxidase as a measure of neutrophilic inflammation, and 3-Chlorotyrosine as a biomarker of the potent oxidant hypochlorous acid, which is formed by myeloperoxidase. Protein carbonyls were also measured as a nonspecific indicator of reactive oxidant production. Myeloperoxidase and 3-Chlorotyrosine levels in BALF from children with CF were 10- and fivefold higher, respectively, than in disease control subjects. There was a strong correlation between myeloperoxidase and 3-Chlorotyrosine. Myeloperoxidase levels were fourfold higher in children with infections in their airways. Median protein carbonyls were elevated by only twofold compared with disease control...
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3 Chlorotyrosine as a marker of protein damage by myeloperoxidase in tracheal aspirates from preterm infants association with adverse respiratory outcome
Pediatric Research, 2003Co-Authors: Hendrikje I Buss, Anthony J Kettle, Revathy Senthilmohan, Brian A Darlow, N Mogridge, Christine C WinterbournAbstract:Oxidative injury is implicated in the development of chronic lung disease in preterm infants with respiratory distress. However, direct evidence of a causal role is limited and the source of reactive oxidants has not been identified. We have previously shown that protein carbonyl levels in tracheal aspirates correlate positively with myeloperoxidase, suggesting that neutrophil oxidants could be the source of this protein injury. We have extended these observations by measuring 3-Chlorotyrosine, a specific biomarker of the neutrophil oxidant, hypochlorous acid, in tracheal aspirate proteins (144 samples) from 69 infants with birth weight <1500 g. 3-Chlorotyrosine levels were higher in these infants than in larger infants without respiratory distress (median 83 compared with 13 μmol/mol tyrosine). They correlated strongly with myeloperoxidase activity (correlation coefficient 0.75, p < 0.0001) and to a lesser extent with protein carbonyls. 3-Chlorotyrosine levels (at 1 wk after birth) correlated negatively with birth weight or gestational age. They were significantly higher in infants who developed chronic lung disease (oxygen requirement at 36 wk postmenstrual age) than in those who did not (median 88 and 49 μmol/mol tyrosine, respectively) and correlated with days of supplemental oxygen. 3-Chlorotyrosine was also significantly higher in infants who had lung infection or were Ureaplasma urealyticum positive. Our results are the first evidence that chlorinated proteins are produced in the lungs of premature infants and that they are higher in infection. The higher 3-Chlorotyrosine levels in infants who develop chronic lung disease suggest that neutrophil oxidants contribute to the pathology of this disease.
Christine C Winterbourn - One of the best experts on this subject based on the ideXlab platform.
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plasma concentrations of myeloperoxidase predict mortality after myocardial infarction
Journal of the American College of Cardiology, 2007Co-Authors: Tessa J. Mocatta, Revathy Senthilmohan, Anna P Pilbrow, Vicky A Cameron, Chris Frampton, Mark A Richards, Christine C WinterbournAbstract:Objectives This study investigated relationships between plasma myeloperoxidase (MPO), protein oxidation markers, and clinical outcome retrospectively in patients after acute myocardial infarction (MI). Background Reactive oxidants are implicated in cardiovascular disease, and elevated plasma MPO is reported to predict adverse outcome in acute coronary syndromes. Methods Detailed demographic information, radionuclide ventriculography, neurohormone measurements, and clinical history were obtained for 512 acute MI patients at hospital admission. Plasma levels of MPO and protein carbonyls were measured in patients and 156 heart-healthy control subjects. 3-Chlorotyrosine was measured in selected patients. Patient mortality was followed for 5 years. Results Plasma MPO and protein carbonyl concentrations were higher in MI patients 24 h to 96 h after admission than in control subjects (medians: MPO 55 ng/ml vs. 39 ng/ml, and protein carbonyls 48 pmol/mg vs. 17 pmol/mg protein, p Conclusions Myeloperoxidase and protein carbonyl levels are elevated in plasma after acute MI, apparently via independent mechanisms. High MPO is a risk factor for long-term mortality and adds prognostic value to LVEF and plasma NT-proBNP measurements.
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3 Chlorotyrosine as a marker of protein damage by myeloperoxidase in tracheal aspirates from preterm infants association with adverse respiratory outcome
Pediatric Research, 2003Co-Authors: Hendrikje I Buss, Anthony J Kettle, Revathy Senthilmohan, Brian A Darlow, N Mogridge, Christine C WinterbournAbstract:Oxidative injury is implicated in the development of chronic lung disease in preterm infants with respiratory distress. However, direct evidence of a causal role is limited and the source of reactive oxidants has not been identified. We have previously shown that protein carbonyl levels in tracheal aspirates correlate positively with myeloperoxidase, suggesting that neutrophil oxidants could be the source of this protein injury. We have extended these observations by measuring 3-Chlorotyrosine, a specific biomarker of the neutrophil oxidant, hypochlorous acid, in tracheal aspirate proteins (144 samples) from 69 infants with birth weight <1500 g. 3-Chlorotyrosine levels were higher in these infants than in larger infants without respiratory distress (median 83 compared with 13 μmol/mol tyrosine). They correlated strongly with myeloperoxidase activity (correlation coefficient 0.75, p < 0.0001) and to a lesser extent with protein carbonyls. 3-Chlorotyrosine levels (at 1 wk after birth) correlated negatively with birth weight or gestational age. They were significantly higher in infants who developed chronic lung disease (oxygen requirement at 36 wk postmenstrual age) than in those who did not (median 88 and 49 μmol/mol tyrosine, respectively) and correlated with days of supplemental oxygen. 3-Chlorotyrosine was also significantly higher in infants who had lung infection or were Ureaplasma urealyticum positive. Our results are the first evidence that chlorinated proteins are produced in the lungs of premature infants and that they are higher in infection. The higher 3-Chlorotyrosine levels in infants who develop chronic lung disease suggest that neutrophil oxidants contribute to the pathology of this disease.
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3-Chlorotyrosine as a Marker of Protein Damage by Myeloperoxidase in Tracheal Aspirates From Preterm Infants: Association With Adverse Respiratory Outcome
Pediatric Research, 2003Co-Authors: I Hendrikje Buss, Anthony J Kettle, Revathy Senthilmohan, Brian A Darlow, N Mogridge, Christine C WinterbournAbstract:Oxidative injury is implicated in the development of chronic lung disease in preterm infants with respiratory distress. However, direct evidence of a causal role is limited and the source of reactive oxidants has not been identified. We have previously shown that protein carbonyl levels in tracheal aspirates correlate positively with myeloperoxidase, suggesting that neutrophil oxidants could be the source of this protein injury. We have extended these observations by measuring 3-Chlorotyrosine, a specific biomarker of the neutrophil oxidant, hypochlorous acid, in tracheal aspirate proteins (144 samples) from 69 infants with birth weight
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Biomarkers of myeloperoxidase-derived hypochlorous acid.
Free Radical Biology and Medicine, 2000Co-Authors: Christine C Winterbourn, Anthony J KettleAbstract:Abstract Hypochlorous acid is the major strong oxidant generated by neutrophils. The heme enzyme myeloperoxidase catalyzes the production of hypochlorous acid from hydrogen peroxide and chloride. Although myeloperoxidase has been implicated in the tissue damage that occurs in numerous diseases that involve inflammatory cells, it has proven difficult to categorically demonstrate that it plays a crucial role in any pathology. This situation should soon be rectified with the advent of sensitive biomarkers for hypochlorous acid. In this review, we outline the advantages and limitations of chlorinated tyrosines, chlorohydrins, 5-chlorocytosine, protein carbonyls, antibodies that recognize HOCl-treated proteins, and glutathione sulfonamide as potential biomarkers of hypochlorous acid. Levels of 3-Chlorotyrosine and 3,5-diChlorotyrosine are increased in proteins after exposure to low concentrations of hypochlorous acid and we conclude that their analysis by gas chromatography and mass spectrometry is currently the best method available for probing the involvement of oxidation by myeloperoxidase in the pathology of particular diseases. The appropriate use of other biomarkers should provide complementary information. Keywords—Free radicals, Myeloperoxidase, Neutrophil oxidant, Hypochlorous acid, Chlorotyrosine, Chlorohydrin, Oxidant biomarker
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Comparison of Mono- and Dichlorinated Tyrosines with Carbonyls for Detection of Hypochlorous Acid Modified Proteins
Archives of biochemistry and biophysics, 2000Co-Authors: Anna L.p. Chapman, Christine C Winterbourn, Revathy Senthilmohan, Anthony J KettleAbstract:Hypochlorous acid is a potent oxidant capable of oxidizing and chlorinating proteins. Based on its indiscriminant reactivity, it is proposed to play a major role in tissue damage associated with a range of inflammatory diseases. We have determined the relative tendencies for formation of protein carbonyls, chlorinated tyrosine residues, and epitopes recognized by an antibody raised against hypochlorous acid oxidized protein (HOP-1) when albumin is treated with hypochlorous acid. We have also tested the specificity of the HOP-1 antibody by measuring how effectively it recognizes proteins oxidized by hypobromous acid. 3-Chlorotyrosine, along with a new marker of hypochlorous acid dependent protein modification, 3, 5-diChlorotyrosine, was formed at the lowest doses of hypochlorous acid that were capable of generating protein carbonyls. Comparatively high doses of hypochlorous acid were needed to generate epitopes recognized by HOP-1, which were also produced by hypobromous acid. Our study demonstrates that it is advantageous to measure protein carbonyls and HOP-1 epitopes in conjunction with chlorinated tyrosines when attempting to identify the oxidants responsible for inflammatory tissue damage.
Steven G. Boxer - One of the best experts on this subject based on the ideXlab platform.
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genetic code expansion in rhodobacter sphaeroides to incorporate noncanonical amino acids into photosynthetic reaction centers
ACS Synthetic Biology, 2018Co-Authors: Jared Bryce Weaver, Steven G. BoxerAbstract:Photosynthetic reaction centers (RCs) are the membrane proteins responsible for the initial charge separation steps central to photosynthesis. As a complex and spectroscopically complicated membrane protein, the RC (and other associated photosynthetic proteins) would benefit greatly from the insight offered by site-specifically encoded noncanonical amino acids in the form of probes and an increased chemical range in key amino acid analogues. Toward that goal, we developed a method to transfer amber codon suppression machinery developed for E. coli into the model bacterium needed to produce RCs, Rhodobacter sphaeroides. Plasmids were developed and optimized to incorporate 3-Chlorotyrosine, 3-bromotyrosine, and 3-iodotyrosine into RCs. Multiple challenges involving yield and orthogonality were overcome to implement amber suppression in R. sphaeroides, providing insights into the hurdles that can be involved in host transfer of amber suppression systems from E. coli. In the process of verifying noncanonical ...
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Genetic Code Expansion in Rhodobacter sphaeroides to Incorporate Noncanonical Amino Acids into Photosynthetic Reaction Centers
2018Co-Authors: Jared Bryce Weaver, Steven G. BoxerAbstract:Photosynthetic reaction centers (RCs) are the membrane proteins responsible for the initial charge separation steps central to photosynthesis. As a complex and spectroscopically complicated membrane protein, the RC (and other associated photosynthetic proteins) would benefit greatly from the insight offered by site-specifically encoded noncanonical amino acids in the form of probes and an increased chemical range in key amino acid analogues. Toward that goal, we developed a method to transfer amber codon suppression machinery developed for E. coli into the model bacterium needed to produce RCs, Rhodobacter sphaeroides. Plasmids were developed and optimized to incorporate 3-Chlorotyrosine, 3-bromotyrosine, and 3-iodotyrosine into RCs. Multiple challenges involving yield and orthogonality were overcome to implement amber suppression in R. sphaeroides, providing insights into the hurdles that can be involved in host transfer of amber suppression systems from E. coli. In the process of verifying noncanonical amino acid incorporation, characterization of this membrane protein via mass spectrometry (which has been difficult previously) was substantially improved. Importantly, the ability to incorporate noncanonical amino acids in R. sphaeroides expands research capabilities in the photosynthetic field
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A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase
Journal of the American Chemical Society, 2016Co-Authors: Steven G. BoxerAbstract:The vibrational Stark effect (VSE) has been used to measure the electric field in the active site of ketosteroid isomerase (KSI). These measured fields correlate with ΔG⧧ in a series of conventional mutants, yielding an estimate for the electrostatic contribution to catalysis (Fried et al. Science 2014, 346, 1510–1513). In this work we test this result with much more conservative variants in which individual Tyr residues in the active site are replaced by 3-Chlorotyrosine via amber suppression. The electric fields sensed at the position of the carbonyl bond involved in charge displacement during catalysis were characterized using the VSE, where the field sensitivity has been calibrated by vibrational Stark spectroscopy, solvatochromism, and MD simulations. A linear relationship is observed between the electric field and ΔG⧧ that interpolates between wild-type and more drastic conventional mutations, reinforcing the evaluation of the electrostatic contribution to catalysis in KSI. A simplified model and ca...
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Dissecting Proton Delocalization in an Enzyme’s Hydrogen Bond Network with Unnatural Amino Acids
Biochemistry, 2015Co-Authors: Stephen D. Fried, Steven G. BoxerAbstract:Extended hydrogen bond networks are a common structural motif of enzymes. A recent analysis proposed quantum delocalization of protons as a feature present in the hydrogen bond network spanning a triad of tyrosines (Y16, Y32, and Y57) in the active site of ketosteroid isomerase (KSI), contributing to its unusual acidity and large isotope shift. In this study, we utilized amber suppression to substitute each tyrosine residue with 3-Chlorotyrosine to test the delocalization model and the proton affinity balance in the triad. X-ray crystal structures of each variant demonstrated that the structure, notably the O–O distances within the triad, was unaffected by 3-Chlorotyrosine substitutions. The changes in the cluster’s acidity and the acidity’s isotope dependence in these variants were assessed via UV–vis spectroscopy and the proton sharing pattern among individual residues with 13C nuclear magnetic resonance. Our data show pKa detuning at each triad residue alters the proton delocalization behavior in the H...
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Dissecting Proton Delocalization in an Enzyme’s Hydrogen Bond Network with Unnatural Amino Acids
2015Co-Authors: Stephen D. Fried, Steven G. BoxerAbstract:Extended hydrogen bond networks are a common structural motif of enzymes. A recent analysis proposed quantum delocalization of protons as a feature present in the hydrogen bond network spanning a triad of tyrosines (Y16, Y32, and Y57) in the active site of ketosteroid isomerase (KSI), contributing to its unusual acidity and large isotope shift. In this study, we utilized amber suppression to substitute each tyrosine residue with 3-Chlorotyrosine to test the delocalization model and the proton affinity balance in the triad. X-ray crystal structures of each variant demonstrated that the structure, notably the O–O distances within the triad, was unaffected by 3-Chlorotyrosine substitutions. The changes in the cluster’s acidity and the acidity’s isotope dependence in these variants were assessed via UV–vis spectroscopy and the proton sharing pattern among individual residues with 13C nuclear magnetic resonance. Our data show pKa detuning at each triad residue alters the proton delocalization behavior in the H-bond network. The extra stabilization energy necessary for the unusual acidity mainly comes from the strong interactions between Y57 and Y16. This is further enabled by Y32, which maintains the right geometry and matched proton affinity in the triad. This study provides a rich picture of the energetics of the hydrogen bond network in enzymes for further model refinement
