Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.     

Distinct HDL subclasses present similar intrinsic susceptibility to oxidation by HOCl

The heme protein myeloperoxidase (MPO) functions as a catalyst for lipoprotein oxidation. Hypochlorous acid (HOCl), a potent two-electron oxidant formed by the MPO-H₂O₂-chloride system of activated phagocytes, modifies antiatherogenic high-density lipoprotein (HDL). The structural heterogeneity and oxidative susceptibility of HDL particle subfractions were probed with HOCl. All distinct five HDL subfraction were modified by HOCl as demonstrated by the consumption of tryptophan residues and free amino groups, cross-linking of apolipoprotein AI, formation of HOCl-modified epitopes, increased electrophoretic mobility and altered content of unsaturated fatty acids in HDL subclasses. Small, dense HDL3 were less susceptible to oxidative modification than large, light HDL2 on a total mass basis at a fixed HOCl:HDL mass ratio of 1:32, but in contrast not on a particle number basis at a fixed HOCl:HDL molar ratio of 97:1. We conclude that structural and physicochemical differences between HDL subclasses do not influence their intrinsic susceptibility to oxidative attack by HOCl.     

Involvement of protein radical, protein aggregation, and effects on NO metabolism in the hypochlorite-mediated oxidation of mitochondrial cytochrome c

Cytochrome c (cyt c)-derived protein radicals, radical adduct aggregates, and protein tyrosine nitration have been implicated in the pro-apoptotic event connecting inflammation to the development of diseases. During inflammation, one of the reactive oxygen species metabolized via neutrophil activation is hypochlorite (HOCl); destruction of the mitochondrial electron transport chain by hypochlorite is considered to be a damaging factor. Previous study has shown that HOCl induces the site-specific oxidation of cyt c at met-80. In this work, we have assessed the hypothesis that exposure of cyt c to physiologically relevant concentrations of HOCl leads to protein-derived radical and consequent protein aggregation, which subsequently affects cyt c's regulation of nitric oxide metabolism. Reaction intermediates, chemical pathways available for protein aggregation, and protein nitration were examined. A weak ESR signal for immobilized nitroxide derived from the protein was detected when a high concentration of cyt c was reacted with hypochlorite in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane. When a low concentration of cyt c was exposed to the physiologically relevant levels of HOCl in the presence of 5,5-dimethyl-pyrroline N-oxide (DMPO), we detected DMPO nitrone adducts derived from both protein and protein aggregate radicals as assessed by Western blot using an antibody raised against the DMPO nitrone adduct. The cyt c-derived protein radicals formed by HOCl were located on lysine and tyrosine residues, with lysine predominating. Cyt c-derived protein aggregates induced by HOCl involved primarily lysine residues and hydrophobic interaction. In addition, HOCl-oxidized cyt c (HOCl-cyt c) exhibited a higher affinity for NO and enhancement of nonenzymatic NO synthesis from nitrite reduction. Furthermore, HOCl-mediated cyt c oxidation also resulted in a significant elevation of cyt c nitration derived from either NO trapping of the cyt c-derived tyrosyl radical or cyt c-catalyzed one-electron oxidation of nitrite.     

Protein chlorination in neutrophil phagosomes and correlation with bacterial killing

Neutrophils ingest and kill bacteria within phagocytic vacuoles. We investigated where they produce hypochlorous acid (HOCl) following phagocytosis by measuring conversion of protein tyrosine residues to 3-chlorotyrosine. We also examined how varying chloride availability affects the relationship between HOCl formation in the phagosome and bacterial killing. Phagosomal proteins, isolated following ingestion of opsonized magnetic beads, contained 11.4 Cl-Tyr per thousand tyrosine residues. This was 12 times higher than the level in proteins from the rest of the neutrophil and ~6 times higher than previously recorded for protein from ingested bacteria. These results indicate that HOCl production is largely localized to the phagosomes and a substantial proportion reacts with phagosomal protein before reaching the microbe. This will in part detoxify the oxidant but should also form chloramines which could contribute to the killing mechanism. Neutrophils were either suspended in chloride-free gluconate buffer or pretreated with formyl-Met-Leu-Phe, a procedure that has been reported to deplete intracellular chloride. These treatments, alone or in combination, decreased both chlorination in phagosomes and killing of Staphylococcus aureus by up to 50%. There was a strong positive correlation between the two effects. Killing was predominantly oxidant and myeloperoxidase dependent (88% inhibition by diphenylene iodonium and 78% by azide). These results imply that lowering the chloride concentration limits HOCl production and oxidative killing. They support a role for HOCl generation, rather than an alternative myeloperoxidase activity, in the killing process.     

Tyr740 and Tyr751 residues of platelet-derived growth factor beta receptor are responsible for the redox regulation of phosphatase and tensin homolog in the cells stimulated with platelet-derived growth factor

Exposure of cells to hydrogen peroxide or platelet-derived growth factor (PDGF) induced Akt phosphorylation and oxidation of phosphatase and tensin homolog (PTEN). The Cys124 and Cys71 residues of PTEN were critical for the formation of a disulfide bond and the intermediate glutathionylation in the process of reduction of the disulfide bond. To determine which specific tyrosine residues of the PDGF beta receptor (PDGFβR) is involved in PDGF-induced PTEN oxidation and Akt phosphorylation, we investigated a kinase activity-deficient mutant and PDGFβR mutants where the tyrosine residues in the binding site for phosphoinositide 3-kinase (PI3K), GTPase-activating protein of Ras, Src homology 2 domain containing protein-tyrosine phosphatase-2, and phospholipase C-1 were replaced by Phe. Both PTEN oxidation and Akt phosphorylation did not occur in response to PDGF in the kinase-deficient mutant and in the PDGFβR mutant with a mutation in the PI3K binding site (Tyr740 and Tyr751). Thus, the kinase activity and the constituent Tyr740 and Tyr751 residues of PDGFβR in the cells stimulated with PDGF are responsible for the oxidation of PTEN and the Akt phosphorylation.     

Development of novel mass spectrometric methods for identifying HOCl‐induced modifications to proteins

Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label‐free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl‐oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS² analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS³ fragment ions from the immonium ions and collisionally‐activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS³ fragment ions were also identified for 2‐hydroxytryptophan and 5‐hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.     

Oxidation of Cu,Zn-superoxide Dismutase by the Myeloperoxidase/Hydrogen Peroxide/Chloride System: Functional and Structural Effects

This study investigated the functional and structural effects of bovine Cu,Zn-superoxide dismutase (Cu,Zn-SOD) oxidation by the myeloperoxidase (MPO)/hydrogen peroxide (H 2 O 2 )/chloride system and reagent hypochlorous acid (HOCl). Exposure to HOCl led to a fast inactivation accompanied by structural alterations. The residual SOD activity depended on the reactants concentration ratio and on the exposure time. The concomitant high consumption of HOCl indicated the presence of multiple targets on the protein. As assessed by SDS/PAGE, HOCl caused the dissociation of the protein into protomers at 16 kDa stable to both SDS and reducing conditions. Results from isoelectric focusing gels showed that exposure to HOCl induced the formation of modified protein derivatives, with a more acidic net electric charge than the parent molecule, consistent with the presence of additional ions observed in the electrospray ionization mass spectra. The reaction of protein with HOCl resulted in changes in protein conformation as assessed by the UV fluorescence and oxidation of the unique methionine and tyrosine, chlorination of several lysines with formation of chloramines. There was no significant formation of dityrosine and carbonyl groups. Exposure to high levels of HOCl resulted in complete enzyme inactivation, loss of additional lysine, histidine and arginine residues and coincident detection of weakly bound zinc and copper using 4-pyridylazaresorcinol. Collectively, the results suggest that the decrease of the dismutase activity is probably related to both dissociation into protomers and unfolding due to extensive oxidative modifications of amino acids.     

Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous and hypobromous acids.

Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, catalyzes the oxidation of halides to hypohalous acids. At plasma concentrations of halides, hypochlorous acid (HOCl) is the major strong oxidant produced. In contrast, the related enzyme eosinophil peroxidase preferentially generates hypobromous acid (HOBr). Since reagent and MPO-derived HOCl converts low-density lipoprotein (LDL) to a potentially atherogenic form, we investigated the effects of HOBr on LDL modification. Compared to HOCl, HOBr caused 2-3-fold greater oxidation of tryptophan and cysteine residues of the protein moiety (apoB) of LDL and 4-fold greater formation of fatty acid halohydrins from the lipids in LDL. In contrast, HOBr was 2-fold less reactive than HOCl with lysine residues and caused little formation of N-bromamines. Nevertheless, HOBr caused an equivalent increase in the relative electrophoretic mobility of LDL as HOCl, which was not reversed upon subsequent incubation with ascorbate, in contrast to the shift in mobility caused by HOCl. Similar apoB modifications were observed with HOBr generated by MPO/H(2)O(2)/Br(-). In the presence of equivalent concentrations of Cl(-) and Br(-), modifications of LDL by MPO resembled those seen in the presence of Br(-) alone. Interestingly, even at physiological concentrations of the two halides (100 mM Cl(-), 100 microM Br(-)), MPO utilized a portion of the Br(-) to oxidize apoB cysteine residues. MPO also utilized the pseudohalide thiocyanate to oxidize apoB cysteine residues. Our data show that even though HOBr has different reactivities than HOCl with apoB, it is able to alter the charge of LDL, converting it into a potentially atherogenic particle.     

Oxidation of Cu,Zn-superoxide dismutase by the myeloperoxidase/hydrogen peroxide/chloride system: functional and structural effects

This study investigated the functional and structural effects of bovine Cu,Zn-superoxide dismutase (Cu,Zn-SOD) oxidation by the myeloperoxidase (MPO)/hydrogen peroxide (H 2 O 2 )/chloride system and reagent hypochlorous acid (HOCl). Exposure to HOCl led to a fast inactivation accompanied by structural alterations. The residual SOD activity depended on the reactants concentration ratio and on the exposure time. The concomitant high consumption of HOCl indicated the presence of multiple targets on the protein. As assessed by SDS/PAGE, HOCl caused the dissociation of the protein into protomers at 16 kDa stable to both SDS and reducing conditions. Results from isoelectric focusing gels showed that exposure to HOCl induced the formation of modified protein derivatives, with a more acidic net electric charge than the parent molecule, consistent with the presence of additional ions observed in the electrospray ionization mass spectra. The reaction of protein with HOCl resulted in changes in protein conformation as assessed by the UV fluorescence and oxidation of the unique methionine and tyrosine, chlorination of several lysines with formation of chloramines. There was no significant formation of dityrosine and carbonyl groups. Exposure to high levels of HOCl resulted in complete enzyme inactivation, loss of additional lysine, histidine and arginine residues and coincident detection of weakly bound zinc and copper using 4-pyridylazaresorcinol. Collectively, the results suggest that the decrease of the dismutase activity is probably related to both dissociation into protomers and unfolding due to extensive oxidative modifications of amino acids.     

Myeloperoxidase-dependent Inactivation of Surfactant Protein D in Vitro and in Vivo

Surfactant protein D (SP-D) plays diverse and important roles in innate immunity and pulmonary homeostasis. Neutrophils and myeloperoxidase (MPO) colocalized with SP-D in a murine bacterial pneumonia model of acute inflammation, suggesting that MPO-derived reactive species might alter the function of SP-D. Exposure of SP-D to the complete MPO-H₂O₂-halide system caused loss of SP-D-dependent aggregating activity. Hypochlorous acid (HOCl), the major oxidant generated by MPO, caused a similar loss of aggregating activity, which was accompanied by the generation of abnormal disulfide-cross-linked oligomers. A full-length SP-D mutant lacking N-terminal cysteine residues and truncation mutants lacking the N-terminal domains were resistant to the oxidant-induced alterations in disulfide bonding. Mass spectroscopy of HOCl-treated human SP-D demonstrated several modifications, but none involved key ligand binding residues. There was detectable oxidation of cysteine 15, but no HOCl-induced cysteine modifications were observed in the C-terminal lectin domain. Together, the findings localize abnormal disulfide cross-links to the N-terminal domain. MPO-deficient mice showed decreased cross-linking of SP-D and increased SP-D-dependent aggregating activity in the pneumonia model. Thus, MPO-derived oxidants can lead to modifications of SP-D structure with associated alterations in its characteristic aggregating activity.     

Collagen type II modification by hypochlorite

Oxidation of proteins is a common phenomenon in the inflammatory process mediated by highly reactive agents such as hypochlorite (HOCl/OCl(-)) produced by activated neutrophils. For instance, in rheumatoid arthritis hypochlorite plays an important role in joint destruction. One of the major targets for HOCl/OCl(-) is collagen type II (CII) - the primary cartilage protein. In our study, HOCl/OCl(-) mediated collagen II modifications were tested using various methods: circular dichroism (CD), HPLC, ELISA, dynamic light scattering (DLS), fluorimetry and spectrophotometry. It was shown that hypochlorite action causes deamination with consecutive carbonyl group formation and transformation of tyrosine residues to dichlorotyrosine. Moreover, it was shown that ammonium chloramine (NH(2)Cl) formed in the reaction mixture reacts with CII. However, in this case the yield of carbonyl groups and dichlorotyrosine is lower than that observed for HOCl/OCl(-) by 50%. CD data revealed that collagen II exists as a random coil in the samples and that chlorination is followed by CII fragmentation. In the range of low HOCl/OCl(-) concentrations (up to 1 mM) 10-90 kDa peptides are predominant whereas massive production of shorter peptides was observed for high (5 mM) hypochlorite concentration. DLS measurements showed that chlorination with HOCl/OCl(-) decreases the radius of collagen II aggregates from 30 to 6.8 nm. Taking into account the fact that chlorinated collagen is partially degraded, the DLS results suggest that smaller micelles are formed of the 10-90 kDa peptide fraction. Moreover, collagen chlorination results in epitope modification which affects CII recognition by anti-CII antibodies. Finally, since in the synovial fluid the plausible hypochlorite concentration is smaller than that used in the model the change of size of molecular aggregates seems to be the best marker of hypochlorite-mediated collagen oxidation.     

Methionine oxidation as a major cause of the functional impairment of oxidized actin

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimer's disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.     

Cytotoxic and genotoxic effects of tert-butyl hydroperoxide on Chinese hamster B14 cells

The organic hydroperoxide, tert-butyl hydroperoxide (t-BHP), is a useful model compound to study mechanisms of oxidative cell injury. In the present work, we examined the features of the interactions of this oxidant with Chinese hamster B14 cells. The aim of our study was to reveal a possible role of structural modifications in membranes and loss of DNA integrity in t-BHP-induced cell injury and death. The tert-butyl hydroperoxide treatment (100-1000 microM, 37 degrees C for 1h) did not decrease cell viability (as measured by cell-specific functional activity with an MTT test), but completely prevented cell growth. We observed intracellular reduced glutathione (GSH) oxidation and total glutathione (GSH+GSSG) depletion, a slight increase in the level of lipid-peroxidation products, an enhancement of membrane fluidity, intracellular potassium leakage and a significant decrease of membrane potential. At oxidant concentrations of 100-1500 microM, a significant damage to DNA integrity was observed as revealed by the Comet assay. The inhibition of cell proliferation (cell-growth arrest) may be explained by genotoxicity of t-BHP, by disturbance of the cellular redox-equilibrium (GSH oxidation) and by structural membrane modifications, which result in ion-non-selective pore formation. The disturbance in passive membrane permeability and the DNA damage may be the most dramatic cell impairments induced by t-BHP treatment. The presence of another oxidant, hypochlorous acid (HOCl), completely prevented t-BHP-induced DNA strand breaks, perhaps due to extracellular oxidation of t-BHP by HOCl.     

Hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin

After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at approximately 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 microM) to reagent HOCl (0-500 microM) or activated human neutrophils (4-40x10(6) cells/ml) yielded oxidized Mb (Mb(ox)) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1-5 mol/mol gave Mb(ox) with up to four additional oxygen atoms. Hydrolysis of Mb(ox) followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55>Met131>Trp7>Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mb(ox) with a composition similar to that determined with HOCl/Mb ratios <2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOCl-modified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation.     

Effects of oxidation of lysozyme by hypohalous acids and haloamines on enzymatic activity and aggregation

Hen egg white lysozyme (HEL), an antibacterial enzyme, is a prototype protein for studying the physical and chemical events that underlie the formation of amyloid fibril aggregates. Here, we studied alterations in enzymatic activity and aggregation provoked by oxidation of HEL by hypochlorous acid (HOCl), hypobromous acid (HOBr), taurine chloramine (Tau-NHCl), taurine monobromamine (Tau-NHBr), and taurine dibromamine (Tau-NBr(2)). Addition of only 4-fold molar excess of Tau-NHBr or Tau-NBr(2) to HEL caused complete depletion of its intrinsic fluorescence, whereas HOCl and HOBr caused 40%-50% bleaching. Tau-NHCl was unable to oxidize lysozyme. The selective effect of bromamines on tryptophan residues had a direct effect on enzymatic activity; bromamines were about two-fold more effective as inhibitors of lysozyme than the acid precursors. The oxidation of HEL by HOCl and HOBr was more effective regarding the aggregation of the protein, which was evidenced by increased turbidity, Rayleigh scattering, and anisotropy. The aggregates presented spectroscopic properties that suggested the formation of amyloid fibrils, as measured by the thioflavin assay. In conclusion, the capacity of Tau-NHBr and Tau-NBr(2) as inhibitors of the bactericidal activity of HEL could represent a role in the exacerbation of pulmonary infection, since leukocytes are rich sources of both taurine and HOBr. Moreover, the oxidation of HEL by just a small excess of hypohalous acids, a condition that could be found in inflammatory sites, may represent a new pathway for initiation of aggregation.     

Relative reactivities of N-chloramines and hypochlorous acid with human plasma constituents

Hypochlorous acid (HOCl), the major strong oxidant produced by the phagocyte enzyme myeloperoxidase, reacts readily with free amino groups to form N-chloramines. Since different N-chloramines have different stabilities and reactivities depending on their structures, we investigated the relative reactivities of three model N-chloramines and HOCl with human plasma constituents. TheN-chloramines studied were N(alpha)-acetyl-lysine chloramine (LysCA, a model of protein-associated N-chloramines), taurine chloramine (TaurCA, the primary N-chloramine produced by activated neutrophils), and monochloramine (MonoCA, a lipophilic N-chloramine). Addition of these chlorine species (100--1000 microM each) to plasma resulted in rapid loss of thiols, with the extent of thiol oxidation decreasing in the order TaurCA = LysCA > MonoCA = HOCl. The single reduced thiol of albumin was the major target. Loss of plasma ascorbate also occurred, with the extent decreasing in the order HOCl > LysCA > TaurCA > MonoCA. Experiments comparing equimolar albumin thiols and ascorbate showed that while HOCl caused equivalent loss of thiols and ascorbate, theN-chloramines reacted preferentially with thiols. The chlorine species also inactivated alpha(1)-antiproteinase, implicating oxidation of methionine residues, and ascorbate provided variable protection depending on the chlorine species involved. Together, our data indicate that in biological fluids N-chloramines react more readily with protein thiols than with methionine residues or ascorbate, and thus may cause biologically relevant, selective loss of thiol groups.     

Hypochlorite-induced oxidation of amino acids, peptides and proteins

Summary. Activated phagocytes generate the potent oxidant hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is known to react with a number of biological targets including proteins, DNA, lipids and cholesterol. Proteins are likely to be major targets for reaction with HOCl within a cell due to their abundance and high reactivity with HOCl. This review summarizes information on the rate of reaction of HOCl with proteins, the nature of the intermediates formed, the mechanisms involved in protein oxidation and the products of these reactions. The predicted targets for reaction with HOCl from kinetic modeling studies and the consequences of HOCl-induced protein oxidation are also discussed.     

Myeloperoxidase inactivates TIMP-1 by oxidizing its N-terminal cysteine residue: an oxidative mechanism for regulating proteolysis during inflammation

An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation.     

Hypochlorous acid-induced oxidative stress in Chinese hamster B14 cells: viability, DNA and protein damage and the protective action of melatonin

This study provides further evidence for the toxicity of hypochlorous acid (HOCl) in mammalian cells. Using the Chinese hamster B14 cell line, a significant decrease in cell viability was demonstrated after exposure to 100-200 microM HOCl for 1 h. Loss of viability was accompanied by a slight increase in DNA damage as shown by the Comet assay and by oxidation of cellular thiols. Exposure of B14 cells, erythrocyte membranes and human serum albumin to HOCl resulted in an extensive protein carbonyl accumulation. Thus, the cytotoxicity of HOCl may be due to both protein damage (carbonyl formation and oxidation of protein thiol groups) and DNA damage. The well-known antioxidant melatonin interacted with the oxidant and significantly protected cells during HOCl exposure, diminishing its cytotoxic effects and reducing protein carbonyl generation.