Generation of intramolecular and intermolecular sulfenamides, sulfinamides, and sulfonamides by hypochlorous acid: a potential pathway for oxidative cross-linking of low-density lipoprotein by myeloperoxidase.

Oxidized low-density lipoprotein (LDL) is implicated in atherogenesis, and human atherosclerotic lesions contain LDL oxidized by myeloperoxidase, a heme protein secreted by activated phagocytes. Using hydrogen peroxide (H(2)O(2)), myeloperoxidase generates hypochlorous acid (HOCl), a powerful oxidant. We now demonstrate that HOCl produces sulfenamides, sulfinamides, and sulfonamides in model peptides, which suggests a potential mechanism for LDL oxidation and cross-linking. When we exposed the synthetic peptide PFKCG to HOCl, the peptide's thiol residue reacted rapidly, generating a near-quantitative yield of products. Tandem mass spectrometric analysis identified the products as the sulfenamide, sulfinamide, and sulfonamide, all formed by intramolecular cross-linking of the peptide's thiol and lysine residues. An intramolecular sulfinamide was also observed after the peptide PFRCG was exposed to HOCl, indicating that the guanidine group of arginine can also form a sulfur-nitrogen cross-link. The synthetic peptide PFVCG, which contains a free thiol residue but lacks nucleophilic amino acid side chains, formed an intermolecular sulfonamide when exposed to HOCl. Tandem mass spectrometric analysis of the dimer revealed that the free N-terminal amino group of one PFVCG molecule cross-linked with the thiol residue of another. This peptide also formed intermolecular sulfonamide cross-links with N(alpha)-acetyllysine after exposure to HOCl, demonstrating that the epsilon-amino group of a lysine residue can undergo a similar reaction. Moreover, human neutrophils used the myeloperoxidase-H(2)O(2) system to generate sulfinamides in model peptides containing lysine or arginine residues. Collectively, our observations raise the possibility that HOCl generated by myeloperoxidase contributes to intramolecular and intermolecular protein cross-linking in the artery wall. Myeloperoxidase might also use this mechanism to form sulfur-nitrogen cross-links in other inflammatory conditions.     

Hypochlorous acid generated by myeloperoxidase modifies adjacent tryptophan and glycine residues in the catalytic domain of matrix metalloproteinase-7 (matrilysin): an oxidative mechanism for restraining proteolytic activity during inflammation

Dysregulation of matrix metalloproteinase (MMP) activity is implicated in tissue destruction under inflammatory conditions. An important mechanism controlling enzymatic activity might involve reactive oxygen species generated by phagocytes. Myeloperoxidase, a heme protein secreted by neutrophils, monocytes, and macrophages, uses hydrogen peroxide to generate hypochlorous acid (HOCl). We demonstrate that HOCl inhibits the activity of human matrilysin (MMP-7) in vitro, suggesting that it might limit proteolytic activity during inflammation. When MMP-7 was exposed to HOCl generated by myeloperoxidase, the proteinase lost activity. High performance liquid chromatographic analysis of the tryptic digest of the HOCl-treated proteinase demonstrated the absence of two peptides that were present in the untreated enzyme. Tandem mass spectrometric analysis revealed that both of the lost peptides contained methionine and tryptophan-glycine residues. The methionine residue of one of the peptides had been oxidized to methionine sulfoxide. In contrast, the major product from the other peptide was 4 atomic mass units smaller than its precursor (WG-4). This novel oxidation product was derived though modification of adjacent tryptophan and glycine residues in the catalytic domain of the enzyme. Loss of proteolytic activity was associated with conversion of the precursor peptide to WG-4 but not with methionine oxidation. In contrast, hydrogen peroxide failed to oxidize MMP-7 or to inactivate the enzyme. Thus, HOCl inactivates MMP-7, perhaps by site-specific conversion of tryptophan-glycine to WG-4. This inactivation mechanism is distinct from the well studied mechanisms involving tissue inhibitors of metalloproteinases. Our findings suggest that local pericellular production of HOCl by phagocytes is a physiological mechanism for governing MMP activity during inflammation.     

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.     

Oxidative cross-linking of tryptophan to glycine restrains matrix metalloproteinase activity: specific structural motifs control protein oxidation

Matrix metalloproteinases (MMPs) function in homeostatic and repair processes, but unregulated catalysis by these extracellular proteinases leads to the pathological destruction of tissue proteins. An important mechanism for controlling enzyme activity might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase system of phagocytes. We have shown that inactivation of MMP-7 (matrilysin) by HOCl coincides with the formation of a novel oxidation product, WG-4, through modification of adjacent tryptophan and glycine residues and loss of 4 atomic mass units. Here, we use mass spectrometry, UV/visible spectroscopy, hydrogen-deuterium exchange, and NMR spectroscopy to investigate the formation and structure of WG-4. For the initial step, HOCl chlorinates the indole ring of tryptophan. The resulting 3-chloroindolenine generates a previously unknown cyclic indole-amide species, in which tryptophan cross-links to the main chain nitrogen of the adjacent glycine residue to form an aromatic six-membered ring. WG-4 kinks and stiffens the peptide backbone, which may hinder the interaction of substrate with the catalytic pocket of MMP-7. Our observations indicate that specific structural motifs are important for controlling protein modification by oxidants and suggest that pericellular oxidant production by phagocytes might limit MMP activity during inflammation.     

Molecular mechanism of the reduction of cysteine sulfinic acid of peroxiredoxin to cysteine by mammalian sulfiredoxin

Among many proteins with cysteine sulfinic acid (Cys-SO2H) residues, the sulfinic forms of certain peroxiredoxins (Prxs) are selectively reduced by sulfiredoxin (Srx) in the presence of ATP. All Srx enzymes contain a conserved cysteine residue. To elucidate the mechanism of the Srx-catalyzed reaction, we generated various mutants of Srx and examined their interaction with PrxI, their ATPase activity, and their ability to reduce sulfinic PrxI. Our results suggest that three surface-exposed amino acid residues, corresponding to Arg50, Asp57, and Asp79 of rat Srx, are critical for substrate recognition. The presence of the sulfinic form (but not the reduced form) of PrxI induces the conserved cysteine of Srx to take the gamma-phosphate of ATP and then immediately transfers the phosphate to the sulfinic moiety of PrxI to generate a sulfinic acid phosphoryl ester (Prx-Cys-S(=O)OPO3(2-)). This ester is reductively cleaved by a thiol molecule (RSH) such as GSH, thioredoxin, and dithiothreitol to produce a disulfide-S-monoxide (Prx-Cys-S(=O)-S-R). The disulfide-S-monoxide is further reduced through the oxidation of three thiol equivalents to complete the catalytic cycle and regenerate Prx-Cys-SH.     

Production of glutathione sulfonamide and dehydroglutathione from GSH by myeloperoxidase-derived oxidants and detection using a novel LC-MS/MS method

GSH is rapidly oxidized by HOCl (hypochlorous acid), which is produced physiologically by the neutrophil enzyme myeloperoxidase. It is converted into, mainly, oxidized glutathione. Glutathione sulfonamide is an additional product that is proposed to be covalently bonded between the cysteinyl thiol and amino group of the gamma-glutamyl residue of GSH. We have developed a sensitive liquid chromatography-tandem MS assay for the detection and quantification of glutathione sulfonamide as well as GSH and GSSG. The assay was used to determine whether glutathione sulfonamide is a major product of the reaction between GSH and HOCl, and whether it is formed by other two-electron oxidants. At sub-stoichiometric ratios of HOCl to GSH, glutathione sulfonamide accounted for up to 32% of the GSH that was oxidized. It was also formed when HOCl was generated by myeloperoxidase and its yield increased with the flux of oxidant. Of the other oxidants tested, only hypobromous acid and peroxynitrite produced substantial amounts of glutathione sulfonamide, but much less than with HOCl. Chloramines were able to generate detectable levels only when at a stoichiometric excess over GSH. We conclude that glutathione sulfonamide is sufficiently selective for HOCl to be useful as a biomarker for myeloperoxidase activity in biological systems. We have also identified a novel oxidation product of GSH with a molecular weight two mass units less than GSH, which we have consequently named dehydroglutathione. Dehydroglutathione represented a few percent of the total products and was formed with all of the oxidants except H2O2.     

Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glutathiolation and detection in aging rats

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H202 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H202 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.     

Selective modification of apoB-100 in the oxidation of low density lipoproteins by myeloperoxidase in vitro

Oxidative modification of LDL may be important in the initiation and/or progression of atherosclerosis, but the precise mechanisms through which low density lipoprotein (LDL) is oxidized are unknown. Recently, evidence for the existence of HOCl-oxidized LDL in human atherosclerotic lesions has been reported, and myeloperoxidase (MPO), which is thought to act through production of HOCl, has been identified in human atherosclerotic lesions. In the present report we describe the formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive modifications in the apolipoprotein (apo) by exposure of LDL to myeloperoxidase in vitro. In contrast with the complex mixture of peptides from oxidation of LDL with reagent HOCl, oxidation with MPO in vitro produced a major tryptic peptide showing absorbance at 365 nm. This peptide was isolated and characterized as VELEVPQL(*C)SFILK..., corresponding to amino acid residues 53-66...on apoB-100. Mass spectrometric analyses of two tryptic peptides from oxidation of LDL by HOCl indicated formation of the corresponding methionine sulfoxide (M=O), cysteinyl azo (*C), RS -N= N-DNP, derivatives of EEL(*C)T(M=O)FIR and LNDLNS VLV(M=O)PTFHVPFTDLQVPS(*C)K, which suggest oxidation to the corresponding sulfinic acids (RSO2H) by HOCl.The present results demonstrate that DNPH-reactive modifications other than aldehydes and ketones can be formed in the oxidation of proteins and illustrate how characterization of specific products of protein oxidation can be useful in assessing the relative contributions of different and unexpected mechanisms to the oxidation of LDL and other target substrates. The data also suggest a direct interaction of the LDL particle with the active site on myeloperoxidase and indicate that effects of the protein microenvironment can greatly influence product formation and stability.     

Reduced nonprotein thiols inhibit activation and function of MMP-9: implications for chemoprevention

Clinical studies demonstrate a positive correlation between the extent of matrix metalloproteinase (MMP) activation and malignant progression of precancerous lesions. Therefore, identification of effective, well-tolerated MMP inhibitors represents a rational chemopreventive strategy. A variety of agents, including proteinases and thiol-oxidizing compounds, activate MMPs by initiating release of the propeptide's cysteine sulfur "blockage" of the MMP active site. Despite the importance of the propeptide's cysteine thiol in preserving MMP latency, limited studies have evaluated the effects of reduced thiols on MMP function. This study investigated the effects of two naturally occurring nonprotein thiols, i.e., glutathione (GSH) and N-acetylcysteine (NAC), on activation, function, and cellular-extracellular matrix interactions of the basement-membrane-degrading gelatinase, MMP-9. Our results reveal that NAC and GSH employ protein S-thiolation to inhibit organomercurial activation of pro-MMP-9. Gelatinase activity assays showed that GSH and NAC significantly inhibited MMP-9 but not MMP-2 function, implying isoform structural specificity. Immunoblot analyses, which suggested GSH interacts with MMP-9's active-site Zn, were corroborated by computational molecular modeling. Cell invasion assays revealed that NAC enhanced endostatin's ability to inhibit human cancer cell invasion. Collectively, these data demonstrate that nonprotein thiols suppress MMP-9 activation and function and introduce the prospect for their use in chemopreventive applications.     

A matrix metalloproteinase-9 activation cascade by hepatic stellate cells in trans-differentiation in the three-dimensional extracellular matrix

Hepatic stellate cells (HSCs) undergo myofibroblastic trans-differentiation in liver fibrogenesis. We previously showed that dual stimulation with three-dimensional type-I collagen and interleukin-1 (IL-1) synergistically induces HSC trans-differentiation in a manner dependent on the activation of matrix metallopreinase-9 (MMP-9). The present study is aimed to determine the mechanism of MMP-9 activation in this model. The pro-MMP-9-converting activities expressed by trans-differentiating HSCs are characterized as secreted factors that are sensitive to MMP inhibitor and have apparent molecular masses of 50 and 25 kDa. This is in sharp contrast to the pro-MMP-9 activator from mouse and human skin, which is a chymotrypsin-like proteinase. Among multiple MMPs induced in HSCs by the dual stimulation, MMP-13 is most conspicuously up-regulated and meets all criteria as the pro-MMP-9 activator. HSC cultured in three-dimensional type-I collagen, but not in Matrigel, IL-1 induces expression of MMP-13 and its matured form at 50 and 25 kDa, respectively. In vitro reconstitution experiment proves that MMP-13, but not its zymogen, activates pro-MMP-9. Further, short hairpin RNA targeting MMP-13 abolishes pro-MMP-9 activation and HSC trans-differentiation. We further demonstrate that pro-MMP-13 activation is facilitated with a membrane-associated factor, inhibited with tissue inhibitor of metalloproteinase-2, and abolished with short hairpin RNA against MMP-14. Moreover, pro-MMP-13 is also activated by a secreted factor, which is absorbed by gelatin-Sepharose and reconstituted with MMP-9. Thus, IL-1-induced trans-differentiation of HSCs in three-dimensional extracellular matrix is facilitated by an MMP activation cascade (MMP-14 > MMP-13 > MMP-9) and a positive feedback loop of MMP-9 > MMP-13, suggesting their critical roles in liver injury and repair.     

Identification of proteins susceptible to thiol oxidation in endothelial cells exposed to hypochlorous acid and N-chloramines

Hypochlorous acid (HOCl) is a potent oxidant produced by the enzyme myeloperoxidase, which is released by neutrophils under inflammatory conditions. Although important in the immune system, HOCl can also damage host tissue, which contributes to the development of disease. HOCl reacts readily with free amino groups to form N-chloramines, which also cause damage in vivo, owing to the extracellular release of myeloperoxidase and production of HOCl. HOCl and N-chloramines react readily with cellular thiols, which causes dysfunction via enzyme inactivation and modulation of redox signaling processes. In this study, the ability of HOCl and model N-chloramines produced on histamine and ammonia at inflammatory sites, to oxidize specific thiol-containing proteins in human coronary artery endothelial cells was investigated. Using a proteomics approach with the thiol-specific probe, 5-iodoacetamidofluorescein, we show that several proteins including peptidylprolyl isomerase A (cyclophilin A), protein disulfide isomerase, glyceraldehyde-3-phosphate dehydrogenase and galectin-1 are particularly sensitive to oxidation by HOCl and N-chloramines formed at inflammatory sites. This will contribute to cellular dysfunction and may play a role in inflammatory disease pathogenesis.     

S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.     

Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ～threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.     

Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

Methionine sulfoxide and proteolytic cleavage contribute to the inactivation of cathepsin G by hypochlorous acid: an oxidative mechanism for regulation of serine proteinases by myeloperoxidase

Using myeloperoxidase and hydrogen peroxide, activated neutrophils produce high local concentrations of hypochlorous acid (HOCl). They also secrete cathepsin G, a serine protease implicated in cytokine release, receptor activation, and degradation of tissue proteins. Isolated cathepsin G was inactivated by HOCl but not by hydrogen peroxide in vitro. We found that activated neutrophils lost cathepsin G activity by a pathway requiring myeloperoxidase, suggesting that oxidants generated by myeloperoxidase might regulate cathepsin G activity in vivo. Tandem mass spectrometric analysis of oxidized cathepsin G revealed that loss of a peptide containing Asp108, which lies in the active site, associated quantitatively with loss of enzymatic activity. Catalytic domain peptides containing Asp108 were lost from the oxidized protein in concert with the conversion of Met110 to the sulfoxide. Release of this peptide was blocked by pretreating cathepsin G with phenylmethylsulfonyl fluoride, strongly implying that oxidation introduced proteolytic cleavage sites into cathepsin G. Model system studies demonstrated that methionine oxidation can direct the regiospecific proteolysis of peptides by cathepsin G. Thus, oxidation of Met110 may contribute to cathepsin G inactivation by at least two distinct mechanisms. One involves direct oxidation of the thioether residue adjacent to the aspartic acid in the catalytic domain. The other involves the generation of new sites that are susceptible to proteolysis by cathepsin G. These observations raise the possibility that oxidants derived from neutrophils restrain pericellular proteolysis by inactivating cathepsin G. They also suggest that methionine oxidation could render cathepsin G susceptible to autolytic cleavage. Myeloperoxidase may thus play a previously unsuspected role in regulating tissue injury by serine proteases during inflammation.     

Production of metalloproteinase-7 (matrilysin) by human myeloma cells and its potential involvement in metalloproteinase-2 activation.

Matrix metalloproteinases (MMPs) play a critical role in bone remodeling and tumor spreading. Multiple myeloma (MM) is a plasma cell malignancy primarily localized within the bone marrow and characterized by its capacity to destroy bone matrix and to disseminate. We have reported recently that human myeloma cells were able to induce the conversion of pro-MMP-2 produced by the tumoral environment in its activated form. In the current study, we have investigated the mechanism involved in this process. We demonstrate that a soluble MMP constitutively produced by myeloma cells was responsible for pro-MMP-2 activation. Furthermore, we show that the soluble MMP, MMP-7, also known as matrilysin, was able to activate the MMP-2 produced in its latent form by bone marrow stromal cells. Finally, we demonstrate that myeloma cells constitutively produce MMP-7 with expected proteolytic activity. Our results suggest that MMP-7 produced by myeloma cells could participate in bone destruction and tumor spreading in MM, on one hand by its own proteolytic activity and on the other hand by its capacity to activate pro-MMP-2. These findings strengthen the idea that inhibition of MMP activity could represent an interesting therapeutic approach in MM.     

Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.