Myeloperoxidase-derived Oxidant Hypochlorous Acid Research Articles

Modification of histones by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) alters their reactivity with vascular smooth muscle cells

In the nucleus, histones are essential in the packaging of DNA and the regulation of gene expression. These histones can also be released to the extracellular space by mechanisms such as necrosis and neutrophil extracellular trap (NET) formation. Histones are cytotoxic and cause sterile inflammation, and as a result, have been implicated in tissue damage in several pathologies, including atherosclerosis. Myeloperoxidase (MPO) is also present on NETs, which is catalytically active and able to produce hypochlorous acid (HOCl). This could modify histones and alter their extracellular reactivity. In this study, we compared the reactivity of histones with and without modification by HOCl with primary human coronary artery smooth muscle cells (HCASMCs). Histones induced a loss in viability and cell death primarily by apoptosis, which was attenuated on modification of the histones by HOCl. Exposure of HCASMCs to histones also resulted in the increased expression of the pro-inflammatory genes monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) and a decrease in intracellular thiols. In addition, there were changes in the expression of the stress related gene heme oxygenase-1 (HO-1). Modification of the histones with HOCl had no significant influence on changes in gene expression or thiol loss, in contrast to the cytotoxicity studies. Together, these studies provide new insight into the pathways by which histones could promote vascular dysfunction, which could be relevant to inflammatory diseases, such as atherosclerosis and sepsis, which are associated with elevated NET release and high circulating histones, respectively.

Mapping the modification of histones by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl)

Histones are critical for the packaging of nuclear DNA and chromatin assembly, which is facilitated by the high abundance of Lys and Arg residues within these proteins. These residues are also the site of a range of post-translational modifications, which influence the regulatory function of histones. Histones are also present in the extracellular environment, following release by various pathways, particularly neutrophil extracellular traps (NETs). NETs contain myeloperoxidase, which retains its enzymatic activity and produces hypochlorous acid (HOCl). This suggests that histones could be targets for HOCl under conditions where aberrant NET release is prevalent, such as chronic inflammation. In this study, we examine the reactivity of HOCl with a mixture of linker (H1) and core (H2A, H2B, H3 and H4) histones. HOCl modified the histones in a dose- and time-dependent manner, resulting in structural changes to the proteins and the formation of a range of post-translational modification products. N-Chloramines are major products following exposure of the histones to HOCl and decompose over 24 h forming Lys nitriles and carbonyls (aminoadipic semialdehydes). Chlorination and dichlorination of Tyr, but not Trp residues, is also observed. Met sulfoxide and Met sulfones are formed, though these oxidation products are also detected albeit at a lower extent, in the non-treated histones. Evidence for histone fragmentation and aggregation was also obtained. These results could have implications for the development of chronic inflammatory diseases, given the key role of Lys residues in regulating histone function.

Mapping the modification of histones by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl)

Mapping the modification of histones by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl)

The effect of HOCl-induced modifications on phosphatase and tensin homologue (PTEN) structure and function

Oxidation by reactive species can cause changes in protein function and affect cell signalling pathways. Phosphatase and tensin homologue (PTEN) is a negative regulator of the PI3K/AKT pathway and is known to be inhibited by oxidation, but its oxidation by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) has not previously been investigated. PTEN-GST was treated with HOCl:protein ratios from 15:1 to 300:1. Decreases in PTEN phosphatase activity were observed at treatment ratios of 60:1 and higher, which correlated with the loss of the intact protein band and appearance of high molecular weight aggregates in SDS-PAGE. LC-MSMS was used to map oxidative modifications (oxPTMs) in PTEN-GST tryptic peptides and label-free quantitative proteomics used to determine their relative abundance. Twenty different oxPTMs of PTEN were identified, of which 14 were significantly elevated upon HOCl treatment in a dose-dependent manner. Methionine and cysteine residues were the most heavily oxidised; the percentage modification depended on their location in the sequence, reflecting differences in susceptibility. Other modifications included tyrosine chlorination and dichlorination, and hydroxylations of tyrosine, tryptophan, and proline. Much higher levels of oxidation occurred in the protein aggregates compared to the monomeric protein for certain methionine and tyrosine residues located in the C2 and C-terminal domains, suggesting that their oxidation promoted protein destabilisation and aggregation; many of the residues modified were classified as buried according to their solvent accessibility. This study provides novel information on the susceptibility of PTEN to the inflammatory oxidant HOCl and its effects on the structure and activity of the protein.

P-12 - The myeloperoxidase-derived oxidant hypochlorous acid induces macrophage extracellular trap release and promotes inflammation

P-12 - The myeloperoxidase-derived oxidant hypochlorous acid induces macrophage extracellular trap release and promotes inflammation

95 - The Myeloperoxidase-Derived Oxidant Hypochlorous Acid Induces Extracellular Trap Formation and Promotes the Inflammatory Response in Primary Human Macrophages

95 - The Myeloperoxidase-Derived Oxidant Hypochlorous Acid Induces Extracellular Trap Formation and Promotes the Inflammatory Response in Primary Human Macrophages

372 - The Myeloperoxidase-Derived Oxidant Hypochlorous Acid Promotes Extracellular Trap Formation and Inflammatory Cytokine Expression in Human Monocyte-Derived Macrophages: A Potential Mechanism of Atherosclerotic Lesion Development

372 - The Myeloperoxidase-Derived Oxidant Hypochlorous Acid Promotes Extracellular Trap Formation and Inflammatory Cytokine Expression in Human Monocyte-Derived Macrophages: A Potential Mechanism of Atherosclerotic Lesion Development

PSS142 - Role of the Myeloperoxidase-Derived Oxidant Hypochlorous Acid in the Induction of Endoplasmic Reticulum Stress in Cells of the Vascular System

PSS142 - Role of the Myeloperoxidase-Derived Oxidant Hypochlorous Acid in the Induction of Endoplasmic Reticulum Stress in Cells of the Vascular System

L-Arginine Chlorination Products Inhibit Endothelial Nitric Oxide Production

The myeloperoxidase-derived oxidant hypochlorous acid (HOCl) is thought to contribute to endothelial dysfunction, but the mechanisms underlying this inhibitory effect are unknown. The present study tested the hypothesis that HOCl and L-arginine (L-Arg) react to form novel compounds that adversely affect endothelial function by inhibiting nitric oxide (NO) formation. Using spectrophotometric techniques, we found that HOCl and L-Arg react rapidly (k = 7.1 x 10(5) m(-1) s(-1)) to form two major products that were identified by mass spectrometry as monochlorinated and dichlorinated adducts of L-Arg. Pretreatment of bovine aortic endothelial cells with the chlorinated L-Arg metabolites (Cl-l-Arg) inhibited the -induced formation of the NO metabolites nitrate (NO(3)(-)) and nitrite (NO(2)(-)) in a concentration-dependent manner. Preincubation of rat aortic ring segments with Cl-L-Arg resulted in concentration-dependent inhibition of acetylcholine-induced relaxation. In contrast, blood vessels relaxed normally to the endothelium-independent vasodilator sodium nitroprusside. In vivo administration of Cl-L-Arg to anesthetized rats increased carotid artery vascular resistance. A greater than 10-fold excess of L-Arg was required to reverse the inhibitory effects of Cl-L-Arg in vivo and in vitro. Reaction of HOCl with D-arginine (D-Arg) did not result in the formation of inhibitory products. These results suggest that HOCl reacts with L-Arg to form chlorinated products that act as nitric-oxide synthase inhibitors.

Dichotomous vasoactive responses to the myeloperoxidase-derived oxidant hypochlorous acid: [formula omitted] C. Reiter, J. P. Eiserich And C.R. White. Center For Free Radical Biology, Univ. Of Alabama At Birmingham, Birmingham, AL 35294

Dichotomous vasoactive responses to the myeloperoxidase-derived oxidant hypochlorous acid: [formula omitted] C. Reiter, J. P. Eiserich And C.R. White. Center For Free Radical Biology, Univ. Of Alabama At Birmingham, Birmingham, AL 35294

The scavenging of oxidants by sulphasalazine and its metabolites: A possible contribution to their anti-inflammatory effects?

Sulphasalazine (Salazopyrin) and its metabolites sulphapyridine and 5-aminosalicylate are powerful scavengers of the hydroxyl radical, determined by pulse radiolysis and confirmed by assays based on deoxyribose degradation by hydroxyl radicals. 5-Aminosalicylate can also protect α 1-anti-protease against attack by the myeloperoxidase-derived oxidant hypochlorous acid. The ability to scavenge oxidants produced at sites of inflammation may contribute to the anti-inflammatory action of sulphasalazine and its metabolites.

Biologically-significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by some anti-inflammatory drugs

Neutrophils contain the enzyme myeloperoxidase, which oxidizes Cl − ions into the powerful oxidant hypochlorous acid (HOCl). HOCl inactivates α 1antiprotease, permitting uncontrolled protease activities. Most anti-inflammatory drugs tested are capable of reacting with HOCl, but the reactions seem insufficiently rapid under physiological conditions to protect α 1-antiprotease against inactivation by HOCl. However, rapid scavenging of HOC1 might contribute to the anti-inflammatory effects of penicillamine, gold sodium thiomalate, phenylbutazone and primaquine.

The antioxidant action of human extracellular fluids. Effect of human serum and its protein components on the inactivation of alpha 1-antiproteinase by hypochlorous acid and by hydrogen peroxide.

The elastase-inhibitory capacity of purified human alpha 1-antiproteinase is inactivated by low concentrations of the myeloperoxidase-derived oxidant hypochlorous acid, but much higher concentrations are required to inhibit the elastase-inhibitory capacity of serum samples. The protective effect of serum appears to be largely due to albumin. High concentrations of H2O2 also inactivate the elastase-inhibitory capacity of alpha 1-antiproteinase, by a mechanism not involving formation of hydroxyl radicals. Serum offers protection against H2O2 inactivation of alpha 1-antiproteinase. The relevance of these results to the tissue damage produced by activated phagocytes is discussed.

Biologically significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by ascorbic acid: Implications for antioxidant protection in the inflamed rheumatoid joint

Ascorbic acid, at physiological concentrations, can scavenge the myeloperoxidase-derived oxidant hypochlorous acid at rates sufficient to protect α 1-antiprotease against inactivation by this molecule. The rapid depletion of ascorbic acid at sites of inflammation, as in the inflamed rheumatoid joint, may therefore facilitate proteolytic damage.

Allopurinol and oxypurinol are hydroxyl radical scavengers

Allopurinol is a scavenger of the highly reactive hydroxyl radical ( k 2 approx. 10 9 M −1s −1). One product of attack of hydroxyl radical upon allopurinol is oxypurinol, which is a major metabolite of allopurinol. Oxypurinol is a better hydroxyl radical scavenger than is allopurinol ( k 2 approx. 4 × 10 9 M −1s −1) and it also reacts with the myeloperoxidase-derived oxidant hypochlorous acid. Hence the protective actions of allopurinol against reperfusion damage after hypoxia need not be entirely due to xanthine oxidase inhibition.

Action of uric acid, allopurinol and oxypurinol on the myeloperoxidase-derived oxidant hypochlorous acid.

Both oxypurinol and uric acid react with the myeloperoxidase-derived oxidant hypochlorous acid at physiological pH, and they can protect the elastase-inhibitory capacity of human alpha 1-antiprotease against inactivation by hypochlorous acid. Allopurinol does not protect alpha 1-antiprotease, possibly because the redox potential of allopurinol at physiological pH is too positive to permit oxidation by hypochlorous acid.