Oxidative Inactivation of Paraoxonase1, an Antioxidant Protein and its Effect on Antioxidant Action

Paraoxonase1 (PON1), one of antioxidant proteins to protect low density lipoprotein (LDL) from the oxidation, is known to lose its activity in the oxidative environment. Here, we attempted to elucidate the possible mechanisms for the oxidative inactivation of PON1, and to examine the capability of hydroxyl radicals-inactivated PON1 to prevent against LDL oxidation. Of various oxidative systems, the ascorbate/Cu²⁺ system was the most potent in inactivating the purified PON1 (PON1) as well as HDL-bound PON1 (HDL-PON1). In contrast to a limited inactivation by Fe²⁺ (2.0?μM), the inclusion of Cu²⁺ (0.1–1.0?μM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.5?mM). A similar result was also obtained with the inactivation of HDL-PON1. The inactivation of PON1 by ascorbate/Cu²⁺ was pevented by catalase, but not general hydroxyl radical scavengers, supporting Cu²⁺-catalyzed oxidative inactivation. In addition, Cu²⁺ alone inactivated PON1, either soluble or HDL-bound, by different mechanisms, concentration-dependent. Separately, there was a reverse relationship between the inactivation of PON1 and its preventive action against LDL oxidation during Cu²⁺-induced oxidation of LDL. Noteworthy, ascorbate/Cu²⁺-inactivated PON1, which was charaterized by the partial loss of histidine residues, expressed a lower protection against Cu²⁺-induced LDL oxidation, compared to native PON1. Based on these results, it is proposed that metal-catalyzed oxidation may be a primary factor to cause the decrease of HDL-associated PON1 activity under oxidative stress, and radicals-induced inactivation of PON1 may lead to the decrease in its antioxidant action against LDL oxidation.     

Effect of 3,4-dihydroxyphenylalanine on Cu2+-induced Inactivation of HDL-associated Paraoxonase1 and Oxidation of HDL; Inactivation of Paraoxonase1 Activity Independent of HDL Lipid Oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Oxidative inactivation of paraoxonase1, an antioxidant protein and its effect on antioxidant action

Paraoxonase1 (PON1), one of antioxidant proteins to protect low density lipoprotein (LDL) from the oxidation, is known to lose its activity in the oxidative environment. Here, we attempted to elucidate the possible mechanisms for the oxidative inactivation of PON1, and to examine the capability of hydroxyl radicals-inactivated PON1 to prevent against LDL oxidation. Of various oxidative systems, the ascorbate/Cu₂₊ system was the most potent in inactivating the purified PON1 (PON1) as well as HDL-bound PON1 (HDL-PON1). In contrast to a limited inactivation by Fe₂₊ (2.0 μM), the inclusion of Cu₂₊ (0.1-1.0 μM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.5 mM). A similar result was also obtained with the inactivation of HDL-PON1. The inactivation of PON1 by ascorbate/Cu₂₊ was pevented by catalase, but not general hydroxyl radical scavengers, supporting Cu₂₊-catalyzed oxidative inactivation. In addition, Cu₂₊ alone inactivated PON1, either soluble or HDL-bound, by different mechanisms, concentration-dependent. Separately, there was a reverse relationship between the inactivation of PON1 and its preventive action against LDL oxidation during Cu₂₊-induced oxidation of LDL. Noteworthy, ascorbate/Cu₂₊-inactivated PON1, which was charaterized by the partial loss of histidine residues, expressed a lower protection against Cu₂₊-induced LDL oxidation, compared to native PON1. Based on these results, it is proposed that metal-catalyzed oxidation may be a primary factor to cause the decrease of HDL-associated PON1 activity under oxidative stress, and radicals-induced inactivation of PON1 may lead to the decrease in its antioxidant action against LDL oxidation.     

Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase

Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The K_i values of the enzyme for Cu²⁺ and Cu⁺ ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF–MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.     

One Enzyme,Two Functions: PON2 PREVENTS MITOCHONDRIAL SUPEROXIDE FORMATION AND APOPTOSIS INDEPENDENT FROM ITS LACTONASE ACTIVITY*

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O₂^˙̄ dismutase activities and cytochrome c expression unaltered, and it did not oxidize O₂^˙̄ but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His¹¹⁴ and His¹³³ are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys³¹¹, was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.     

Deficiency of the cystine-transporter gene, xCT, does not exacerbate the deleterious phenotypic consequences of SOD1 knockout in mice

Both α-lipoic acid (LA) and ascorbic acid (vitamin C) have been shown to improve endothelial dysfunction, a precursor of atherosclerosis. Since oxidant stress can cause endothelial dysfunction, we tested the interaction and efficacy of these antioxidants in preventing oxidant damage to lipids due to both intra- and extracellular oxidant stresses in EA.hy926 endothelial cells. LA spared intracellular ascorbate in culture and in response to an intracellular oxidant stress induced by the redox cycling agent menadione. Extracellular oxidant stress generated by incubating cells for 2 h in with 0.2 mg/ml LDL and 5 μM Cu²⁺ caused a time-dependent increase of the lipid peroxidation product malondialdehyde in both cells and LDL, preceded by rapid disappearance of` α-tocopherol in LDL. α-Lipoic acid at concentrations of 40–80 μM blunted these effects. Similarly, intracellular ascorbate concentrations of 1–2 mM also prevented Cu²⁺-induced lipid peroxidation in LDL and cells. Cu²⁺-dependent oxidation of LDL in the presence of ascorbate-loaded cells decreased intracellular ascorbate by 20%, but this decrease was not reversed by LA. Both LA and ascorbate protect endothelial cells and LDL from either intra- or extracellular oxidant stress, but that LA does not spare ascorbate in oxidatively stressed cells.     

Cu2+-catalyzed oxidative degradation of thyroglobulin

Thyroglobulin (Tg) was subjected to metal-catalyzed oxidation, and the oxidative degradation was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. In contrast to no effect of hydrogen peroxide (H₂O₂) alone on the Tg degradation, the inclusion of Cu²⁺ (30 μM), in combination with 2 mM H₂O₂, caused a remarkable degradation of Tg, time- and concentration-dependent. The action of Cu²⁺ was not mimicked by Fe²⁺, suggesting that Tg may interact selectively with Cu²⁺. A similar degradation of Tg was also observed with Cu²⁺corbate system, and the concentration of Cu²⁺ (5–10 μM), in combination with ascorbate, required for the effective degradation was smaller than that of Cu²⁺ (10–30 μM) in combination with H₂O₂. In support of involvement of H₂O₂ in the Cu²⁺ corbate action, catalase expressed a complete protection. However, hydroxyl radical scavengers such as dimethylsulfoxide or mannitol failed to prevent the oxidation of Tg whereas phenolic compounds, which can interact with Cu²⁺, diminished the oxidative degradation, presumably consistent with the mechanism for Cu²⁺-catalyzed oxidation of protein. Moreover, the amount of carbonyl groups in Tg was increased as the concentration (3–100 μM) of Cu²⁺ was enhanced, while the formation of acid-soluble peptides was not remarkable in the presence of Cu²⁺ up to 200 μM. In further studies, Tg pretreated with heat or trichloroacetic acid seemed to be somewhat resistant to Cu²⁺-catalyzed oxidation, implying a possible involvement of protein conformation in the susceptibility to the oxidation. Based on these observations, it is proposed that Tg could be degraded non-enzymatically by Cu²⁺-catalyzed oxidation.     

A biphasic U-shape effect of cellular oxidative stress on the macrophage anti-oxidant paraoxonase 2 (PON2) enzymatic activity

Expression of macrophage paraoxonase 2 (PON2), a cellular lactonase with anti-oxidant and anti-atherogenic properties, was shown to be upregulated under high oxidative stress. The aim of the present study was to analyze the relationship between the extent of cellular oxidative stress in J774A.1 macrophage and PON2 lactonase activity under various levels of oxidation, obtained by cell incubation with either anti-oxidants or oxidants. PON2 activity exhibited a U-shape response curve. In the oxidative stress range below that of control untreated cells, PON2 activity decreased upon increasing macrophage oxidative state, whereas in the range over that of control untreated cells, PON2 activity increased. The biphasic effect of oxidative stress on macrophage PON2 activity could be related to PON2 inactivation (decreased enzymatic activity) under oxidative stress induction at its low range, whereas at high range of oxidative stress, macrophage anti-oxidant compensatory mechanism up-regulates PON2 (increased protein expression), in order to cope with oxidative burden.     

Effect of 3,4-dihydroxyphenylalanine on Cu(2+)-induced inactivation of HDL-associated paraoxonasel and oxidation of HDL; inactivation of paraoxonasel activity independent of HDL lipid oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Genetic deletion of apolipoprotein A-I increases airway hyperresponsiveness,inflammation, and collagen deposition in the lung

The relationship between high-density lipoprotein and pulmonary function is unclear. To determine mechanistic relationships we investigated the effects of genetic deletion of apolipoprotein A-I (apoA-I) on plasma lipids, paraoxonase (PON1), pro-inflammatory HDL (p-HDL), vasodilatation, airway hyperresponsiveness and pulmonary oxidative stress, and inflammation. ApoA-I null (apoA-I^−/−) mice had reduced total and HDL cholesterol but increased pro-inflammatory HDL compared with C57BL/6J mice. Although PON1 protein was increased in apoA-I^−/− mice, PON1 activity was decreased. ApoA-I deficiency did not alter vasodilatation of facialis arteries, but it did alter relaxation responses of pulmonary arteries. Central airway resistance was unaltered. However, airway resistance mediated by tissue dampening and elastance were increased in apoA-I^−/− mice, a finding also confirmed by positive end-expiratory pressure (PEEP) studies. Inflammatory cells, collagen deposition, 3-nitrotyrosine, and 4-hydroxy-2-nonenal were increased in apoA-I^−/− lungs but not oxidized phospholipids. Colocalization of 4-hydroxy-2-nonenal with transforming growth factor β-1 (TGFβ-1 was increased in apoA-I^−/− lungs. Xanthine oxidase, myeloperoxidase and endothelial nitric oxide synthase were increased in apoA-I^−/− lungs. Dichlorodihydrofluorescein-detectable oxidants were increased in bronchoalveolar lavage fluid (BALF) in apoA-I^−/− mice. In contrast, BALF nitrite+nitrate levels were decreased in apoA-I^−/− mice. These data demonstrate that apoA-I plays important roles in limiting pulmonary inflammation and oxidative stress, which if not prevented, will decrease pulmonary artery vasodilatation and increase airway hyperresponsiveness.     

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu²⁺ ions either alone or in the presence of ascorbate (Cu²⁺/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu²⁺ alone displayed Cu²⁺-binding, which was significantly increased in the presence of Zn²⁺, Hg²⁺, Cd²⁺, Ag⁺¹ and As³⁺. Treatment of microsomes with Cu²⁺ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu²⁺ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu²⁺ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu²⁺ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.     

Paraoxonase 1 in endothelial cells impairs vasodilation induced by arachidonic acid lactone metabolite

IntroductionParaoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated lactonase, which is known for its antiatherogenic properties. Previous studies in PON1 knockout (PON1KO) mice revealed that PON1KO mice have low blood pressure, which is inversely correlated with the renal levels of the cytochrome P450 -derived arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET). Our previous studies revealed that 5,6-EET is unstable, transforming to the δ-lactone isomer 5,6-δ-DHTL, an endothelium-derived hyperpolarizing factor (EDHF) that mediates vasodilation, and it is a potential substrate for PON1.AimTo elucidate the role of PON1 in the modulation of vascular resistance via the regulation of the lactone-containing metabolite 5,6-δ-DHTL.ResultsIn mouse resistance arteries, PON1 was found to be present and active in the endothelial layer. Vascular reactivity experiments revealed that 5,6-δ-DHTL dose-dependently dilates PON1KO mouse mesenteric arteries significantly more than wild type (w.t.) resistance arteries. Pre-incubation with HDL or rePON1 reduced 5,6-δ-DHTL-dependent vasodilation. FACS analyses and confocal microscopy experiments revealed that fluorescence-tagged rePON1 penetrates into human endothelial cells' (ECs') in both dose- and time- dependent manner, accumulate in the perinuclear compartment, and retains its lactonase activity in the cells. The presence of rePON1, but not the presence of PON1 loss-of-lactonase-activity mutant, reduced the Ca²⁺ influx in the ECs mediated by 5,6-δ-DHTL.ConclusionPON1 lactonase activity in the endothelium affects vascular dilation by regulating Ca²⁺ influx mediated by the lactone-containing EDHF 5,6-δ-DHTL.     

Oxidative Inactivation of Brain Ecto-5′-Nucleotidase by Thiols/Fe2+ System

5-Nucleotidase, responsible for the conversion of adenosine-5-monophosphate into adenosine, was purified from bovine brain membranes, and subjected to oxidative inactivation. The 5-nucleotidase activity decreased slightly after the exposure to either glutathione or Fe²⁺. The glutathione-mediated inactivation of 5-nucleotidase was potentiated remarkably by Fe²⁺, but not Cu²⁺, in a concentration-dependent manner. Similarly, glutathione exhibited a concentration-dependent enhancement of the Fe²⁺-mediated inactivation. In comparison, the glutathione/Fe²⁺ system was much more effective than the ascorbate/Fe²⁺ system in inactivating the enzyme. In support of an intermediary role of superoxide ions or H₂O₂ in the action of glutathione/Fe²⁺ system, superoxide dismutase and catalase expressed a substantial protection against the inactivation by the glutathione/Fe²⁺ system. Meanwhile, hydroxyl radical scavangers such as mannitol, benzoate or ethanol were incapable of preventing the inactivation, excluding the participation of extraneous hydroxyl radicals. Whereas adenosine 5-monophosphate as substrate exhibited a modest protection against the glutathione/Fe²⁺ action, a remarkable protection was expressed by divalent metal ions such as Zn²⁺ or Mn²⁺. Structure-activity study with a variety of thiols indicates that the inactivating action of thiols in combination with Fe²⁺ resides in the free sulfhydyl group and amino group of thiols. Overall, thiols, expressing more inhibitory effect on the activity of 5-nucleotidase, were found to be more effective in potentiating the Fe²⁺- mediated inactivation. Further, kinetic analyses indicate that Fe²⁺ and thiols inhibit the 5-nucleotidase in a competitive or uncompetitive manner, respectively. These results suggest that ecto-5-nucleotidase from brain membrane is one of proteins susceptible to thiols/ Fe²⁺-catalyzed oxidation, and the oxidative inactivation may be related to the selective association of Fe²⁺ and thiols to the enzyme molecule.     

Preferable stimulation of PON1 arylesterase activity by phosphatidylcholines with unsaturated acyl chains or oxidized acyl chains at sn-2 position

To examine the effect of phospholipids on PON1 activities, purified PON1 was exposed to phospholipids prior to the determination of arylesterase and paraoxonase activities. Phosphatidylcholines with saturated acyl chains (C10-C16) showed a stimulation of both activities, chain length-dependent, with a greater stimulation of arylesterase activity, suggesting the implication of lipid bilayer in the stimulatory action. Such a preferable stimulation of arylesterase activity was more remarkable with phosphatidylcholines with polyunsaturated acyl chains or oxidized chains at sn-2 position, implying that the packing degree of acyl chain may be also important for the preferable stimulation of arylesterase activity. Separately, 1-palmitoyl-lysoPC also stimulated arylesterase activity preferably, indicating that the micellar formation of lipids around PON1 also contributes to the stimulatory action. Additionally, phosphatidylglycerols slightly enhanced arylesterase activity, but not paraoxonase activity. In contrast, phosphatidylserine and phosphatidic acid (≥0.1 mM) inhibited both activities Further, such a preferable stimulation of arylesterase activity by phosphatidylcholines was also reproduced with VLDL-bound PON1, although to a less extent. These data indicate that phosphatidylcholines with polyunsaturated acyl chains or oxidized chain, or lysophosphatidylcholine cause a preferable stimulation of arylesterase activity, thereby contributing to the decrease in the ratio of paraoxonase activity to arylesterase activity.     

A Common Mutation in Paraoxonase-2 Results in Impaired Lactonase Activity

Paraoxonases (PONs) are a family of lactonases with promiscuous enzyme activity that has been implicated in multiple diseases. PON2 is intracellularly located, is the most ubiquitously expressed PON, and has the highest lactonase activity of the PON family members. Whereas some single-nucleotide polymorphisms (SNPs) in PON1 have resulted in altered enzymatic activity in serum, to date the functional consequences of SNPs on PON2 function remain unknown. We hypothesized that a common PON2 SNP would result in impaired lactonase activity. Substitution of cysteine for serine at codon 311 in recombinant PON2 resulted in normal protein production and localization but altered glycosylation and decreased lactonase activity. Moreover, we screened 200 human lung samples for the PON2 Cys³¹¹ variant and found that in vivo this mutation impaired lactonase activity. These data suggest that impaired lactonase activity may play a role in innate immunity, atherosclerosis, and other diseases associated with the PON2 311 SNP.     

Salinity and Copper-Induced Oxidative Damage and Changes in the Antioxidative Defence Systems of Anabaena doliolum

Summary This study provides first-hand information on the salinity and copper-induced oxidative damage and its protection in Anabaena doliolum by the antioxidant defence system. Oxidative damage measured in terms of lipid peroxidation, electrolyte leakage and H₂O₂ production was induced by different concentrations of NaCl and Cu²⁺. A greater electrolyte leakage by NaCl than Cu²⁺ supported the hypothesis of salinity being more injurious than copper. To explore the survival strategies of A. doliolum under NaCl and Cu stress, enzymatic antioxidant activities e.g. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) and nonenzymatic antioxidant contents such as glutathione reduced (GSH), ascorbate, α-tocopherol, and carotenoid were measured. A general induction in SOD and APX activities as well as ascorbate and α-tocopherol contents was found under NaCl and Cu²⁺ stress. In contrast to this, an appreciable decline in GR activity, GSH pool and carotenoid content under Cu²⁺ and an increase under NaCl stress were observed. CAT activity was completely inhibited at high doses of NaCl but stimulated following Cu²⁺ treatment. The above results suggest the involvement of APX and CAT in the scavenging of H₂O₂ under Cu²⁺ stress. In contrast to this, only APX was involved in H₂O₂ scavenging under salt stress. Our postulate of Cu²⁺-mediated antagonism of salt stress can be explained by a conceivable reversion of Na⁺-induced disturbance of cellular homeostasis by redox active Cu²⁺.     

Selective Cu2+ binding, redox silencing, and cytoprotective effects of the small heat shock proteins alphaA- and alphaB-crystallin

Oxidative stress and Cu²⁺ have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu²⁺, is also known to induce the expression of the small heat shock proteins α-crystallins. However, the role of α-crystallins in oxidative stress and in Cu²⁺-mediated processes is not clearly understood. We demonstrate using fluorescence and isothermal titration calorimetry that α-crystallins (αA- and αB-crystallin and its phosphorylation mimic, 3DαB-crystallin) bind Cu²⁺ with close to picomolar range affinity. The presence of other tested divalent cations such as Zn²⁺, Mg²⁺, and Ca²⁺ does not affect Cu²⁺ binding, indicating selectivity of the Cu²⁺-binding site(s) in α-crystallins. Cu²⁺ binding induces structural changes and increase in the hydrodynamic radii of α-crystallins. Cu²⁺ binding increases the stability of α-crystallins towards guanidinium chloride-induced unfolding. Chaperone activity of αA-crystallin increases significantly upon Cu²⁺ binding. α-Crystallins rescue amyloid beta peptide, Aβ_1-40, from Cu²⁺-induced aggregation in vitro. α-Crystallins inhibit Cu²⁺-induced oxidation of ascorbate and, hence, prevent the generation of reactive oxygen species. Interestingly, α-synuclein, a Cu²⁺-binding protein, does not inhibit this oxidation process significantly. We find that the Cu²⁺-sequestering (or redox-silencing) property of α-crystallins confers cytoprotection. To the best of our knowledge, this is the first study to reveal high affinity (close to picomolar) for Cu²⁺ binding and redox silencing of Cu²⁺ by any heat shock protein. Thus, our study ascribes a novel functional role to α-crystallins in Cu²⁺ homeostasis and helps in understanding their protective role in neurodegenerative diseases and cataract.     

Broad-spectrum antioxidant peptides derived from His residue-containing sequences present in human paraoxonase 1

Hydroxyl or peroxyl radicals and hypochlorous acid (HOCl) are known to cause the oxidation of lipoproteins. Here, we examined Cu²⁺-binding property of paraoxonase 1 (PON1), and antioxidant actions of peptides, resembling His residue-containing sequences in PON1, against oxidations by Cu²⁺, peroxyl radicals or HOCl. When Cu²⁺-binding property of PON1 was examined spectrophotometrically, the maximal Cu²⁺ binding was achieved at 1:1 molar ratio of PON1: Cu²⁺. Additionally, Cu²⁺-catalyzed oxidative inactivation of PON1 was prevented by Ca²⁺-depleted PON1 at 1:1 ratio, but not diethylpyrocarbonate (DEPC)-modified PON1, suggesting the participation of His residue in Cu²⁺-binding. When His-containing peptides were examined for antioxidant actions, those with either His residue at N-terminal position 2 or 3, or His-Pro sequence at C-terminal remarkably prevented Cu²⁺-mediated low density lipoprotein (LDL) oxidation and PON1 inactivation. Especially, FHKALY, FHKY or NHP efficiently prevented Cu²⁺-induced LDL oxidation (24 h), indicating a tight binding of Cu²⁺ by peptides. In support of this, the peptide/Cu²⁺ complexes exhibited a superoxide-scavenging activity. Separately, in oxidations by 2,2'-azobis-2-amidinopropane hydrochloride or HOCl, the presence of Tyrosine (Tyr) or Cysteine (Cys) residue markedly enhanced antioxidant action of His-containing peptides. These results indicate that His-containing peptides with Tys or Cys residues correspond to broad spectrum antioxidants in oxidation models employing Cu²⁺, 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) or HOCl.     

Myeloperoxidase-induced modification of HDL by isolevuglandins inhibits paraoxonase-1 activity

Reduced activity of paraoxonase 1 (PON1), a high-density lipoprotein (HDL)-associated enzyme, has been implicated in the development of atherosclerosis. Post-translational modifications of PON1 may represent important mechanisms leading to reduced PON1 activity. Under atherosclerotic conditions, myeloperoxidase (MPO) is known to associate with HDL. MPO generates the oxidants hypochlorous acid and nitrogen dioxide, which can lead to post-translational modification of PON1, including tyrosine modifications that inhibit PON1 activity. Nitrogen dioxide also drives lipid peroxidation, leading to the formation of reactive lipid dicarbonyls such as malondialdehyde and isolevuglandins, which modify HDL and could inhibit PON1 activity. Because isolevuglandins are more reactive than malondialdehyde, we used in vitro models containing HDL, PON1, and MPO to test the hypothesis that IsoLG formation by MPO and its subsequent modification of HDL contributes to MPO-mediated reductions in PON1 activity. Incubation of MPO with HDL led to modification of HDL proteins, including PON1, by IsoLG. Incubation of HDL with IsoLG reduced PON1 lactonase and antiperoxidation activities. IsoLG modification of recombinant PON1 markedly inhibited its activity, while irreversible IsoLG modification of HDL before adding recombinant PON1 only slightly inhibited the ability of HDL to enhance the catalytic activity of recombinant PON1. Together, these studies support the notion that association of MPO with HDL leads to lower PON1 activity in part via IsoLG-mediated modification of PON1, so that IsoLG modification of PON1 could contribute to increased risk for atherosclerosis, and blocking this modification might prove beneficial to reduce atherosclerosis.     

A comparative study of antioxidative defense system in the copper and temperature acclimated strains of <Emphasis Type="Italic">Anabaena doliolum</Emphasis>

This study provides first hand comparative account of growth and antioxidative defense system of the wild type, Cu²⁺ and temperature treated wild type and acclimated strains of Anabaena doliolum Bharadwaja against Cu²⁺ and high temperature. The acclimated strains showed perceptible growth at 250 μM Cu²⁺ and 47°C temperatures, respectively. In contrast to this the wild type strain on exposure to 50 μM Cu²⁺ and 47°C temperature depicted almost complete inhibition of growth. However, the peroxide content was significantly higher in the acclimated strains than the wild type. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) showed maximum activity at high temperature followed by Cu²⁺ acclimated and minimum in the wild type strains. The ascorbate (ASC) and glutathione (GSH) contents were increased by 2.3 and 43.3, and 15.5 and 36.5-fold in Cu²⁺ and 47°C acclimated strains, respectively. However, when the wild type strain was subjected to Cu²⁺ and temperature all antioxidative enzymes except SOD showed inhibition of their activity. In case of wild type the GSH content was inhibited by 0.39-fold at 50 μM Cu²⁺ but the ASC content registered increase by 2 and 2.7-fold on subjecting to Cu²⁺ and temperature, respectively. Thus increased activity of enzymatic antioxidants as well as accumulation of ascorbate and glutathione in both the acclimated strains suggests that enzymatic and non-enzymatic antioxidants help in the acclimation of A. doliolum Bharadwaja against Cu²⁺ and high temperature. However, inhibition of antioxidative defense system of wild type under Cu²⁺ and heat stress appears to be the reason for its non survival. In view of the appreciable increase in the level of antioxidants as well as greater inhibition of specific growth rate in temperature than Cu²⁺ acclimated strains, temperature (47°C) is proposed to be is more deleterious to the organism than copper (250 μM).