Regulation of peroxiredoxins by nitric oxide in immunostimulated macrophages

Reactive oxygen species and nitric oxide (NO) are capable of both mediating redox-sensitive signal transduction and eliciting cell injury. The interplay between these messengers is quite complex, and intersection of their signaling pathways as well as regulation of their fluxes requires tight control. In this regard, peroxiredoxins (Prxs), a recently identified family of six thiol peroxidases, are central because they reduce H2O2, organic peroxides, and peroxynitrite. Here we provide evidence that endogenously produced NO participates in protection of murine primary macrophages against oxidative and nitrosative stress by inducing Prx I and VI expression at mRNA and protein levels. We also show that NO prevented the sulfinylation-dependent inactivation of 2-Cys Prxs, a reversible overoxidation that controls H2O2 signaling. In addition, studies using macrophages from sulfiredoxin (Srx)-deficient mice indicated that regeneration of 2-Cys Prxs to the active form was dependent on Srx. Last, we show that NO increased Srx expression and hastened Srx-dependent recovery of 2-Cys Prxs. We therefore propose that modulation by NO of Prx expression and redox state, as well as up-regulation of Srx expression, constitutes a novel pathway that contributes to antioxidant response and control of H2O2-mediated signal transduction in mammals.     

Influence of iNOS and COX on peroxiredoxin gene expression in primary macrophages

Peroxiredoxins (Prxs) are a family of multifunctional antioxidant thiol-dependent peroxidases. This study aimed to examine the regulatory mechanisms of Prx gene expression in murine bone marrow-derived macrophages (BMMs) using standardized serum-free conditions. Stimulation with LPS and IFNγ increased mRNA levels of Prx 1, 2, 4, 5, and 6 in BMMs of both C57BL/6 and BALB/c mice, with Prx 1, 2, 4, and 6 more strongly induced in C57BL/6 BMMs. Further investigations on signaling pathways in C57BL/6 BMMs demonstrated that up-regulation of Prx 5 and 6 by LPS and IFNγ was associated with the activation of multiple protein kinases, most notably JAK2, PI3K, and p38 MAPK. Our experiments also revealed a contribution of inducible NO synthase-derived nitric oxide to the increase in Prx 1, 2, 4, and 6 mRNA expression, whereas NADPH oxidase-derived superoxide was not involved. Furthermore, we could show that LPS- and IFNγ-induced gene expression of Prx 6 was also regulated in an NO-independent manner by cyclooxygenases and prostaglandin E₂. Taken together our results indicate a possible role for Prxs in defense mechanisms of activated macrophages against oxidative stress during inflammation or infection.     

Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization

S-nitrosation of thiols in key proteins in cell signaling pathways is thought to be an important contributor to nitric oxide (NO)-dependent control of vascular (patho)physiology. Multiple metabolic enzymes are targets of both NO and S-nitrosation, including those involved in glycolysis and oxidative phosphorylation. Thus it is important to understand how these metabolic pathways are integrated by NO-dependent mechanisms. Here, we compared the effects of NO and S-nitrosation on both glycolysis and oxidative phosphorylation in bovine aortic endothelial cells using extracellular flux technology to determine common and unique points of regulation. The compound S-nitroso-L-cysteine (L-CysNO) was used to initiate intracellular S-nitrosation since it is transported into cells and results in stable S-nitrosation in vitro. Its effects were compared with the NO donor DetaNONOate (DetaNO). DetaNO treatment caused only a decrease in the reserve respiratory capacity; however, L-CysNO impaired both this parameter and basal respiration in a concentration-dependent manner. In addition, DetaNO stimulated extracellular acidification rate (ECAR), a surrogate marker of glycolysis, whereas L-CysNO stimulated ECAR at low concentrations and inhibited it at higher concentrations. Moreover, a temporal relationship between NO- and S-nitrosation-mediated effects on metabolism was identified, whereby NO caused a rapid impairment in mitochondrial function, which was eventually overwhelmed by S-nitrosation-dependent processes. Taken together, these results suggest that severe pharmacological nitrosative stress may differentially regulate metabolic pathways through both intracellular S-nitrosation and NO-dependent mechanisms. Moreover, these data provide insight into the role of NO and related compounds in vascular (patho)physiology.     

Variable overoxidation of peroxiredoxins in human lung cells in severe oxidative stress

Peroxiredoxins (Prxs) are a group of thiol containing proteins that participate both in signal transduction and in the breakdown of hydrogen peroxide (H(2)O(2)) during oxidative stress. Six distinct Prxs have been characterized in human cells (Prxs I-VI). Prxs I-IV form dimers held together by disulfide bonds, Prx V forms intramolecular bond, but the mechanism of Prx VI, so-called 1-Cys Prx, is still unclear. Here we describe the regulation of all six Prxs in cultured human lung A549 and BEAS-2B cells. The cells were exposed to variable concentrations of H(2)O(2), menadione, tumor necrosis factor-alpha or transforming growth factor-beta. To evoke glutathione depletion, the cells were furthermore treated with buthionine sulfoximine. Only high concentrations (300 microM) of H(2)O(2) caused a minor increase (<28%, 4 h) in the expression of Prxs I, IV, and VI. Severe oxidant stress (250-500 microM H(2)O(2)) caused a significant increase in the proportion of the monomeric forms of Prxs I-IV; this was reversible at lower H(2)O(2) concentrations (< or =250 microM). This recovery of Prx overoxidation differed among the various Prxs; Prx I was recovered within 24 h, but recovery required 48 h for Prx III. Overall, Prxs are not significantly modulated by mild oxidant stress or cytokines, but there is variable, though reversible, overoxidation in these proteins during severe oxidant exposure.     

Targeted disruption of iNOS prevents LPS-induced S-nitrosation of IRbeta/IRS-1 and Akt and insulin resistance in muscle of mice

We have previously demonstrated that the insulin resistance associated with inducible nitric oxide synthase (iNOS) induction in two different models of obesity, diet-induced obesity and the ob/ob mice, is mediated by S-nitrosation of proteins involved in insulin signal transduction: insulin receptor beta-subunit (IRbeta), insulin receptor substrate 1(IRS-1), and Akt. S-nitrosation of IRbeta and Akt impairs their kinase activities, and S-nitrosation of IRS-1 reduces its tissue expression. In this study, we observed that LPS-induced insulin resistance in the muscle of wild-type mice, as demonstrated by reduced insulin-induced tyrosine phosphorylation of IRbeta and IRS-1, reduced IRS-1 expression and reduced insulin-induced serine phosphorylation of Akt. This resistance occurred in parallel with enhanced iNOS expression, which was accompanied by S-nitrosation of IRbeta/IRS-1 and Akt. In the muscle of iNOS(-/-) mice, we did not observe enhanced iNOS expression or any S-nitrosation of IRbeta/IRS-1 and Akt after LPS treatment. Moreover, insulin resistance was not present. The preservation of insulin-induced tyrosine phosphorylation of IRbeta and IRS-1, of IRS-1 protein expression, and of insulin-induced serine phosphorylation of Akt observed in LPS-treated iNOS(-/-) mice strongly suggests that the insulin resistance induced by LPS is iNOS mediated, probably through S-nitrosation of proteins of early steps of insulin signaling.     

Mechanism and kinetics of inducible nitric oxide synthase auto-S-nitrosation and inactivation

Nitric oxide (NO), the product of the nitric oxide synthase (NOS) reaction, was previously shown to result in S-nitrosation of the NOS Zn(2+)-tetrathiolate and inactivation of the enzyme. To probe the potential physiological significance of NOS S-nitrosation, we determined the inactivation time scale of the inducible NOS isoform (iNOS) and found it directly correlates with an increase in the level of iNOS S-nitrosation. A kinetic model of NOS inactivation in which arginine is treated as a suicide substrate was developed. In this model, NO synthesized at the heme cofactor is partitioned between release into solution (NO release pathway) and NOS S-nitrosation followed by NOS inactivation (inactivation pathway). Experimentally determined progress curves of NO formation were fit to the model. The NO release pathway was perturbed through addition of the NO traps oxymyoglobin (MbO(2)) and β2 H-NOX, which yielded partition ratios between NO release and inactivation of ~100 at 4 μM MbO(2) and ~22000 at saturating trap concentrations. The results suggest that a portion of the NO synthesized at the heme cofactor reacts with the Zn(2+)-tetrathiolate without being released into solution. Perturbation of the inactivation pathway through addition of the reducing agent GSH or TCEP resulted in a concentration-dependent decrease in the level of iNOS S-nitrosation that directly correlated with protection from iNOS inactivation. iNOS inactivation was most responsive to physiological concentrations of GSH with an apparent K(m) value of 13 mM. NOS turnover that leads to NOS S-nitrosation might be a mechanism for controlling NOS activity, and NOS S-nitrosation could play a role in the physiological generation of nitrosothiols.     

Analysis of peroxiredoxin decreasing oxidative stress in hypertensive aortic smooth muscle

To determine the role of peroxiredoxin (Prx) in response to oxidative stress and during hypertension in the vasculature, we identified Prx proteins and analyzed their antioxidant effects. Rat aortic smooth muscle contains all six Prxs (I-VI). Prx I, II, and VI shifted to its acidic site on two-dimensional polyacrylamide gel electrophoresis after exposure to H(2)O(2). The total expression of Prx I and VI was increased in response to H(2)O(2). The expression of Prx I, but not that of Prx II and VI, increases and the acidic form of Prx I and the sulfonic acid form of Prx (SO(3)H-Prx) are more strongly expressed in the aortic smooth muscle of hypertensive rats than in that of normotensive control rats. Prxs were also found in the mesenteric artery, heart, and kidney. The expression levels of Prx I and VI were increased in mesenteric artery, but not heart and kidney, from hypertensive rats compared with that from normotensive rats. These results suggest that Prxs play a crucial role against oxidative stress in vascular smooth muscles during hypertension.     

Persistent S-nitrosation of complex I and other mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide or peroxynitrite: implications for the interaction of nitric oxide with mitochondria

S-nitrosation of mitochondrial proteins has been proposed to contribute to the pathophysiological interactions of nitric oxide (NO) and its derivatives with mitochondria but has not been shown directly. Furthermore, little is known about the mechanism of formation or the fate of these putative S-nitrosothiols. Here we have determined whether mitochondrial membrane protein thiols can be S-nitrosated on exposure to free NO from 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) by interaction with S-nitrosoglutathione or S-nitroso-N-acetylpenicillamine (SNAP) and by the NO derivative peroxynitrite. S-Nitrosation of protein thiols was measured directly by chemiluminescence detection. S-Nitrosoglutathione and S-nitroso-N-acetylpenicillamine led to extensive protein thiol oxidation, with about 30% of the modified protein thiols persistently S-nitrosated. In contrast, there was no protein thiol oxidation or S-nitrosation on exposure to 3,3-bis (aminoethyl)-1-hydroxy-2-oxo-1-triazene. Peroxynitrite extensively oxidized protein thiols but produced negligible amounts of S-nitrosothiols. Therefore, mitochondrial membrane protein thiols are S-nitrosated by preformed S-nitrosothiols but not by NO or by peroxynitrite. These S-nitrosated protein thiols were readily reduced by glutathione, so S-nitrosation will only persist when the mitochondrial glutathione pool is oxidized. Respiratory chain complex I was S-nitrosated by S-nitrosothiols, consistent with it being an important target for S-nitrosation during nitrosative stress. The S-nitrosation of complex I correlated with a significant loss of activity that was reversed by thiol reductants. S-Nitrosation was also associated with increased superoxide production from complex I. These findings point to a significant role for complex I S-nitrosation and consequent dysfunction during nitrosative stress in disorders such as Parkinson disease and sepsis.     

Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins

Cysteine residues of certain peroxiredoxins (Prxs) undergo reversible oxidation to sulfinic acid (Cys-SO2H) and the reduction reaction is catalyzed by sulfiredoxin (Srx). Specific Cys residues of various other proteins are also oxidized to sulfinic acid, suggesting that formation of Cys-SO2H might be a novel posttranslational modification that contributes to regulation of protein function. To examine the susceptibility of sulfinic forms of proteins to reduction by Srx, we prepared such forms of all six mammalian Prx isoforms and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Purified sulfiredoxin reduced the sulfinic forms of the four 2-Cys members (Prx I to Prx IV) of the Prx family in vitro, but it did not affect those of Prx V, Prx VI, or GAPDH. Furthermore, Srx bound specifically to the four 2-Cys Prxs in vitro and in cells. Sulfinic forms of Prx I and Prx II, but not of Prx VI or GAPDH, present in H2O2-treated A549 cells were gradually reduced after removal of H2O2; overexpression of Srx increased the rate of the reduction of Prx I and Prx II but did not induce that of Prx VI or GAPDH. These results suggest that reduction of Cys-SO2H by Srx is specific to 2-Cys Prx isoforms. For proteins such as Prx VI and GAPDH, sulfinic acid formation might be an irreversible process that causes protein damage.     

Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis

Nitric oxide, generated by endogenous nitric oxide synthases or nitric oxide donors, can promote or prevent apoptosis induced by diverse pro-apoptotic stimuli in cell culture models. Both mitochondrial-dependent and -independent apoptotic signaling pathways mediate this dichotomous cellular response to nitric oxide. The molecular mechanisms behind these effects are complex and involve a number of nitrogen oxide-related species that are more reactive than nitric oxide itself. The local cellular environment plays a dynamic role in determining the nature and concentration of these species. Important components of the microenvironment include: the cellular redox state, glutathione, transition metals and the presence of other oxygen- and nitrogen-centered radicals. In particular, redox-sensitive nitrosating species are favorably generated under physiological conditions and capable of modifying multiple cell signaling pathways through reversible S-nitrosation reactions. Cytochrome c release from mitochondria is an important mechanism for the activation of caspase-3 and the initiation of cell death in response to 'intrinsic' pro-apoptotic stimuli, including oxidative and nitrosative stress. In turn, caspases and mitogen associated protein kinases may modulate cytochrome c release through their effects on the Bcl-2 family of proteins. This review will focus on (i) the importance of the cellular environment in determining the fate of nitric oxide and (ii) the ability of S-nitrosation to regulate mitochondrial-dependent apoptosis at the level of mitochondrial bioenergetics, cytochrome c release, caspases, mitogen associated protein kinases, and the Bcl-2 family of proteins.     

Nitric oxide and hydrogen cyanide as regulating factors of enzymatic antioxidant system in germinating apple embryos

Short-term (3 or 6 h) pre-treatment of apple (Malus domestica Borkh.) embryos with nitric oxide (NO) or hydrogen cyanide (HCN) induces transient accumulation of reactive oxygen species (ROS) leading to dormancy removal and germination. We demonstrated that enhanced NO emission by apple embryos during early phase of germination “sensu stricto” is required for seed transition from dormant into non-dormant state, and may be described by the model of “nitrosative door”, analogous to “oxidative window”. Cellular ROS concentration, resulting from NO or HCN embryo pre-treatment, seems to be under severe control of antioxidant system. Activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX) and total peroxidases (Prxs) was determined during NO and HCN-mediated germination “sensu stricto” of embryos. CAT and SOD activity increased transiently 24 h after embryos pre-treatment, while GR and Prx activity was stimulated mainly after 96 h. The most evident alterations were detected in GPX activity, being more than threefold stimulated by NO or HCN. Based on this results, we conclude that these reactive molecules act simultaneously crossing their signaling pathways and we propose that ROS, reactive nitrogen species, HCN at accurate level are essential during seed germination as signaling factors.     

S-Nitrosation and regulation of inducible nitric oxide synthase

The inducible isoform of nitric oxide synthase (iNOS) and three zinc tetrathiolate mutants (C104A, C109A, and C104A/C109A) were expressed in Escherichia coli and purified. The mutants were found by ICP-AES and the zinc-specific PAR colorimetric assay to be zinc free, whereas the wild-type iNOS zinc content was 0.38 +/- 0.01 mol of Zn/mol of iNOS dimer. The cysteine mutants (C104A and C109A) had an activity within error of wild-type iNOS (2.24 +/- 0.12 micromol of NO min(-1) mg(-1)), but the double cysteine mutant had a modestly decreased activity (1.75 +/- 0.14 micromol of NO min(-1) mg(-1)). To determine if NO could stimulate release of zinc and dimer dissociation, wild-type protein was allowed to react with an NO donor, DEA/NO, followed by buffer exchange. ICP-AES of samples treated with 10 microM DEA/NO showed a decrease in zinc content (0.23 +/- 0.01 to 0.09 +/- 0.01 mol of Zn/mol of iNOS dimer) with no loss of heme iron. Gel filtration of wild-type iNOS treated similarly resulted in approximately 20% more monomeric iNOS compared to a DEA-treated sample. Only wild-type iNOS had decreased activity (42 +/- 2%) after reaction with 50 microM DEA/NO compared to a control sample. Using the biotin switch method under the same conditions, only wild-type iNOS had increased levels of S-biotinylation. S-Biotinylation was mapped to C104 and C109 on wild-type iNOS using LysC digestion and MALDI-TOF/TOF MS. Immunoprecipitation of iNOS from the mouse macrophage cell line, RAW-264.7, and the biotin switch method were used to confirm endogenous S-nitrosation of iNOS. The data show that S-nitrosation of the zinc tetrathiolate cysteine results in zinc release from the dimer interface and formation of inactive monomers, suggesting that this mode of inhibition might occur in vivo.     

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys: IMPLICATIONS FOR ACTIVITY,ALLOSTERY, AND REGULATION*

Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS₄ site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions.     

Nitrosative capacity of macrophages is dependent on nitric-oxide synthase induction signals

Nitrosative stress can occur when reactive nitric oxide (NO) species compromise the function of biomolecules via formation of NO adducts on critical amine and thiol residues. The capacity of inducible nitric-oxide synthase (iNOS) to generate nitrosative stress was investigated in the murine macrophage line ANA-1. Sequential activation with the cytokines IFN-gamma and either tumor necrosis factor-alpha or interleukin-1beta resulted in the induction of iNOS and production of nitrite (20 nM/min) but failed to elicit nitrosation of extracellular 2,3-diaminonapthalene. Stimulation with IFN-gamma and bacterial lipopolysaccharide increased the relative level of iNOS protein and nitrite production of ANA-1 cells 2-fold; however, a substantial level of NO in the media was also observed, and nitrosation of 2,3-diaminonapthalene was increased greater than 30-fold. Selective scavenger compounds suggested that the salient nitrosating mechanism was the NO/O(2) reaction leading to N(2)O(3) formation. These data mimicked the pattern observed with a 5 microM concentration of the synthetic NO donor (Z)-1-[N-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium -1,2-diolate (PAPA/NO). The NO profiles derived from iNOS can be distinct and depend on the inductive signal cascades. The diverse consequences of NO production in macrophages may reside in the cellular mechanisms that control the ability of iNOS to form N(2)O(3) and elicit nitrosative stress.     

As(III) inhibits ultraviolet radiation-induced cyclobutane pyrimidine dimer repair via generation of nitric oxide in human keratinocytes

Inorganic arsenic enhances skin tumor formation when combined with other carcinogens including ultraviolet radiation (UVR). The inhibition of DNA damage repair by arsenic has been hypothesized to contribute to the cocarcinogenic activities of arsenic observed in vivo. Cyclobutane pyrimidine dimers (CPDs) are an important mutagenic UVR photoproduct and implicated in the genesis of nonmelanoma skin cancer. The current study demonstrates that low concentrations of arsenite (As(III)) inhibit UVR-induced CPD repair in a human keratinocyte cell line via nitric oxide (NO) and inducible nitric oxide synthase (iNOS). Following As(III) treatment, NO production and iNOS expression are elevated. Little is known about regulation of iNOS by As(III) and further investigations indicated that p38 mitogen-activated protein kinase (p38 MAPK) and NF-kappaB are required for As(III) induction of iNOS expression. This As(III)-stimulated signaling cascade was involved in inhibition of UVR-induced CPD repair as disruption of p38 MAPK activity and NF-kappaB nuclear translocation counteracted the effects of As(III) on CPD repair. Selective inhibition of iNOS ameliorated As(III) inhibition of CPD repair, thereby suggesting that iNOS is a downstream mediator of As(III) activity. These findings provide evidence that an As(III)-stimulated signal transduction cascade culminating in elevated iNOS expression and NO generation is an underlying mechanism for inhibition of UVR-induced DNA damage repair by arsenic.     

A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection

Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster S2 and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito.     

Cyclophilin a binds to peroxiredoxins and activates its peroxidase activity

Six distinct peroxiredoxin (Prx) proteins (Prx I-VI) from distinct genes have been identified in mammalian tissues. Prxs are members of a group of peroxidases that have conserved reactive cysteine residue(s) in the active site(s). An immediate physiological electron donor for the peroxidase catalysis for five Prx proteins (Prx I-V) has been identified as thioredoxin (Trx), but that for Prx VI (1-Cys Prx) is still unclear. To identify an immediate electron donor and a binding protein for Prx VI, we performed a Prx VI protein overlay assay. A 20-kDa binding protein was identified by the Prx VI protein overlay assay with flow-through fractions from a High-Q column with rat lung crude extracts. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MS-Fit, we identified the 20-kDa Prx VI-binding protein as a cyclophilin A (CyP-A). The binding of recombinant human CyP-A (hCyP-A) to Prx VI was confirmed by using the hCyP-A protein overlay assay and Western immunoblot analysis with hCyP-A-specific antibodies. hCyP-A enhanced the antioxidant activity of Prx VI, as well as the other known mammalian Prx isotypes. hCyP-A supported antioxidant activity of Prx II and Prx VI both against thiol (dithiothreitol)-containing metal-catalyzed oxidation (MCO) systems and ascorbate-containing MCO systems. Prx II was reduced by hCyP-A without help from any other reductant, and the reduction was cyclosporin A-independent. These results strongly suggest that CyP-A not only binds to Prx proteins but also supports its peroxidase activity as an immediate electron donor. In addition, Cys(115) and Cys(161) of hCyP-A were found to be involved in the activation and the reduction of Prx.     

Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells

Nitric oxide (NO) release upon microglial cell activation has been implicated in the tissue injury and cell death in many neurodegenerative diseases. Recent studies have indicated the ability of interferon-gamma (IFNgamma) and lipopolysaccharides (LPS) to independently induce type II nitric oxide synthase (iNOS) expression and NO production in BV-2 microglial cells. However, a detailed comparison between the signaling pathways activating iNOS by these two agents has not been accomplished. Analysis of PKC isoforms revealed mainly the presence of PKCdelta, iota and lambda in BV-2 cells. Although both IFNgamma and LPS could specifically enhance the tyrosine phosphorylation of PKCdelta, treatment with IFNgamma induced a steady increase of phospho-PKCdelta for up to 1h, whereas treatment with LPS elevated phospho-PKCdelta levels only transiently, with peak activity at 5 min. Rottlerin, a specific inhibitor for PKCdelta, dose-dependently inhibited IFNgamma- and LPS-induced NO production. Despite the common involvement of PKCdelta, IFNgamma- but not LPS-induced NO production involved extracellular signal-regulated kinases (ERK1/2) cascade and IFNgamma-induced phosphorylation of ERK1/2 was mediated through PKC. On the other hand, LPS- but not IFNgamma-induced NO production was through stimulation of NF-kappaB activation and nuclear translocation to interact with DNA. These results demonstrated distinct signaling pathways for induction of iNOS by IFNgamma and LPS in BV-2 microglial cells.