The Experts below are selected from a list of 1920 Experts worldwide ranked by ideXlab platform
Achille Iolascon - One of the best experts on this subject based on the ideXlab platform.
-
Peroxiredoxin 2 a novel regulator of iron homeostasis in ineffective erythropoiesis
Antioxidants & Redox Signaling, 2018Co-Authors: Alessandro Matte, Luigia De Falco, Achille Iolascon, Enrica Federti, Anna Cozzi, Sonia Levi, Narla Mohandas, Alberto Zamo, Mariasole Bruno, Christophe LebouefAbstract:Abstract Aims: Iron overload (IO) is a life-threatening complication of chronic hemolytic disorders such as β-thalassemia. IO results in severe cellular oxidative damage, leading to organ failure. Peroxiredoxin-2 (Prx2), a typical 2-cysteine-(Cys)-Peroxiredoxin, is an important component of the cytoprotective system, but its response to IO is still to be fully defined. Results: We studied the effects of IO on Prx2-knockout mice (Prx2−/−). The absence of Prx2 enhanced toxicity due to IO on erythropoiesis. We found that IO failed to induce the typical hepcidin (Hamp) upregulation in Prx2−/− mice due to its failure to activate the signal transducer and activator of transcription-3 (STAT3) with intact Jak2 signaling. In Prx2−/− mice, the loss of Hamp response was also observed after administration of a single dose of oral iron. When lipopolysaccharide (LPS) was used to explore IL6-STAT3 activation in Prx2−/− mice, STAT3 activation and Hamp upregulation were once again defective. Treatment with PEP-fusion-reco...
-
Peroxiredoxin 2 a novel factor involved in iron homeostasis
Blood, 2015Co-Authors: Alessandro Matte, Luigia De Falco, Achille Iolascon, Enrica Federti, Anne Janin, Sonia Levi, Narla Mohandas, Mariasole Bruno, Giorgia Federico, Christophe LeboeufAbstract:Peroxiredoxin-2 (Prx2), a typical 2-cysteine (Cys) Peroxiredoxin, is a key anti-oxidant system in both normal and pathological erythropoiesis characterized by oxidative stress such as b-thalassemia. We recently showed that the absence of Prx2 worsens s-thalassemic erythropoiesis and related iron-overload (Matte A et al. Antioxid Redox Signal, 2015). Here, we studied the effects of iron overload in a mouse model genetically lacking Prx2 (Prx2-/-). Two months old female wild-type (WT) and Prx2-/- pure bred mice were fed with a diet containing 2.5% carbonyl-iron compared to standard diet treated mice. We evaluated hematologic parameters, red cell indices and reticulocyte count in both mouse strains at baseline and at 30, 49, 60 and 90 days of treatment with carbonyl-iron. We observed a rapid drop in Hct and reticulocyte count in Prx2-/- mice compared to wild-type mice between 30-49 days of iron supplementation with the appearance of severe hyporegenerative anemia at 60 days of treatment in Prx2-/- characterized by a significant reduction in CD44+TER119+Fsc high cells. This was associated with marked increases in apoptotic Prx2-/- orthochromatic erythroblast compared to either baseline values or WT treated mice. In sorted erythroid precursors from iron overload WT mice, Prx2 expression was significantly increased compared to WT under standard diet. We observed a modulation of Erythropherrone (Erfe) expression during erythropoiesis with upregulation of Erfe in WT orthochromatic erythroblast compared to Prx2-/- erythroblasts. In liver from Prx2-/- mice exposed to iron-overload, we found liver iron content similar to WT mice but Pearls stain analysis showed differential iron distribution in cellular components of liver. While iron accumulated in hepatocytes and Kuppfer cells in Prx2-/- mice, iron-deposits were present only in hepatocytes of WT liver. Oxyblot analysis and liver MDA levels were significantly higher in Prx2-/- mice indicating that the absence of Prx2 promotes a severe liver oxidative stress. In WT liver, Prx2 expression was marked increased in a time depend way during iron supplementation, indicating that Prx2 is part of an adaptive cellular response to iron overload. This is in agreement with increased levels of ferritin-H in Prx2-/- mice compared to WT mice. Hepcidin (HAMP) expression was markedly increased in iron-overload WT mice compared to untreated control group, while no major changes were observed in Prx2-/- mice. Tfr2 expression was significantly increased only in livers of iron-overload WT mice, whereas phospho smad 1-5 was significantly increased in both mouse strains in response to iron-overload. The activation of the signaling pathway through Erk-1/2 only in iron-overload WT mice but not in Prx2-/- mice is most likely related to severe oxidative stress in Prx2-/- resulting in switching off of the Erks pathway. Importantly, administration of PEP1-Prx2 fusion protein rescue almost completely the hematologic phenotype with modulation of Erk signaling pathway towards Tfr2 and the smad system, validating our hypothesis of a role for Erk signaling for the observed phenoytpes. Our data highlight Prx2 as novel factor involved in iron homestasis through the control of oxidative stress modulating signaling pathway towards hepcidin expression. Disclosures No relevant conflicts of interest to declare.
-
oxidative stress modulates heme synthesis and induces Peroxiredoxin 2 as a novel cytoprotective response in β thalassemic erythropoiesis
Haematologica, 2011Co-Authors: Lucia De Franceschi, Mariarita Bertoldi, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Franco Turrini, Alessandra Colancecco, Clara Camaschella, Maria Domenica Cappellini, Achille IolasconAbstract:Background β-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to β-thalassemic anemia is only partially understood. Design and Methods We studied erythroid precursors from normal and β-thalassemic peripheral CD34+ cells in two-phase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of δ-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced Peroxiredoxin-2 to H2O2 and generated a recombinant Peroxiredoxin-2 for kinetic measurements in the presence of H2O2 or hemin. Results In β-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of Peroxiredoxin-2. In agreement with these observations in β-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, δ-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant δ-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in β-thalassemic cells. We then addressed the question of the protective role of Peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing Peroxiredoxin-2 in human erythroleukemia K562 cells. We found that Peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that Peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of Peroxiredoxin-2 in β-thalassemia. Conclusions In β-thalassemic erythroid cells the reduction of δ-aminolevulinate synthase-2 activity and the increased expression of Peroxiredoxin-2 might represent two novel stress-response protective systems.
-
OXIDATIVE STRESS MODULATES HEME LEVELS and INDUCES Peroxiredoxin-2 IN β THALASSEMIC ERYTHROPOIESIS as NOVEL CYTOPROTECTIVE RESPONSE
Blood, 2010Co-Authors: Lucia De Franceschi, Mariarita Bertoldi, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Franco Turrini, Alessandra Colancecco, Clara Camaschella, Maria Domenica Cappellini, Achille IolasconAbstract:Abstract 4266 Beta thalassemia (β-thal) syndromes are worldwide distributed congenital red cell disorders. Increased levels of reactive-oxygen-species (ROS) have been reported to contribute to anemia in β-thal but the mechanism(s) involved in cell protection against ROS damage has only partially investigated. Here, we studied in vitro normal and β-thal erythropoiesis in erythroid cell cultures from CD34+ cells isolated from peripheral blood from adult normal volunteers and from homozygous (bcod39) b-thalassemia patients. We showed increased ROS production in β-thal erythropoiesis and we evaluated the effects of ROS on normal and β-thal erythropoiesis. We carried out a proteomic comparative study, validated by coupling Quantitative-Real time PCR and immunoblot analysis of the differently expressed proteins. We found down-regulation in expression of enzymes involved in heme catabolism such as biliverdin reductase (BVR) and heme-oxygenase-1 (HO-1) and up-regulation of two new cytoprotective cysteine-based-systems: Peroxiredoxin-2 (Prx2) and heat-shock-protein-27 (HSP27), while catalase was similarly expressed in both cell models, suggesting a specific pattern of Px2 and HSP27 in β-thal erythroid precursors. We then measured heme levels and during b-thal-erythropoiesis and found that the synthesis of heme was biphasic displaying an increase of heme levels in early phase followed by a decrease in late phase in comparison to controls. Since heme synthesis depends on the erythroid δ-aminolevulinate-synthase isoform (ALAS-2), we evaluated ALAS-2 expression that resulted similar in normal and β-thal erythroid cells. We then showed that ALAS-2 activity was inhibited by both ROS and hemin, suggesting a possible role of heme and ROS levels in regulation of heme biosynthesis in β-thal cells. Since it has been reported that oxidative stress can up-regulate Prx2 expression and that genetically modified cells over-expressing Prx2 are generally more protected from severe oxidative stress (Phalen TJ et al 2006; Rabilloud T et al 2002; Kang SW et al 1998; Zhang P et al 1997), we have hypothesized a cytoprotective role of Prx2 in b-thal-erythropoiesis. We determined that the anti-oxidant Prx2 specifically binds hemin with high and affinity, most likely involving Prx2 cysteine residues. In order to look for the structural determinants to the binding, we noted that both ALAS-2 and Prx2 possess one and two cys-pro motifs, respectively. This motif is generally considered a heme sensor for many proteins able to bind heme and we propose that it could be responsible for heme binding in both enzymes. These data suggest a wider role of Prx2 as both anti-oxidant and heme-binding protein in protective stress-response-systems in β-thal erythropoiesis. Disclosures: No relevant conflicts of interest to declare.
Christine C Winterbourn - One of the best experts on this subject based on the ideXlab platform.
-
The High Reactivity of Peroxiredoxin 2 with H 2 O 2 Is Not Reflected in Its Reaction with Other Oxidants and
2020Co-Authors: Alexander V Peskin, Mark B Hampton, Louise N Paton, Ghassan J Maghzal, Christine C WinterbournAbstract:Peroxiredoxin 2 is a member of the mammalian Peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte Peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect Peroxiredoxin 2 against oxidation by added H2O2. Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and Peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 10 7 M 1 s 1 for the Peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for Peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with Peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with Peroxiredoxin 2. These results demonstrate that Peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.
-
intra dimer cooperativity between the active site cysteines during the oxidation of Peroxiredoxin 2
bioRxiv, 2020Co-Authors: Alexander V Peskin, Christine C Winterbourn, Flavia Carla Meotti, L F De Souza, Robert F Anderson, Armindo SalvadorAbstract:Peroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites. We have observed positive cooperativity when H2O2 is added as a bolus and oxidation of the second site occurs while the first site is present as a sulfenic acid. Modelling of this reaction showed that the second site reacts 2.2 {+/-} 0.1 times faster. In contrast, when H2O2 was generated slowly and the first active site condensed to a disulfide before the second site reacted, no cooperativity was evident. Conversion of the sulfenic acid to the disulfide showed negative cooperativity, with modelling of the exponential rise in tryptophan fluorescence yielding a rate constant of 0.75 {+/-} 0.08 s-1 when the alternate active site was present as a sulfenic acid and 2.29 {+/-} 0.08-fold lower when it was a disulfide. No difference in the rate of hyperoxidation at the two sites was detected. Our findings imply that oxidation of one active site affects the conformation of the second site and influences which intermediate forms of the protein are favored under different cellular conditions.
-
glutathionylation of the active site cysteines of Peroxiredoxin 2 and recycling by glutaredoxin
Journal of Biological Chemistry, 2016Co-Authors: Alexander V Peskin, Louise N Paton, Paul Pace, Jessica B Behring, Marjolein Soethoudt, Markus Bachschmid, Christine C WinterbournAbstract:: Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H2O2 and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m(-1) s(-1)) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.
-
Accumulation of oxidized Peroxiredoxin 2 in red blood cells and its prevention
Transfusion, 2015Co-Authors: Simone B. Bayer, Mark B Hampton, Christine C WinterbournAbstract:Background The thiol protein Peroxiredoxin 2 (Prx2) is a major red blood cell (RBC) antioxidant that breaks down hydroperoxides and in the process is converted to an oxidized disulfide. Our objective was to determine whether Prx2 becomes oxidized during storage of RBCs, to understand the underlying mechanism, and to find ways of preventing the accumulation of the oxidized form. Study design and Methods RBCs were stored for up to 6 weeks under simulated blood banking conditions and Prx2 oxidation was monitored by nonreducing gel electrophoresis. The ability of the cells to reverse Prx2 oxidation after storage and to respond to added hydrogen peroxide was also evaluated. Results Prx2 remained predominantly reduced during the first 3 weeks of storage, and then the oxidized form accumulated progressively. In contrast to fresh cells, oxidation was not reversed by incubation with glucose. Storage of RBCs in a high-pH, low-chloride, and high-phosphate/bicarbonate buffer (EAS-76v6) largely prevented accumulation of oxidized Prx for at least 6 weeks, and dihydrolipoic acid (DHLA), but not Rejuvesol, N-acetylcysteine, or α-lipoic acid, was able to reverse or protect against Prx2 oxidation. Additional, Prx2 oxidation occurred when hydrogen peroxide was added. However, this was reversible, suggesting that the reductive capacity was compromised in some but not in all cells. Conclusion Prx2 remains mostly reduced in a high-pH storage solution with buffering capacity. Addition of DHLA to stored RBCs might be advantageous. Prx2 redox status could be used as a biomarker for the quality of stored RBCs.
-
Increased basal oxidation of Peroxiredoxin 2 and limited Peroxiredoxin recycling in glucose-6-phosphate dehydrogenase-deficient erythrocytes from newborn infants
The FASEB Journal, 2014Co-Authors: Fook-choe Cheah, Alexander V Peskin, Fei-liang Wong, Azlin Ithnin, Ainoon Othman, Christine C WinterbournAbstract:Erythrocytes require glucose-6-phosphate dehydrogenase (G6PD) to generate NADPH and protect themselves against hemolytic anemia induced by oxidative stress. Peroxiredoxin 2 (Prx2) is a major antioxidant enzyme that requires NADPH to recycle its oxidized (disulfide-bonded) form. Our aims were to determine whether Prx2 is more highly oxidized in G6PD-deficient erythrocytes and whether these cells are able to recycle oxidized Prx2 after oxidant challenge. Blood was obtained from 61 Malaysian neonates with G6PD deficiency (average 33% normal activity) and 86 controls. Prx2 redox state was analyzed by Western blotting under nonreducing conditions. Prx2 in freshly isolated blood was predominantly reduced in both groups, but the median level of oxidation was significantly higher (8 vs 3%) and the range greater for the G6PD-deficient population. When treated with reagent H2O2, the G6PD-deficient erythrocytes were severely compromised in their ability to recycle oxidized Prx2, with only 27 or 4% reduction after 1 ...
Lucia De Franceschi - One of the best experts on this subject based on the ideXlab platform.
-
The novel role of Peroxiredoxin-2 in red cell membrane protein homeostasis and senescence.
Free Radical Biology and Medicine, 2014Co-Authors: Alessandro Matte, Mariarita Bertoldi, Franco Turrini, Angela Siciliano, Antonella Pantaleo, Emanuela Ferru, Francesca Lupo, Chae Ho Zoon, Lucia De FranceschiAbstract:Abstract Peroxiredoxin-2 (Prx2), a typical two-cysteine Peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2−/− mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2−/− red cells and reduced Prx2−/− red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2−/− mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2−/− mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2−/− mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.
-
oxidative stress modulates heme synthesis and induces Peroxiredoxin 2 as a novel cytoprotective response in β thalassemic erythropoiesis
Haematologica, 2011Co-Authors: Lucia De Franceschi, Mariarita Bertoldi, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Franco Turrini, Alessandra Colancecco, Clara Camaschella, Maria Domenica Cappellini, Achille IolasconAbstract:Background β-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to β-thalassemic anemia is only partially understood. Design and Methods We studied erythroid precursors from normal and β-thalassemic peripheral CD34+ cells in two-phase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of δ-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced Peroxiredoxin-2 to H2O2 and generated a recombinant Peroxiredoxin-2 for kinetic measurements in the presence of H2O2 or hemin. Results In β-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of Peroxiredoxin-2. In agreement with these observations in β-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, δ-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant δ-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in β-thalassemic cells. We then addressed the question of the protective role of Peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing Peroxiredoxin-2 in human erythroleukemia K562 cells. We found that Peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that Peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of Peroxiredoxin-2 in β-thalassemia. Conclusions In β-thalassemic erythroid cells the reduction of δ-aminolevulinate synthase-2 activity and the increased expression of Peroxiredoxin-2 might represent two novel stress-response protective systems.
-
OXIDATIVE STRESS MODULATES HEME LEVELS and INDUCES Peroxiredoxin-2 IN β THALASSEMIC ERYTHROPOIESIS as NOVEL CYTOPROTECTIVE RESPONSE
Blood, 2010Co-Authors: Lucia De Franceschi, Mariarita Bertoldi, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Franco Turrini, Alessandra Colancecco, Clara Camaschella, Maria Domenica Cappellini, Achille IolasconAbstract:Abstract 4266 Beta thalassemia (β-thal) syndromes are worldwide distributed congenital red cell disorders. Increased levels of reactive-oxygen-species (ROS) have been reported to contribute to anemia in β-thal but the mechanism(s) involved in cell protection against ROS damage has only partially investigated. Here, we studied in vitro normal and β-thal erythropoiesis in erythroid cell cultures from CD34+ cells isolated from peripheral blood from adult normal volunteers and from homozygous (bcod39) b-thalassemia patients. We showed increased ROS production in β-thal erythropoiesis and we evaluated the effects of ROS on normal and β-thal erythropoiesis. We carried out a proteomic comparative study, validated by coupling Quantitative-Real time PCR and immunoblot analysis of the differently expressed proteins. We found down-regulation in expression of enzymes involved in heme catabolism such as biliverdin reductase (BVR) and heme-oxygenase-1 (HO-1) and up-regulation of two new cytoprotective cysteine-based-systems: Peroxiredoxin-2 (Prx2) and heat-shock-protein-27 (HSP27), while catalase was similarly expressed in both cell models, suggesting a specific pattern of Px2 and HSP27 in β-thal erythroid precursors. We then measured heme levels and during b-thal-erythropoiesis and found that the synthesis of heme was biphasic displaying an increase of heme levels in early phase followed by a decrease in late phase in comparison to controls. Since heme synthesis depends on the erythroid δ-aminolevulinate-synthase isoform (ALAS-2), we evaluated ALAS-2 expression that resulted similar in normal and β-thal erythroid cells. We then showed that ALAS-2 activity was inhibited by both ROS and hemin, suggesting a possible role of heme and ROS levels in regulation of heme biosynthesis in β-thal cells. Since it has been reported that oxidative stress can up-regulate Prx2 expression and that genetically modified cells over-expressing Prx2 are generally more protected from severe oxidative stress (Phalen TJ et al 2006; Rabilloud T et al 2002; Kang SW et al 1998; Zhang P et al 1997), we have hypothesized a cytoprotective role of Prx2 in b-thal-erythropoiesis. We determined that the anti-oxidant Prx2 specifically binds hemin with high and affinity, most likely involving Prx2 cysteine residues. In order to look for the structural determinants to the binding, we noted that both ALAS-2 and Prx2 possess one and two cys-pro motifs, respectively. This motif is generally considered a heme sensor for many proteins able to bind heme and we propose that it could be responsible for heme binding in both enzymes. These data suggest a wider role of Prx2 as both anti-oxidant and heme-binding protein in protective stress-response-systems in β-thal erythropoiesis. Disclosures: No relevant conflicts of interest to declare.
-
Peroxiredoxin 2 expression is increased in β thalassemic mouse red cells but is displaced from the membrane as a marker of oxidative stress
Free Radical Biology and Medicine, 2010Co-Authors: Alessandro Matte, Mariarita Bertoldi, Franco Turrini, Angela Siciliano, Antonella Pantaleo, Maria Estela Campanella, Daniela Spano, Lucia De FranceschiAbstract:Peroxiredoxin 2 (Prx2), the third most abundant cytoplasmic protein in red blood cells (RBCs), is involved in the defense against oxidative stress. Although much is known about Prx2 in healthy RBCs, its role in pathological RBCs remains largely unexplored. Here, we show that the expression and net content of Prx2 are markedly increased in RBCs from two mouse models of β-thalassemia (β-thal; Hbbth/th and Hbbth3/+ strains). We also demonstrate that the increased expression of Prx2 correlates with the severity of the disease and that the amount of Prx2 bound to the membrane is markedly reduced in β-thal mouse RBCs. To explore the impact of oxidative stress on Prx2 membrane association, we examined Prx2 dimerization and membrane translocation in murine RBCs exposed to various oxidants (phenylhydrazine, PHZ; diamide; H2O2). PHZ-treated RBCs, which mimic the membrane damage in β-thal RBCs, exhibited a kinetic correlation among Prx2 membrane displacement, intracellular methemoglobin levels, and hemichrome membrane association, suggesting the possible masking of Prx2 docking sites by membrane-bound hemichromes, providing a possible mechanism for the accumulation of oxidized/dimerized Prx2 in the cytoplasm and the increased membrane damage in β-thal RBCs. Thus, reduced access of Prx2 to the membrane in β-thal RBCs represents a new factor that could contribute to the oxidative damage characterizing the pathology.
Franco Turrini - One of the best experts on this subject based on the ideXlab platform.
-
The novel role of Peroxiredoxin-2 in red cell membrane protein homeostasis and senescence.
Free Radical Biology and Medicine, 2014Co-Authors: Alessandro Matte, Mariarita Bertoldi, Franco Turrini, Angela Siciliano, Antonella Pantaleo, Emanuela Ferru, Francesca Lupo, Chae Ho Zoon, Lucia De FranceschiAbstract:Abstract Peroxiredoxin-2 (Prx2), a typical two-cysteine Peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2−/− mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2−/− red cells and reduced Prx2−/− red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2−/− mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2−/− mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2−/− mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.
-
membrane association of Peroxiredoxin 2 in red cells is mediated by the n terminal cytoplasmic domain of band 3
Free Radical Biology and Medicine, 2013Co-Authors: Alessandro Matte, Mariarita Bertoldi, Angela Siciliano, Narla Mohandas, Xiuli An, Antonella Bugatti, Anna Maria Brunati, Marco Rusnati, Elena Tibaldi, Franco TurriniAbstract:Abstract Band 3 (B3), the anion transporter, is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In this study, using a variety of experimental approaches including cross-linking studies, fluorescence and dichroic measurements, surface plasmon resonance analysis, and proteolytic digestion assays, we document that the antioxidant protein Peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in the interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine of RBCs prevented membrane association of PRDX2, implying overlapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes, in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acids of this domain are crucial for PRDX2 membrane association in intact RBCs. These findings imply yet another important role for B3 in regulating red cell membrane function.
-
oxidative stress modulates heme synthesis and induces Peroxiredoxin 2 as a novel cytoprotective response in β thalassemic erythropoiesis
Haematologica, 2011Co-Authors: Lucia De Franceschi, Mariarita Bertoldi, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Franco Turrini, Alessandra Colancecco, Clara Camaschella, Maria Domenica Cappellini, Achille IolasconAbstract:Background β-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to β-thalassemic anemia is only partially understood. Design and Methods We studied erythroid precursors from normal and β-thalassemic peripheral CD34+ cells in two-phase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of δ-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced Peroxiredoxin-2 to H2O2 and generated a recombinant Peroxiredoxin-2 for kinetic measurements in the presence of H2O2 or hemin. Results In β-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of Peroxiredoxin-2. In agreement with these observations in β-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, δ-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant δ-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in β-thalassemic cells. We then addressed the question of the protective role of Peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing Peroxiredoxin-2 in human erythroleukemia K562 cells. We found that Peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that Peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of Peroxiredoxin-2 in β-thalassemia. Conclusions In β-thalassemic erythroid cells the reduction of δ-aminolevulinate synthase-2 activity and the increased expression of Peroxiredoxin-2 might represent two novel stress-response protective systems.
-
OXIDATIVE STRESS MODULATES HEME LEVELS and INDUCES Peroxiredoxin-2 IN β THALASSEMIC ERYTHROPOIESIS as NOVEL CYTOPROTECTIVE RESPONSE
Blood, 2010Co-Authors: Lucia De Franceschi, Mariarita Bertoldi, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Franco Turrini, Alessandra Colancecco, Clara Camaschella, Maria Domenica Cappellini, Achille IolasconAbstract:Abstract 4266 Beta thalassemia (β-thal) syndromes are worldwide distributed congenital red cell disorders. Increased levels of reactive-oxygen-species (ROS) have been reported to contribute to anemia in β-thal but the mechanism(s) involved in cell protection against ROS damage has only partially investigated. Here, we studied in vitro normal and β-thal erythropoiesis in erythroid cell cultures from CD34+ cells isolated from peripheral blood from adult normal volunteers and from homozygous (bcod39) b-thalassemia patients. We showed increased ROS production in β-thal erythropoiesis and we evaluated the effects of ROS on normal and β-thal erythropoiesis. We carried out a proteomic comparative study, validated by coupling Quantitative-Real time PCR and immunoblot analysis of the differently expressed proteins. We found down-regulation in expression of enzymes involved in heme catabolism such as biliverdin reductase (BVR) and heme-oxygenase-1 (HO-1) and up-regulation of two new cytoprotective cysteine-based-systems: Peroxiredoxin-2 (Prx2) and heat-shock-protein-27 (HSP27), while catalase was similarly expressed in both cell models, suggesting a specific pattern of Px2 and HSP27 in β-thal erythroid precursors. We then measured heme levels and during b-thal-erythropoiesis and found that the synthesis of heme was biphasic displaying an increase of heme levels in early phase followed by a decrease in late phase in comparison to controls. Since heme synthesis depends on the erythroid δ-aminolevulinate-synthase isoform (ALAS-2), we evaluated ALAS-2 expression that resulted similar in normal and β-thal erythroid cells. We then showed that ALAS-2 activity was inhibited by both ROS and hemin, suggesting a possible role of heme and ROS levels in regulation of heme biosynthesis in β-thal cells. Since it has been reported that oxidative stress can up-regulate Prx2 expression and that genetically modified cells over-expressing Prx2 are generally more protected from severe oxidative stress (Phalen TJ et al 2006; Rabilloud T et al 2002; Kang SW et al 1998; Zhang P et al 1997), we have hypothesized a cytoprotective role of Prx2 in b-thal-erythropoiesis. We determined that the anti-oxidant Prx2 specifically binds hemin with high and affinity, most likely involving Prx2 cysteine residues. In order to look for the structural determinants to the binding, we noted that both ALAS-2 and Prx2 possess one and two cys-pro motifs, respectively. This motif is generally considered a heme sensor for many proteins able to bind heme and we propose that it could be responsible for heme binding in both enzymes. These data suggest a wider role of Prx2 as both anti-oxidant and heme-binding protein in protective stress-response-systems in β-thal erythropoiesis. Disclosures: No relevant conflicts of interest to declare.
-
Peroxiredoxin 2 expression is increased in β thalassemic mouse red cells but is displaced from the membrane as a marker of oxidative stress
Free Radical Biology and Medicine, 2010Co-Authors: Alessandro Matte, Mariarita Bertoldi, Franco Turrini, Angela Siciliano, Antonella Pantaleo, Maria Estela Campanella, Daniela Spano, Lucia De FranceschiAbstract:Peroxiredoxin 2 (Prx2), the third most abundant cytoplasmic protein in red blood cells (RBCs), is involved in the defense against oxidative stress. Although much is known about Prx2 in healthy RBCs, its role in pathological RBCs remains largely unexplored. Here, we show that the expression and net content of Prx2 are markedly increased in RBCs from two mouse models of β-thalassemia (β-thal; Hbbth/th and Hbbth3/+ strains). We also demonstrate that the increased expression of Prx2 correlates with the severity of the disease and that the amount of Prx2 bound to the membrane is markedly reduced in β-thal mouse RBCs. To explore the impact of oxidative stress on Prx2 membrane association, we examined Prx2 dimerization and membrane translocation in murine RBCs exposed to various oxidants (phenylhydrazine, PHZ; diamide; H2O2). PHZ-treated RBCs, which mimic the membrane damage in β-thal RBCs, exhibited a kinetic correlation among Prx2 membrane displacement, intracellular methemoglobin levels, and hemichrome membrane association, suggesting the possible masking of Prx2 docking sites by membrane-bound hemichromes, providing a possible mechanism for the accumulation of oxidized/dimerized Prx2 in the cytoplasm and the increased membrane damage in β-thal RBCs. Thus, reduced access of Prx2 to the membrane in β-thal RBCs represents a new factor that could contribute to the oxidative damage characterizing the pathology.
Luigia De Falco - One of the best experts on this subject based on the ideXlab platform.
-
Peroxiredoxin 2 a novel regulator of iron homeostasis in ineffective erythropoiesis
Antioxidants & Redox Signaling, 2018Co-Authors: Alessandro Matte, Luigia De Falco, Achille Iolascon, Enrica Federti, Anna Cozzi, Sonia Levi, Narla Mohandas, Alberto Zamo, Mariasole Bruno, Christophe LebouefAbstract:Abstract Aims: Iron overload (IO) is a life-threatening complication of chronic hemolytic disorders such as β-thalassemia. IO results in severe cellular oxidative damage, leading to organ failure. Peroxiredoxin-2 (Prx2), a typical 2-cysteine-(Cys)-Peroxiredoxin, is an important component of the cytoprotective system, but its response to IO is still to be fully defined. Results: We studied the effects of IO on Prx2-knockout mice (Prx2−/−). The absence of Prx2 enhanced toxicity due to IO on erythropoiesis. We found that IO failed to induce the typical hepcidin (Hamp) upregulation in Prx2−/− mice due to its failure to activate the signal transducer and activator of transcription-3 (STAT3) with intact Jak2 signaling. In Prx2−/− mice, the loss of Hamp response was also observed after administration of a single dose of oral iron. When lipopolysaccharide (LPS) was used to explore IL6-STAT3 activation in Prx2−/− mice, STAT3 activation and Hamp upregulation were once again defective. Treatment with PEP-fusion-reco...
-
Peroxiredoxin 2 a novel factor involved in iron homeostasis
Blood, 2015Co-Authors: Alessandro Matte, Luigia De Falco, Achille Iolascon, Enrica Federti, Anne Janin, Sonia Levi, Narla Mohandas, Mariasole Bruno, Giorgia Federico, Christophe LeboeufAbstract:Peroxiredoxin-2 (Prx2), a typical 2-cysteine (Cys) Peroxiredoxin, is a key anti-oxidant system in both normal and pathological erythropoiesis characterized by oxidative stress such as b-thalassemia. We recently showed that the absence of Prx2 worsens s-thalassemic erythropoiesis and related iron-overload (Matte A et al. Antioxid Redox Signal, 2015). Here, we studied the effects of iron overload in a mouse model genetically lacking Prx2 (Prx2-/-). Two months old female wild-type (WT) and Prx2-/- pure bred mice were fed with a diet containing 2.5% carbonyl-iron compared to standard diet treated mice. We evaluated hematologic parameters, red cell indices and reticulocyte count in both mouse strains at baseline and at 30, 49, 60 and 90 days of treatment with carbonyl-iron. We observed a rapid drop in Hct and reticulocyte count in Prx2-/- mice compared to wild-type mice between 30-49 days of iron supplementation with the appearance of severe hyporegenerative anemia at 60 days of treatment in Prx2-/- characterized by a significant reduction in CD44+TER119+Fsc high cells. This was associated with marked increases in apoptotic Prx2-/- orthochromatic erythroblast compared to either baseline values or WT treated mice. In sorted erythroid precursors from iron overload WT mice, Prx2 expression was significantly increased compared to WT under standard diet. We observed a modulation of Erythropherrone (Erfe) expression during erythropoiesis with upregulation of Erfe in WT orthochromatic erythroblast compared to Prx2-/- erythroblasts. In liver from Prx2-/- mice exposed to iron-overload, we found liver iron content similar to WT mice but Pearls stain analysis showed differential iron distribution in cellular components of liver. While iron accumulated in hepatocytes and Kuppfer cells in Prx2-/- mice, iron-deposits were present only in hepatocytes of WT liver. Oxyblot analysis and liver MDA levels were significantly higher in Prx2-/- mice indicating that the absence of Prx2 promotes a severe liver oxidative stress. In WT liver, Prx2 expression was marked increased in a time depend way during iron supplementation, indicating that Prx2 is part of an adaptive cellular response to iron overload. This is in agreement with increased levels of ferritin-H in Prx2-/- mice compared to WT mice. Hepcidin (HAMP) expression was markedly increased in iron-overload WT mice compared to untreated control group, while no major changes were observed in Prx2-/- mice. Tfr2 expression was significantly increased only in livers of iron-overload WT mice, whereas phospho smad 1-5 was significantly increased in both mouse strains in response to iron-overload. The activation of the signaling pathway through Erk-1/2 only in iron-overload WT mice but not in Prx2-/- mice is most likely related to severe oxidative stress in Prx2-/- resulting in switching off of the Erks pathway. Importantly, administration of PEP1-Prx2 fusion protein rescue almost completely the hematologic phenotype with modulation of Erk signaling pathway towards Tfr2 and the smad system, validating our hypothesis of a role for Erk signaling for the observed phenoytpes. Our data highlight Prx2 as novel factor involved in iron homestasis through the control of oxidative stress modulating signaling pathway towards hepcidin expression. Disclosures No relevant conflicts of interest to declare.
-
oxidative stress modulates heme synthesis and induces Peroxiredoxin 2 as a novel cytoprotective response in β thalassemic erythropoiesis
Haematologica, 2011Co-Authors: Lucia De Franceschi, Mariarita Bertoldi, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Franco Turrini, Alessandra Colancecco, Clara Camaschella, Maria Domenica Cappellini, Achille IolasconAbstract:Background β-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to β-thalassemic anemia is only partially understood. Design and Methods We studied erythroid precursors from normal and β-thalassemic peripheral CD34+ cells in two-phase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of δ-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced Peroxiredoxin-2 to H2O2 and generated a recombinant Peroxiredoxin-2 for kinetic measurements in the presence of H2O2 or hemin. Results In β-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of Peroxiredoxin-2. In agreement with these observations in β-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, δ-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant δ-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in β-thalassemic cells. We then addressed the question of the protective role of Peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing Peroxiredoxin-2 in human erythroleukemia K562 cells. We found that Peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that Peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of Peroxiredoxin-2 in β-thalassemia. Conclusions In β-thalassemic erythroid cells the reduction of δ-aminolevulinate synthase-2 activity and the increased expression of Peroxiredoxin-2 might represent two novel stress-response protective systems.
-
OXIDATIVE STRESS MODULATES HEME LEVELS and INDUCES Peroxiredoxin-2 IN β THALASSEMIC ERYTHROPOIESIS as NOVEL CYTOPROTECTIVE RESPONSE
Blood, 2010Co-Authors: Lucia De Franceschi, Mariarita Bertoldi, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Franco Turrini, Alessandra Colancecco, Clara Camaschella, Maria Domenica Cappellini, Achille IolasconAbstract:Abstract 4266 Beta thalassemia (β-thal) syndromes are worldwide distributed congenital red cell disorders. Increased levels of reactive-oxygen-species (ROS) have been reported to contribute to anemia in β-thal but the mechanism(s) involved in cell protection against ROS damage has only partially investigated. Here, we studied in vitro normal and β-thal erythropoiesis in erythroid cell cultures from CD34+ cells isolated from peripheral blood from adult normal volunteers and from homozygous (bcod39) b-thalassemia patients. We showed increased ROS production in β-thal erythropoiesis and we evaluated the effects of ROS on normal and β-thal erythropoiesis. We carried out a proteomic comparative study, validated by coupling Quantitative-Real time PCR and immunoblot analysis of the differently expressed proteins. We found down-regulation in expression of enzymes involved in heme catabolism such as biliverdin reductase (BVR) and heme-oxygenase-1 (HO-1) and up-regulation of two new cytoprotective cysteine-based-systems: Peroxiredoxin-2 (Prx2) and heat-shock-protein-27 (HSP27), while catalase was similarly expressed in both cell models, suggesting a specific pattern of Px2 and HSP27 in β-thal erythroid precursors. We then measured heme levels and during b-thal-erythropoiesis and found that the synthesis of heme was biphasic displaying an increase of heme levels in early phase followed by a decrease in late phase in comparison to controls. Since heme synthesis depends on the erythroid δ-aminolevulinate-synthase isoform (ALAS-2), we evaluated ALAS-2 expression that resulted similar in normal and β-thal erythroid cells. We then showed that ALAS-2 activity was inhibited by both ROS and hemin, suggesting a possible role of heme and ROS levels in regulation of heme biosynthesis in β-thal cells. Since it has been reported that oxidative stress can up-regulate Prx2 expression and that genetically modified cells over-expressing Prx2 are generally more protected from severe oxidative stress (Phalen TJ et al 2006; Rabilloud T et al 2002; Kang SW et al 1998; Zhang P et al 1997), we have hypothesized a cytoprotective role of Prx2 in b-thal-erythropoiesis. We determined that the anti-oxidant Prx2 specifically binds hemin with high and affinity, most likely involving Prx2 cysteine residues. In order to look for the structural determinants to the binding, we noted that both ALAS-2 and Prx2 possess one and two cys-pro motifs, respectively. This motif is generally considered a heme sensor for many proteins able to bind heme and we propose that it could be responsible for heme binding in both enzymes. These data suggest a wider role of Prx2 as both anti-oxidant and heme-binding protein in protective stress-response-systems in β-thal erythropoiesis. Disclosures: No relevant conflicts of interest to declare.