Expression Of Prx Research Articles

Peroxiredoxin-2 (Prx-2) Shows a Dual Role as Antioxidant and Chaperone in Red Cells from a β Thalassemic Mouse Model.

Peroxiredoxins are a ubiquitous family of anti-oxidant enzymes, which have been recently involved in different cellular functions such as cell proliferation or intracellular signaling. In red blood cells (RBCs) Prx-2 is present as scavenger of the reactive oxygen species generated by hemoglobin auto-oxidation as supported also by the evidences in mouse RBCs genetically lacking Prx-2. β thalassemia (thal) is an inherited RBC disorder due to defective β-globin chain synthesis, leading to ineffective erythropoiesis and chronic hemolytic anemia related to severe RBCs membrane oxidative damage. Here, we evaluated Prx-2 expression and localization in RBCs from a mouse model of β thal, which shows biological and clinical features similar to human β thal intermedia. We studied βthal (Hbbth/th) and wild-type mice (WT) at different ages (2- 6 months old), divided into groups of 10 mice each. Since β thal is characterized by high reticulocyte count due to the chronic hemolytic anemia, we divided RBCs into reticulocyte enriched fraction (F1) and dense RBCs fraction (F2), corresponding to the oldest cells. Then, RBCs from F1 and F2 were studied as total RBCs lysate and as RBCs membrane fraction. No significant differences in Prx-2 expression related to animals aging were present within mouse strains. βthal total RBCs lysates showedi.increase expression of Prx-2 as monomers and dimers in both F1 and F2 fractions compared to WT ones;ii.increase dimerization of Prx-2 in F1 compared to F2, while no differences were present between F1 and F2 in WT RBCs, suggesting an upregulation of Prx-2 synthesis in β thal reticulocytes, since it is already present, and a Prx-2 hyperoxidation in circulating β thal RBCs.Since a chaperone activity for Prx-2 has been recently shown in yeasts and bacteria and being the assistance to denaturated proteins one of the major activity of chaperones, we evaluated Prx-2 membrane localization in β thal RBCs, characterized by severe membrane oxidative damage. Prx-2, as monomers and dimers, was recruited to the membrane in both mouse strains, suggesting a possible dual role of Prx-2 as antioxidant and chaperone. We also observed that the amount of Prx-2 bound to the RBCs membrane was higher in WT than in β thal mice, most likely related to either removal of the more severely damaged β thal RBCs from circulation or to perturbation of Prx-2 function in β thal RBCs. In order to evaluate Prx-2 functions we exposed WT RBCs to either Phenylhydrazine (PHZ 10–20 μM), mimicking β thal RBCs membrane damage or H2O2 at low concentration (5 μM). PHZ treatment significantly reduced the amount of Prx-2 monomers and dimers bound to the membrane in a dose dependent manner, whereas H2O2 did not significantly modify them, indicating that PHZ and H2O2 differently affect Prx-2 membrane recruitment. Since Prx-2 functions seem to be related to phosphorylation events, we analysed Prx-2 localization in RBCs lacking protein tyrosin phosphatase- ε (PTP-ε). In the absence of PTP-ε we observed a lower amount of Prx-2 bound to the membrane as monomers, with undetectable Prx-2 dimers compared to WT ones. When PTP-ε RBCs were treated with PHZ, Prx-2 was almost undetectable on RBCs membrane, suggesting a possible role of PTP-ε in changes of Prx-2 oligomeric state. In conclusion, in β thal mouse RBCs Prx-2 expression is up-regulated, is present in both monomeric and dimeric state, and is modulated by oxidative damage and phosphorylation events supporting a dual role of Prx-2 as antioxidant and chaperone.

Heat-Shock Protein-70, -27 and Peroxiredoxin-2 Show Chaperone Function in Sickle Red Cells: Evidences from Transgenic Sickle Cell Mouse Model.

Sickle cell disease (SCD) is a worldwide-distributed hereditary red cell (RBCs) disorder characterized by the pathologic hemoglobin-S (HbS). The acute clinical manifestations of SCD are strictly related to HbS polymerization, to generation of dense RBCs, to their interaction with the abnormal activated vascular endothelial cells and to amplified inflammatory response. To identify new biomarkers associated with acute SCD events, we studied the RBCs membrane proteome from human SCD erythrocytes (n=20) fractioned according to density compared to normal RBCs (n=20), we divided RBCs into 2 fractions: fraction 1 (F1) corresponding to the least dense RBCs (density < 1.074) and fraction 2 (F2) corresponding to the densest RBCs (density > 1.095). None of the patients were under hydroxyurea treatment and they did not receive transfusion in the sixmonths before the study. Bi-dimensional electrophoresis (2-DE) followed by MS-analysis of RBCs F1 and F2 from both control and SCD subjects was carried out and 65 proteins differently expressed were identified. We focused on molecular chaperones. In SCD RBCs, we found that the amount of HSP27, HSP70 and Prx-2 recruited to the membrane was higher than in controls, Prx-2 was present as monomers and dimmers that were more abundant in SCD RBCs than in normal controls. Then, we exposed F1 and F2 fractions from normal and SCD patients to in vitro cellular stress (deoxygenation). In sickle RBCs, deoxygenation induced1.increase membrane recruitment of HSP27 in F1,2.modulation of HSP70 membrane binding in F2;3.reduction of Prx-2 monomers with increase Prx-2 dimerization in F1, but slightly changes in Prx-2 monomers in F2.These data suggest that in SCD RBCs HSP-27, -70 and Prx-2 are recruited to the membrane in response to cellular stress, acting as molecular chaperones to assist proteins in regaining their functional conformation. In order to evaluate their role in vivo and because increase serum levels of HSP70 has been recently reported in few SCD patients, we used transgenic sickle cell SAD mice as a model for SCD and exposed to hypoxia (8%-O2) respectively for 2–12–48 hours, mimicking acute human SCD vaso-occlusive-crisis (VOCs). We studies SAD mice and wild-type mice (C57/B6, WT) divided into groups of 6 animals each. We evaluated HSP70, HSP27 and Prx-2 expression on RBCs membrane, hematological parameters, RBCs density, and cation content. We found that HSP70 and HSP27 bound to RBCs membrane respectively after 12 hours and 48 hours (hrs) of hypoxia, while Prx-2 membrane binding was modulated during hypoxia. At the different time schedule, we observed a marked reduction in red cell K+ at 12 and 48 hrs hypoxia, associated with significant increase of the dense RBCs at 48 hrs hypoxia. Our data indicate that in SCD, HSP70, HSP27 and Prx-2 membrane recruitment is modulated in vivo during acute VOCs, supporting their novel role as RBCs membrane protein protectors and as new markers of severity of RBCs membrane damage during acute sickle VOCs.

Increase in Expression Levels and Resistance to Sulfhydryl Oxidation of Peroxiredoxin Isoforms in Amyloid β-Resistant Nerve Cells

Peroxiredoxins (Prxs) are a ubiquitously expressed family of thiol peroxidases that reduce hydrogen peroxide, peroxynitrite, and hydroperoxides using a highly conserved cysteine. There is substantial evidence that oxidative stress elicited by amyloid beta (Abeta) accumulation is a causative factor in the pathogenesis of Alzheimer disease (AD). Here we show that Abeta-resistant PC12 cell lines exhibit increased expression of multiple Prx isoforms with reduced cysteine oxidation. Abeta-resistant PC12 cells also display higher levels of thioredoxin and thioredoxin reductase, two enzymes critical for maintaining Prx activity. PC12 cells and rat primary hippocampal neurons transfected with wild type Prx1 exhibit increased Abeta resistance, whereas mutant Prx1, lacking a catalytic cysteine, confers no protection. Using an antibody that specifically recognizes sulfinylated and sulfonylated Prxs, it is demonstrated that primary rat cortical nerve cells exposed to Abeta display a time-dependent increase in cysteine oxidation of the catalytic site of Prxs that can be blocked by the addition of the thiol-antioxidant N-acetylcysteine. In support of previous findings, expression of Prx1 is higher in post-mortem human AD cortex tissues than in age-matched controls. In addition, two-dimensional gel electrophoresis and mass spectrometry analysis revealed that Prx2 exists in a more oxidized state in AD brains than in control brains. These findings suggest that increased Prx expression and resistance to sulfhydryl oxidation in Abeta-resistant nerve cells is a compensatory response to the oxidative stress initiated by chronic pro-oxidant Abeta exposure.

Tumor suppressor Prdx1 is a prognostic factor in esophageal squamous cell carcinoma patients

Peroxiredoxins (Prdxs) are a family of antioxidant enzymes that are also known as scavengers of peroxide in mammalian cells. Some reports have shown that the overexpression of Prdx1, which is one of the peroxiredoxins that is a ubiquitously expressed protein, was related to a poor prognosis in several types of human cancers. In this study, we investigated the expression levels of Prdx1 in esophageal squamous cell carcinoma by immunohistochemistry, and the correlation between the Prdx1 expression and the clinical status was elucidated. Immunohistochemical staining was performed in 114 samples which were collected from surgical esophageal cancer specimens. Cytoplasmic staining of Prdx1 was evaluated based on the following scoring criteria: Grade I, negative or weak staining; Grade II, moderate staining; and Grade III, strong staining. The percentage of patients with a Grade I expression of Prx1 was 20% (23 of 114), 44% had Grade II (50 of 114), and 36% had Grade III (41 of 114). The Prdx1 immunoreactivity showed an inverse significant correlation with T-category (P<0.0001), lymph node metastasis (P=0.048), and stage (P=0.001). In addition, the patients with tumors exhibiting a reduced Prdx1 expression had shorter overall survival (P=0.022) in comparison to the patients with tumors which had a higher Prdx1 expression. Currently, Prdx1 has been shown to act as a tumor suppressor. Our results provide strong evidence that the reduced Prdx1 expression is an important factor in esophageal squamous cancer progression and could serve as a useful prognostic marker.

Isoform-specific downregulation of peroxiredoxin in human failing myocardium

Peroxiredoxins (Prx) are a family of antioxidant thioredoxin or glutathione dependent peroxidases. The major functions of Prx comprise modulation of signalling cascades that apply hydrogen peroxide (H 2O 2) and cellular protection against oxidative stress. Nothing is known about Prx isoforms in human myocardium. We investigated the protein expression of Prx isoforms 1–6 in human non-failing (NF, donor hearts, n = 6, male, age: 53.3 ± 2.1 years) and failing myocardium (DCM, orthotopic heart transplantation, dilated cardiomyopathy, n = 15, male, 57.0 ± 1.7 years). In addition, we performed immunohistochemical stainings and measured Prx 4 mRNA expression levels (RNAse protection assay). The protein expression of Prx 1–2 was similar in NF and DCM. The protein expression of Prx 3–6 and the mRNA-expression of Prx 4 were decreased in DCM. Immunohistochemical analyses provided evidence that all Prx isoforms are present in cardiomyocytes and endothelial cells. Whereas Prx 1–5 staining was more pronounced in endothelial cells, Prx6 staining was more evident in cardiomyocytes. This study provides evidence that Prx are differentially regulated in DCM. The selective downregulation of peroxiredoxin 3–6 isoforms may point towards a subcellular specific dysregulation of the antioxidative defence during the development of DCM.

Elevated Peroxiredoxin 1, but not NF-E2–Related Factor 2, Is an Independent Prognostic Factor for Disease Recurrence and Reduced Survival in Stage I Non–Small Cell Lung Cancer

Lung cancer is the leading cause of cancer death with chance of survival restricted to a subset of non-small cell lung cancer (NSCLC) patients able to undergo surgical resection. However, the recurrence rate of NSCLC after surgery remains high with few prognostic indicators of clinical outcome. Peroxiredoxin1 (Prx1) is shown to be elevated in various cancers and confers an aggressive survival phenotype. We recently cloned the prx1 promoter and found that NF-E2-related factor 2 (Nrf2) is a key transcription factor for prx1 up-regulation. Previous studies suggest that Nrf2 may be constitutively activated in NSCLC. Based on the above information, we investigated whether Prx1 and/or Nrf2 levels have prognostic significance in stage I NSCLC. Immunohistochemical expression of Prx1 and Nrf2 was evaluated in paraffin-embedded tissues from 90 patients who underwent a curative surgical resection. Increased expression of cytosolic Prx1 (66.7%) and nuclear Nrf2 (61.8%) was observed in this series. Prx1 elevation, but not Nrf2, correlated with reduced recurrence-free survival and overall survival on univariate (P = 0.01 and P = 0.03) and multivariate (P = 0.003 and P = 0.005) analyses. This is the first study to test the prognostic significance of Prx1 and Nrf2 in human cancers. Our results show that Prx1 expression status predicts for recurrence and shorter survival in stage I NSCLC after surgery. Considering the possible role of Prx1 and Nrf2 in radioresistance/chemoresistance, it warrants future investigation to evaluate whether elevated Prx1 and/or Nrf2 levels are predictive of treatment response in advanced lung cancer and other malignancies.

Role of Cdk5-Mediated Phosphorylation of Prx2 in MPTP Toxicity and Parkinson's Disease

We reported previously that calpain-mediated Cdk5 activation is critical for mitochondrial toxin-induced dopaminergic death. Here, we report a target that mediates this loss. Prx2, an antioxidant enzyme, binds Cdk5/p35. Prx2 is phosphorylated at T89 in neurons treated with MPP+ and/or MPTP in animals in a calpain/Cdk5/p35-dependent manner. This phosphorylation reduces Prx2 peroxidase activity. Consistent with this, p35-/- neurons show reduced oxidative stress upon MPP+ treatment. Expression of Prx2 and Prx2T89A, but not the phosphorylation mimic Prx2T89E, protects cultured and adult neurons following mitochondrial insult. Finally, downregulation of Prx2 increases oxidative stress and sensitivity to MPP+. We propose a mechanistic model by which mitochondrial toxin leads to calpain-mediated Cdk5 activation, reduced Prx2 activity, and decreased capacity to eliminate ROS. Importantly, increased Prx2 phosphorylation also occurs in nigral neurons from postmortem tissue from Parkinson's disease patients when compared to control, suggesting the relevance of this pathway in the human condition.

RNA-binding Protein RBMS3 Is Expressed in Activated Hepatic Stellate Cells and Liver Fibrosis and Increases Expression of Transcription Factor Prx1

Hepatic stellate cells (HSCs) are mesenchymal cells of the liver, activation of which is responsible for excessive synthesis of extracellular matrix, including type I collagen, and development of liver fibrosis. The activation of HSCs is driven by transcription factors and pair-related homeobox transcription factor Prx1 was identified as one of the transcription factors involved in this process, because transcription of collagen α1(I) gene is stimulated by Prx1 in HSCs and in the liver. Here, we show that expression of the RNA-binding protein RBMS3 is upregulated in the activation of HSCs and fibrotic livers. Immunoprecipitation followed by differential display identified Prx1 mRNA as one of the mRNAs interacting with RBMS3. The RBMS3 sequence-specific binding site was mapped to 60 nt located 1946 nt 3′ of the stop codon of Prx1 mRNA. Ectopic expression of RBMS3 in quiescent HSCs, which express trace amounts of type I collagen, increased expression of Prx1 mRNA and collagen α1(I) mRNA. Expression of reporter Prx1 mRNA containing the RBMS3 binding site was higher than the mRNA lacking this site. Over-expression of RBMS3 further increased the steady-state level of the reporter mRNA-containing RBMS3 binding site, but had no effect on the mRNA lacking this site. Binding of RBMS3 to the Prx1 3′ UTR increased the half-life of this mRNA, resulting in increased protein synthesis. These results suggest that RBMS3, by binding Prx1 mRNA in a sequence-specific manner, controls Prx1 expression and indirectly collagen synthesis. This is the first description of the function of RBMS3, as a key regulator of profibrotic potential of HSCs, representing a novel mechanism by which activated HSCs contribute to liver fibrosis.

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 2O 2. The total expression of Prx I and VI was increased in response to H 2O 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 3H-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.

Expression of immune-related genes in larval stages of the giant tiger shrimp, Penaeus monodon

Shrimp undergo several morphologically different stages during development and therefore the expression of some immune-related genes such as prophenoloxidase ( proPO), peroxinectin ( Prx), crustin ( Crus), penaeidin ( Pen), transglutaminase ( TGase), haemocyanin ( Hc) and astakine ( Ak) were determined during larval development of the shrimp ( Penaeus monodon), i.e. nauplius 4 (N4), protozoea 1 and 3 (Z1 and 3), mysis 3 (My 3), post-larvae 3 (PL3) and also in haemocytes of juveniles. Semi-quantitative RT-PCR analysis showed that all transcripts were already present in the early larval stage of N4 but at different levels. The transcript of proPO was found to be extremely low or even absent at N4, whereas Prx, Crus, Pen, TGase, Hc and Ak were significantly expressed at all larval stages. Up to now expression of proPO and Prx has only been reported from haemocytes in crustaceans and in this study Prx also appeared to be expressed in stages which appear to lack haemocytes. Thus, this may suggest that Prx is expressed in other cells than haemocytes. It is well known among invertebrates that the proPO system plays a crucial role as an immune effector molecule against microbes. However, in this study, the transcript of proPO was low during the larval stages and hardly present at all at N4. This might indicate that the development of immune-competent haemocytes during the larval stages is not completed and as a consequence they are likely to be more susceptible to infectious diseases during these stages.

Differential protein expression of peroxiredoxin I and II by benzo(a)pyrene and quercetin treatment in 22Rv1 and PrEC prostate cell lines

Mechanisms of benzo(a)pyrene (BaP)-mediated toxicity and chemopreventative potential of quercetin in prostate cancer are poorly understood. Two-dimensional gel electrophoresis was used to map the differences in protein expression in BaP (1 microM)- and quercetin (5 microM)-treated 22Rv1 human prostate cancer cells. As compared to DMSO, 26 proteins in BaP and 41 proteins in quercetin were found to be differentially expressed (+/-2-fold). Western blots confirmed that BaP increased peroxiredoxin (Prx) Prx I and decreased Prx II in 22Rv1 cells. Similar results were found in PrEC normal prostate epithelial cells. Quercetin (up to 10 microM) upregulated Prx II without altering Prx I levels in 22Rv1 cells whereas in PrEC cells, it did not alter the constitutive protein expression of Prx I or II. The lack of quercetin-mediated changes in Prx expression suggests that quercetin does not interfere with H(2)O(2) levels, and thus may have no deleterious effect in normal prostate cells. Quercetin inhibited both BaP-mediated effects on Prx I and II in 22Rv1 cells. In PrEC cells, quercetin inhibited BaP-mediated upregulation of Prx I and had tendency to neutralize BaP-mediated downregulation of Prx II. Quercetin also inhibited BaP-induced concentrations of reactive oxygen species in both 22Rv1 and PrEC cells. These results suggest that Prx I and II may be involved in BaP-mediated toxicity and the potential chemopreventative mechanisms of quercetin.

Expression of 1-Cys Peroxiredoxin in the Corneal Wound-Healing Process

To study the regulation by mitomycin C (MMC) of 1-cys peroxiredoxin (Prx) expression during the corneal wound-healing process and its induction pathway in cultured bovine keratocytes (BKs). Rat corneas were excised at 4 hours, 12 hours, 1 day, 3 days, and 7 days after photorefractive keratectomy (PRK). Expression of 1-cys Prx in the corneas was examined by Northern blot and immunoblot analyses. Cultured BKs were exposed to 0.02% MMC for 5 minutes and maintained under normal culture conditions for different time periods. Subsequently, levels of 1-cys Prx and extracellular signal-regulated kinase (ERK)1/2 expression were measured by immunoblot analysis using polyclonal 1-cys Prx, ERK1/2, or phospho-ERK1/2 antibodies. To inhibit ERK1/2 activation, the BKs were pretreated with 50 micromol/mL PD98059 for 1 hour before MMC exposure and incubated in complete medium with or without PD98059 for 24 hours. MMC-induced cytotoxicity was determined by colorimetric cell-counting kit-8 assay. Increased levels of 1-cys Prx expression were seen in wounded rat corneas at 12 hours after injury and reached the highest level between 1 and 3 days, during which time active proliferation occurred. Induction of 1-cys Prx expression was obvious in proliferating BKs stimulated by growth factors. MMC treatment in cultured BKs resulted in increased expression of 1-cys Prx and phospho-ERK1/2 in a time-dependent manner. Treatment with 50 micromol/mL PD98059 significantly inhibited the active ERK1/2 and 1-cys Prx expression induced by MMC, leading to synergistic cytotoxicity in the BKs. 1-Cys Prx may function as an important enzyme in cell proliferation during the postinjury corneal wound-healing process. Furthermore, the induction of 1-cys Prx expression through the ERK1/2 signaling pathway may contribute to cellular defense against cytotoxic agents, thus playing an important role in cell survival.

Tenascin-C is induced by mutated BMP type II receptors in familial forms of pulmonary arterial hypertension

Familial forms of human pulmonary arterial hypertension (FPAH) have been linked to mutations in bone morphogenetic protein (BMP) type II receptors (BMPR2s), yet the downstream targets of these receptors remain obscure. Here we show that pulmonary vascular lesions from patients harboring BMPR2 mutations express high levels of tenascin-C (TN-C), an extracellular matrix glycoprotein that promotes pulmonary artery (PA) smooth muscle cell (SMC) proliferation. To begin to define how TN-C is regulated, PA SMCs were cultured from normal subjects and from those with FPAH due to BMPR2 mutations. FPAH SMCs expressed higher levels of TN-C than normal SMCs. Similarly, expression of Prx1, a factor that drives TN-C transcription, was elevated in FPAH vascular lesions and SMCs derived thereof. Furthermore, Prx1 and TN-C promoter activities were significantly higher in FPAH vs. normal SMCs. To delineate how BMPR2s control TN-C, we focused on receptor (R)-Smads, downstream effectors activated by wild-type BMPR2s. Nuclear localization and phosphorylation of R-Smads was greater in normal vs. FPAH SMCs. As well, indirect blockade of R-Smad signaling with a kinase-deficient BMP receptor Ib upregulated TN-C in normal SMCs. Because ERK1/2 MAPKs inhibit the transcriptional activity of R-Smads, and because ERK1/2 promotes TN-C transcription, we determined whether ERK1/2 inhibits R-Smad signaling in FPAH SMCs and whether this activity is required for TN-C transcription. Indeed, ERK1/2 activity was greater in FPAH SMCs, and inhibition of ERK1/2 resulted in nuclear localization of R-Smads and inhibition of TN-C. These studies define a novel signaling network relevant to PAH underscored by BMPR2 mutations.

The function of peroxiredoxins in plant organelle redox metabolism

In 1996, cDNA sequences referred to as plant peroxiredoxins (Prx), i.e. a 1-Cys Prx and a 2-Cys Prx, were reported from barley. Ten years of research have advanced our understanding of plant Prx as thiol-based peroxide reductases with a broad substrate specificity, ranging from hydrogen peroxide to alkyl hydroperoxides and peroxinitrite. Prx have several features in common. (i) They are abundant proteins that are routinely detected in proteomics approaches. (ii) They interact with proteins such as glutaredoxins, thioredoxins, and cyclophilins as reductants, but also non-dithiol-disulphide exchange proteins. By work with transgenic plants, their activity was shown to (iii) affect metabolic integrity, (iv) protect DNA from damage in vitro and as shown here in vivo, and (v) modulate intracellular signalling related to reactive oxygen species and reactive nitrogen species. (vi) In all organisms Prx are encoded by small gene families that are of particular complexity in higher plants. A comparison of the Prx gene families in rice and Arabidopsis thaliana supports previous suggestions on Prx function in specific subcellular and metabolic context. (vii) Prx gene expression and activity are subjected to complex regulation realized by an integration of various signalling pathways. 2-Cys Prx expression depends on redox signals, abscisic acid, and protein kinase cascades. Besides these general properties, the chloroplast Prx have acquired specific roles in the context of photosynthesis. The thioredoxin-dependent peroxidase activity can be measured in crude plant extracts and contributes significantly to the overall H(2)O(2) detoxification capacity. Thus organellar Prx proteins enable an alternative water-water cycle for detoxification of photochemically produced H(2)O(2), which acts independently from the ascorbate-dependent Asada-Halliwell-Foyer cycle. 2-Cys Prx and Prx Q associate with thylakoid membrane components. The mitochondrial PrxII F is essential for root growth under stress. Following a more general introduction, the paper summarizes present knowledge on plant organellar Prx, addressing Prx in signalling, and also suggests some lines for future research.

Impairment of mitochondrial anti-oxidant defence in SOD1-related motor neuron injury and amelioration by ebselen

There is now compelling evidence of mitochondrial dysfunction in motor neuron disease (MND), but the molecular basis of these abnormalities is unknown. It is also unclear whether the observed mitochondrial dysfunction plays a central role in disease pathogenesis, and if so, whether its amelioration might present therapeutic opportunities. We adopted a candidate generation approach using proteomics to screen for changes in mitochondrial protein expression in a well-validated cell-culture model of superoxide dismutase 1 (SOD1) related familial MND (fMND). Changed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy. Protein candidates included apoptotic regulators, anti-oxidants and components of the electron transport chain. Confirmatory Western blotting was performed, and validated protein expression changes were further investigated. Peroxiredoxin 3 (Prx3), a mitochondrial thioredoxin-dependent hydroperoxidase, is downregulated in the presence of mutant SOD1 in both our cell-culture model and in the spinal cord mitochondria of mutant SOD1 transgenic mice. We confirm the expression of Prx3 within the mitochondria of spinal motor neurons in mouse and humans by immunohistochemistry. Using quantitative real-time PCR (Q-PCR), we show that Prx3 is also downregulated in spinal motor neurons from patients with both sporadic (sMND) and SOD1-related fMND. In a disease characterized by oxidative stress, this represents a potentially important deficit in mitochondrial anti-oxidant defence. Recent evidence suggests that oxidative stress from aberrant copper chemistry may not play a major part in the pathogenesis of SOD1-related fMND. From the results of this study we propose disruption of mitochondrial anti-oxidant defence as an alternative mechanism whereby mutant SOD1 may generate oxidative stress within motor neurons. We further demonstrate that ebselen, an anti-oxidant drug already safely used in human studies and that acts as a Prx mimic, is able to ameliorate the toxicity of mutant SOD1 in our cell-culture model. We conclude by showing that ebselen is capable of inducing transcription of the anti-oxidant response element (ARE) and postulate that ebselen may act both by the transcriptional upregulation of anti-oxidant proteins, and directly as an anti-oxidant in its own right.

Oxidative/nitrosative stress and peroxiredoxin 2 are associated with grade and prognosis of human renal carcinoma

Peroxiredoxins (Prxs) 1-6 were assessed in 138 renal cell carcinomas (RCC) using immunohistochemistry and selected samples by Western blotting analysis. Oxidative/nitrosative damage was evaluated using nitrotyrosine immunoreactivity. The expressions of Prxs were correlated with tumor grade and survival and nitrotyrosine reactivity. Non-malignant kidney tubular cells showed positivity with variable intensity for all six Prxs. In RCCs, most cases were positive for Prxs 1 and 2, while only 15-20% of tumors showed expression for Prxs 3 and 4. Prx 2 was associated with tumors of a lower grade (p=0.009) and with a lower frequency of distant metastases (p=0.046). Patients with tumors expressing Prx2 had better prognosis (p=0.027). Instead, nitrotyrosine was significantly associated with high grade tumors (p=0.001). Compared with the non-malignant kidney tubular cells, low Prx expression in the tumor cells can make them more susceptible to oxidative damage. Prx 2 was more abundantly expressed in low grade tumors, suggesting that this protein could play a role in preventing the development of oxidative damage, which in turn can lead to the activation of pathways leading to aggressive tumors.

High Expression of Antioxidant Proteins in Dendritic Cells

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

A putative proteinase gene is involved in regulation of landomycin E biosynthesis inStreptomyces globisporus1912

The prx gene, which is highly homologous to putative proteinases, has been identified by sequencing in the vicinity of the biosynthetic gene cluster for landomycin E (LaE) biosynthesis (lnd) in Streptomyces globisporus 1912. The S. globisporus Pro6 gene, deficient in prx, produced fivefold less LaE than the parental strain. The expression of prx in S. globisporus Pro6 restored LaE production to wild-type levels, whereas expression of the pathway-specific regulatory gene lndI did not. The introduction of additional copies of prx into the wild-type strain using a pSG5-based plasmid, pKC1139, led to a 2.7-fold increase in LaE production. These results indicate that prx is a novel regulatory gene for LaE biosynthesis.

The Inhibition of Human Telomerase Reverse Transcriptase Expression by Peroxiredoxin I and c-Myc in Prostatic Cancer Cells

Purpose: We evaluated the hypothesis that the telomerase expression is associated with c-Myc and peroxiredoxin I (Prx I) in patients with prostate cancer. The study determined the link between Prx I, c-Myc and human telomerase reverse transcriptase (hTERT) in prostate cancer cells. Materials and Methods: The cDNA of the Prx I gene was obtained by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification. Cotransfections were performed by using a hTERT luciferase reporter plasmid and each expression vector as indicated (c-Myc or Prx I). Empty vectors were used as controls for determining the basal promoter activity. RT-PCR was performed to evaluate the effect of the DEM-induced Prx I mRNA expression. Luciferase assay was performed to evaluate the inhibitory effect of transfected Prx I and the DEM induced Prx I on the transcriptional activity of hTERT in the human prostatic cancer cell lines PC-3 and DU-145. Results: In this study, we found that Prx I could inhibit hTERT expression through direct interaction with c-Myc protein in the prostate cancer cell lines. In addition, it was obvious that Prx I could interact with c-Myc protein. We also found that DEM could induce upregulation of the Prx I mRNA expression and that the increased expression of Prx I could downregulate the expression of hTERT. Conclusions: Our results demonstrated a direct link between Prx I, c-Myc and hTERT, and we suggest that Prx I regulates cellular immortalization through c-Myc and hTERT, which is activation step in carcinogenesis. (Korean J Urol 2006;47:418-425)

Regulating Peroxide Signaling

Platelet-derived growth factor (PDGF) stimulates the production of H 2 O 2 , which acts as a second messenger to regulate the tyrosine phosphorylation of various proteins. Choi et al . found that, in mouse embryonic fibroblasts (MEFs) lacking the cellular peroxidase 2-Cys peroxiredoxin type II (Prx II -/- MEFs), PDGF elicited twice the peak H 2 O 2 production that it elicited from wild-type MEFs. PDGF-induced total tyrosine phosphorylation and phosphorylation of tyrosine residues of phospholipase Cγ1 (PLC-γ1) that are required for its activation were also enhanced in Prx II -/- MEFs. Retroviral expression of human Prx II in Prx II -/- MEFs inhibited PDGF-induced tyrosine phosphorylation and inositol-1,4,5-trisphosphate production, as well as attenuating PDGF-stimulated cell proliferation, migration, and adhesion. The PDGF receptor (PDGFR) in Prx II -/- MEFs was hyperphosphorylated on residues involved in the activation of PDGFR kinase activity (an effect that was inhibited by expression of Prx II) and showed an increased in vitro phosphorylation of PLC-γ1. PDGF promoted the association of Prx II with PDGFR-β, where it appeared to attenuate the oxidative inactivation of protein tyrosine phosphatases. In a mouse model of restenosis (in which vascular smooth muscle cells undergo PDGF-dependent proliferation and migration), Prx II -/- mice showed increased thickening of the innermost layer of injured carotid arteries compared with wild-type mice. Thus, Prx II appears to regulate PDGF signaling and to play a role in cardiovascular lesions that occur in atherosclerosis or following injury. M. H. Choi, I. K. Lee, G. W. Kim, B. U. Kim, Y.-H. Han, D.-Y. Yu, H. S. Park, K. Y. Kim, J. S. Lee, C. Choi, Y. S. Bae, B. I. Lee, S. G. Rhee, S. W. Kang, Regulation of PDGF signalling and vascular remodelling by peroxiredoxin II. Nature 435 , 347-353 (2005). [PubMed]