Unraveling the effects of peroxiredoxin 2 nitration; role of C-terminal tyrosine 193.

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of CP, which occurs when a second molecule of peroxide reacts with the CP in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the CP sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients.

PURPOSE: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. METHODS: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. RESULTS: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). CONCLUSION: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.

Peroxiredoxins are important for the regulation of hydrogen peroxide concentrations in ticks and tick cell line.

Ticks are obligate hematophagous ectoparasites, as they need to feed blood from vertebrate hosts for development. Host blood contains high levels of iron. Host-derived iron may lead to high levels of reactive oxygen species (ROS), including hydrogen peroxide (H2O2). Since a high concentration of H2O2 causes serious damage to organisms, this molecule is known to be a harmful chemical compound for aerobic organisms. On the other hand, the transparent method is compatible with chemical fluorescent probes. Therefore, we tried to establish the visualizing method for H2O2 in unfed tick tissues. The combination method of a chemical fluorescent probe (BES-H2O2-Ac) with the transparent method, Scale, demonstrated in unfed tick tissues that H2O2 and paraquat could induce oxidative stress in the tissues, such as the midgut and ovary. In addition, an H2O2 detection method using BES-H2O2-Ac was established in Ixodes scapularis embryo-derived cell line (ISE6) in vitro to evaluate the antioxidant activity of peroxiredoxins (PRXs), H2O2 scavenging enzymes, against H2O2 in the cells. The effects of paraquat in ISE6 cells were also observed in the PRXs gene-silenced ISE6 cells. A high intensity of H2O2 fluorescence induced by paraquat was observed in the PRX gene-knockdowned cells. These results suggest that H2O2 and paraquat act as an H2O2 inducer, and PRX genes are important for the regulation of the H2O2 concentration in unfed ticks and ISE6 cells. Therefore, this study contributes to the search for H2O2 visualization in ticks and tick cell line and furthers understanding of the tick's oxidative stress induced by H2O2.

The role of peroxiredoxin I in cisplatin-induced ototoxicity.

OBJECTIVE: Peroxiredoxin (Prx) is a new family of antioxidative proteins. Prx I is ubiquitously expressed in various tissues and is important in the defense of tissues from increases in reactive oxygen species (ROS). The present study was designed to examine the expression of Prx subtypes in the mouse cochlea and to show the possible involvement of Prx I in protecting the cochlea against cisplatin ototoxicity. METHODS: Postnatal-day-3-to-5 wildtype mice and Prx I-deficient mice were used. Prx expression in the cochlea was assessed by real-time PCR assay. Prx I protein expression was examined by immunofluorescence staining. Cochlear explants were exposed to 2, 5, and 10-µM cisplatin for 48h, and the cochlear hair cell losses of the wildtype and Prx I-deficient mice were compared. In addition, the histologic features of the cochlear lateral wall were examined after cisplatin incubation. RESULTS: mRNAs of all Prx subtypes were expressed in the mouse cochlea. Prx I was one of the abundant subtypes and was upregulated after 48-h exposure to 5-µM cisplatin. Immunofluorescence staining showed the ubiquitous expression of Prx I in the cochlea. No difference in cochlear hair cell loss induced by cisplatin was found between the wildtype mice and the Prx I-deficient mice. However, spiral ligament fibrocytes of Prx I-deficient mice were significantly sensitive to cisplatin at 20-µM or lower. CONCLUSION: Prx I is important for protection of at least the spiral ligament fibrocytes of the cochlear lateral wall in cisplatin ototoxicity.

Oxidative stress and altered expression of peroxiredoxin genes family (PRDXS) and sulfiredoxin-1 (SRXN1) in human lung tissue following exposure to sulfur mustard.

BACKGROUND: Sulfur mustard (SM) is a potent and mutagenic agent that targets human lung tissue. PURPOSE OF THE STUDY: The purpose of this investigation is to characterize the expression of sulfiredoxin-1 (SRXN1) and peroxiredoxin (PRDXs) genes and oxidative stress (OS) status in human lung after exposure to SM. MATERIALS AND METHODS: Lung biopsy specimens bronchoalveolar lavage (BAL) fluids were provided from SM-exposed patients (n = 6) and controls (n = 5). Changes in gene expression were measured using RT(2) Profiler PCR Array. OS was considered by measuring BAL fluid levels of malondialdehyde (MDA) and protein carbonyls (PC). RESULTS: Mean of MDA and PC values in BAL fluid of patients (0.6467 ± 0.05922 nmol/l and 1.391 ± 0.421 nmol/mg, respectively) was higher than in controls (0.486 ± 0.04615 nmol/l and 0.949 ± 0.149 nmol/mg, respectively). Expression of all examined genes was in the order PRDX1> PRDX3> PRDX6> SRXN1> PRDX2> PRDX4> PRDX5. Among the most upregulated genes was the PRDX1, which was overexpressed by 10.1029-fold (p = 0.000634). SM-exposed individuals demonstrated expression of PRDX3 4.6231 (p = 0.000134), PRDX6 3.4964 (p = 0.001102), SRXN1 3.3719 (p < 0.0001) and PRDX2 2.7725-folds (p = 0.000383) higher than those of controls that reveal. CONCLUSIONS: Upregulation of PRDXs and SRXN1 genes may be because of reactive oxygen species (ROS) production and OS in lung tissue of patients after SM exposure. Expression of SRXN1 and PRDXNs genes, especially I, II, III, and VI is increased in SM-injured lungs, suggesting the induction of cellular responses to increased production of ROS and OS in lung of the patients. Therefore, sulfiredoxin and peroxiredoxins can be targeted as biomarkers of OS in these patients.

ARF7 increases the endogenous contents of castasterone through suppression of BAS1 expression in Arabidopsis thaliana.

Homeostasis of brassinosteroids (BRs) maintained by the balance between their biosynthesis and inactivation is important to coordinate the diverse physiological and developmental responses of plants. Although BR signaling regulates the endogenous levels of BRs via negative feedback regulation, it remains largely unknown how the biosynthesis and inactivation of BR are triggered. BAS1 encodes CYP734A1, which inactivates the biologically active BRs via C-26 hydroxylation and is down-regulated by a BR-responsive transcription factor, BZR1. Here it is demonstrated that the expression of the BAS1 gene is regulated by auxin response factors (ARFs) in Arabidopsis thaliana. Two successive E-box motifs on the BAS1 promoter function as BZR1 binding sites and are essential for BR-regulated BAS1 expression. The expression of BAS1 is increased in the arf7 and arf7arf19 mutants. The endogenous level of bioactive BR, castasterone, is greatly decreased in those mutants. ARF7 can bind to the E-box motifs of the BAS1 promoter where BZR1 binds, suggesting that ARF7 and BZR1 mutually compete for the same cis-element of the BAS1 promoter. Additionally, ARF7 directly interacts with BZR1, which inhibits their DNA binding activities and regulation of BAS1 expression. In conclusion, auxin signaling via ARF7 directly modulates the expression of BAS1 by competition with BZR1, thereby increasing the level of castasterone and promoting growth and development in A. thaliana.

Stem Cell-Derived Bioactive Materials Accelerate Development of Porcine In Vitro-Fertilized Embryos.

Stem cells show the capability to proliferate in an undifferentiated state with long-term self-renewal, which gives the cells advantages for use as bioactive material (BM) for embryo culture in vitro. The objective of this experiment was to investigate the effect of two BMs-human adipose tissue-derived mesenchymal stem cell BM (hAT-MSC-BM) and human embryonic stem cell-derived BM (hESC-BM)-on porcine embryo development compared to commonly used bovine serum albumin (BSA) or serum treatment groups. In vitro-fertilized (IVF) embryos were cultured in PZM-5 with 4 mg/mL BSA until day 4 and equally divided into four groups. Starting from day 4 (until day 6), each group was treated with the following protein additives: 4 mg/mL BSA (control), 10% fetal bovine serum (FBS), 10% hAT-MSC-BM, or 10% hESC-BM. Our results show FBS- and two other BM-treated groups showed significant increases in blastocyst formation rate, hatching rate, and total cell number compared with the control group (p<0.05). The hAT-MSC-BM and hESC-BM treatment groups presented better-quality embryo development, especially from the middle expanding stage to hatching. In particular, the hAT-MSC-BM-treated group showed the highest developmental potential of all groups and formed the most expanding-stage blastocysts. The relative expression of reprogramming-related transcription factor (POU5F1, SOX2, DPPA5, and CDH1), antioxidant (PRDX5), and apoptosis (BCL2L1 and BIRC5) genes also increased in two types of BMs compared to the control. In addition, we investigated the protein synthesis of the tight junction- and gap junction-related genes, connexin 43 and zonula occludens-1 (ZO-1); these increased more than in the control. These results demonstrate that stem cell-derived BMs accelerate porcine preimplantation embryo development and that the BMs would be helpful in the development of preimplantation embryos.

[Effects of dioscin on apoptosis in pancreatic cancer MiaPaCa-2 cells and its mechanism].

OBJECTIVE: The aim of this study was to observe the effects of dioscin on apoptosis and on expression of PRDX1 in pancreatic cancer MiaPaCa-2 cells in vitro. METHODS: MTT assay was used to detect the growth rate among the medication groups treated with different concentrations of dioscin. The apoptosis rate was determined by annexin V-fluorescein isothiocyanate/propidium iodide double staining and flow cytometry. Western blot analysis was used to assay the expression of PRDX1 and apoptotic proteins in the cells. Reactive oxygen species (ROS) formation was measured by 2'7'-dichlorofluorescein diacetate (DCFH-DA). RESULTS: Dioscin considerably inhibited the proliferation of MiaPaCa-2 cells in vitro. The inhibitory action was enhanced in a dose-dependent manner. The levels of intracellular ROS detected with DCFH-DA were highly increased after dioscin treatment. The flow cytometry analysis using annexin V-PI staining showed that compared with the apoptotic rate of control group [(3.5 ± 0.7)%], 2.5 µmol/L and 5 µmol/L dioscin induced apoptosis in (28.4 ± 0.9)% and (49.6 ± 2.7)% MiaPaCa-2 cells, and Western blot analysis showed that apoptotic proteins Bax and cleaved caspase-3 expressions were increased and antiapoptotic protein Bcl-2 expression was decreased. In addition, these effects could be blocked by antioxidant N-acetylcysteine (NAC) administration, and the apoptotic rates decreased to (10.8 ± 2.3)% and (18.8 ± 3.0)%, respectively. We further observed the decrease of PRDX1 expression after dioscin treatment. Moreover, after PRDX1 overexpression, dioscin treatment no longer induced high levels of ROS and apoptosis, and the apoptotic rate was decreased to (21.3 ± 5.9)%. CONCLUSION: Dioscin can down-regulate the PRDX1 expression, and then induces ROS-mediated apoptosis in cancer cells.

p10, the N-terminal domain of p35, protects against CDK5/p25-induced neurotoxicity.

Cyclin-dependent kinase 5(CDK5) in complex with its activator, p35 (protein of 35 kDa), is essential for early neurodevelopment in mammals. However, endogenous cleavage of p35 to p25 is associated with neuron death and neurodegenerative disease. Here we show that a peptide (p10') encoding the N-terminal domain of p35 protects against CDK5/p25-induced toxicity in neurons. p10' also prevented the death of neurons treated with the neurotoxin, 1-methyl-4-phenylpyridinium (MPP(+)), which induces conversion of endogenous p35 to p25, and Parkinson disease (PD)-like symptoms in animals. MPP(+) induces CDK5/p25-dependent phosphorylation of peroxiredoxin 2 (Prx2), resulting in inhibition of its peroxireductase activity and accumulation of reactive oxygen species (ROS). We found that p10' expression inhibited both Prx2 phosphorylation and ROS accumulation in neurons. In addition, p10' inhibited the p25-induced appearance of antigen of the Ki67 antibody (Ki67) and phosphohistone H2AX (γH2AX), classic markers of cell cycle activity and DNA double-strand breakage, respectively, associated with neuron death. Our results suggest that p10 (protein of 10 kDa) is a unique prosurvival domain in p35, essential for normal CDK5/p35 function in neurons. Loss of the p10 domain results in CDK5/p25 toxicity and neurodegeneration in vivo.

The complement regulator CD46 is bactericidal to Helicobacter pylori and blocks urease activity.

BACKGROUND & AIMS: CD46 is a C3b/C4b binding complement regulator and a receptor for several human pathogens. We examined the interaction between CD46 and Helicobacter pylori (a bacterium that colonizes the human gastric mucosa and causes gastritis), peptic ulcers, and cancer. METHODS: Using gastric epithelial cells, we analyzed a set of H pylori strains and mutants for their ability to interact with CD46 and/or influence CD46 expression. Bacterial interaction with full-length CD46 and small CD46 peptides was evaluated by flow cytometry, fluorescence microscopy, enzyme-linked immunosorbent assay, and bacterial survival analyses. RESULTS: H pylori infection caused shedding of CD46 into the extracellular environment. A soluble form of CD46 bound to H pylori and inhibited growth, in a dose- and time-dependent manner, by interacting with urease and alkyl hydroperoxide reductase, which are essential bacterial pathogenicity-associated factors. Binding of CD46 or CD46-derived synthetic peptides blocked the urease activity and ability of bacteria to survive in acidic environments. Oral administration of one CD46 peptide eradicated H pylori from infected mice. CONCLUSIONS: CD46 is an antimicrobial agent that can eradicate H pylori. CD46 peptides might be developed to treat H pylori infection.

The intracellular redox stress caused by hexavalent chromium is selective for proteins that have key roles in cell survival and thiol redox control.

Hexavalent chromium [Cr(VI)] compounds (e.g. chromates) are strong oxidants that readily enter cells where they are reduced to reactive Cr intermediates that can directly oxidize some cell components and can promote the generation of reactive oxygen and nitrogen species. Inhalation is a major route of exposure which directly exposes the bronchial epithelium. Previous studies with non-cancerous human bronchial epithelial cells (BEAS-2B) demonstrated that Cr(VI) treatment results in the irreversible inhibition of thioredoxin reductase (TrxR) and the oxidation of thioredoxins (Trx) and peroxiredoxins (Prx). The mitochondrial Trx/Prx system is somewhat more sensitive to Cr(VI) than the cytosolic Trx/Prx system, and other redox-sensitive mitochondrial functions are subsequently affected including electron transport complexes I and II. Studies reported here show that Cr(VI) does not cause indiscriminant thiol oxidation, and that the Trx/Prx system is among the most sensitive of cellular protein thiols. Trx/Prx oxidation is not unique to BEAS-2B cells, as it was also observed in primary human bronchial epithelial cells. Increasing the intracellular levels of ascorbate, an endogenous Cr(VI) reductant, did not alter the effects on TrxR, Trx, or Prx. The peroxynitrite scavenger MnTBAP did not protect TrxR, Trx, Prx, or the electron transport chain from the effects of Cr(VI), implying that peroxynitrite is not required for these effects. Nitration of tyrosine residues of TrxR was not observed following Cr(VI) treatment, further ruling out peroxynitrite as a significant contributor to the irreversible inhibition of TrxR. Cr(VI) treatments that disrupt the TrxR/Trx/Prx system did not cause detectable mitochondrial DNA damage. Overall, the redox stress that results from Cr(VI) exposure shows selectivity for key proteins which are known to be important for redox signaling, antioxidant defense, and cell survival.

The role of peroxiredoxin V in (-)-epigallocatechin 3-gallate-induced multiple myeloma cell death.

(-)-Epigallocatechin 3-gallate (EGCG) is a potent antioxidant polyphenol in green tea that acts as an anticancer agent via both direct and indirect pathways. Although the relationship between EGCG's anticancer effects and its antioxidant activity is not fully understood, it is known that EGCG stimulates production of reactive oxygen species (ROS), which induce oxidative stress leading to cell death. In IM9 multiple myeloma cells, EGCG acted in a dose- and time-dependent manner to induce apoptotic cell death. Among the antioxidant enzymes expressed in IM9 cells, levels of peroxiredoxin V (PrdxV) were selectively and significantly reduced by EGCG. Moreover, the ROS scavenger NAC completely inhibited EGCG-induced apoptosis and PrdxV reduction, while overexpression of PrdxV, but not a Prdx(VC48S) mutant, protected IM9 cells from EGCG-induced apoptosis. EGCG-induced reductions in cell viability and PrdxV levels were also observed in primary CD138+ multiple myeloma cells from patients. These results suggest that PrdxV is a key target via which EGCG mediates its anticancer effects.

Differential roles of OxyR-controlled antioxidant enzymes alkyl hydroperoxide reductase (AhpCF) and catalase (KatB) in the protection of Pseudomonas aeruginosa against hydrogen peroxide in biofilm vs. planktonic culture.

The role of the Pseudomonas aeruginosa OxyR-controlled antioxidants alkyl hydroperoxide reductase CF (AhpCF) and catalase B (KatB) was evaluated in biofilm vs. planktonic culture upon exposure to hydrogen peroxide. AhpCF was found to be critical for survival of biofilm bacteria while KatB was more important for survival of planktonic free-swimming organisms.

Silencing of peroxiredoxin 3 and peroxiredoxin 5 reveals the role of mitochondrial peroxiredoxins in the protection of human neuroblastoma SH-SY5Y cells toward MPP+.

Peroxiredoxins (PRDXs) are a family of peroxidases well conserved throughout evolution. Human PRDX3 and PRDX5, two mitochondrial PRDXs, have been implicated in several pathologies associated with oxidative stress. However, the individual role of PRDX3 and PRDX5 in cellular antioxidant defense has never been well established due to their overlapping peroxidatic activities. We investigated the expression and function of mitochondrial PRDXs in human neuroblastoma SH-SY5Y cells. Our results show that PRDX3 and PRDX5 are expressed constitutively in these neuronal cells. To examine further the function of mitochondrial PRDXs, we silenced the expression of PRDX3 and/or PRDX5 using small hairpin RNAs. Our results show that mitochondrial PRDX-depleted cells are more prone to oxidative damages and apoptosis induced by MPP(+), a complex I inhibitor which provides an experimental paradigm of Parkinson's disease.