Peroxiredoxin II Research Articles

Small interfering RNA targeting of peroxiredoxinⅡ gene enhances formaldehyde-induced toxicity in bone marrow cells isolated from BALB/c mice

BackgroudsFormaldehyde (FA) is an important chemicals that can induce sick house syndrome and may be an incentive of childhood leukemia, however the exact mechanism is unclear. Oxidative stress may be an underlying reason of cancer occurring, while diverse antioxidants can protect the bone marrow cells (BMCs) from damaged. PeroxiredoxinⅡ (PrxⅡ) is an important member of the peroxiredoxin family, can remove reactive oxygen species (ROS), and is closely related with the occurrence of tumor. The present study aimed to detect a possible relationship between PrxⅡ gene and FA-induced bone marrow toxicity. MethodsThe BMCs were taken out from BALB/c mice, then exposed to control and different doses of FA (50, 100, 200 μmol/L). The cell viability, ROS level and expressions of PrxⅡ gene were examined. Afterwards, we used a small interfering RNA (siRNA) to inhibit the expression of PrxⅡ gene, and chose 100 μmol/L FA for exposure dose, to examine the cell viability, ROS level, cell cycle, apoptotic rate, expressions of PrxⅡ gene in BMCs. ResultsAfter a 24 h exposure to different doses of FA, the cell viability, expressions of PrxⅡ gene were decreased with the increasing of FA concentration, while the ROS level was increased. Inhibiting PrxⅡ gene's expression could enhance above FA-induced events. Additionally, siRNA targeting of PrxⅡcould aggravate cell cycle arrest to inhibit cell's growth and development, as well as increase apoptotic rates induced by FA. ConclusionThese results demonstrated that PrxⅡ gene was involved in FA-induced bone marrow toxicity, and siRNA targeting of PrxⅡcould enhance this toxic process.

232 - Peroxiredoxin II Deficiency Contributes to Contraction-Induced Force Loss in the mdx Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is a lethal, degenerative muscle disease caused by loss of the protein dystrophin. Eccentric contraction-induced force loss is a hallmark of dystrophin-deficient muscle, although the underlying cause of force loss remains obscure. Here, we employed iTRAQ proteomics to identify proteins that were differentially expressed in skeletal muscle of dystrophin-deficient mdx mice overexpressing γ cyto -actin (mdx/Actg1-TG), a model which displays partial protection from eccentric contraction-induced force loss. We identified peroxiredoxin II (PrxII) as significantly decreased in mdx muscle compared to WT, and recovered to WT levels in mdx/Actg1-TG muscle. PrxII is an antioxidant enzyme that reduces reactive oxygen species (ROS) in the form of hydrogen peroxide to water. Because the sarcolemmal NAPDH oxidase 2 (NOX2) complex exhibits increased expression and activity in mdx mice, and inhibition of NOX2 also partially protects mdx muscle against eccentric contraction-induced force loss, we investigated the relationship between PrxII and NOX2 in WT and mdx muscle. Like NOX2, PrxII was enriched at the sarcolemma. Knockout of the p47 phox subunit of NOX2 in mdx muscle restored PrxII levels back to WT, suggesting that elevated NOX2 activity may drive PrxII hyperoxidation and proteasomal degradation. Most interestingly, force production by mdx/PrxII -/- double knockout muscle exhibited increased sensitivity to eccentric contraction, while transgenic overexpression of PrxII protected mdx muscle from eccentric contraction to a similar extent measured in mdx/Actg1-TG muscle. Our results suggest that PrxII functions as the “off-switch” in the sarcolemmal NOX2 signaling pathway and its loss from dystrophic muscle contributes to pathogenesis associated with excessive ROS production.

Threonine modulates immune response, antioxidant status and gene expressions of antioxidant enzymes and antioxidant-immune-cytokine-related signaling molecules in juvenile blunt snout bream (Megalobrama amblycephala).

A 9-week feeding trial was conducted to investigate the effects of graded dietary threonine (Thr) levels (0.58-2.58%) on the hematological parameters, immune response, antioxidant status and hepatopancreatic gene expression of antioxidant enzymes and antioxidant-immune-cytokine-related signaling molecules in juvenile blunt snout bream. For this purpose, 3 tanks were randomly arranged and assigned to each experimental diet. Fish were fed with their respective diet to apparent satiation 4 times daily. The results indicated that white blood cell, red blood cell and haemoglobin significantly responded to graded dietary Thr levels, while hematocrit didn't. Complement components (C3 and C4), total iron-binding capacity (TIBC), immunoglobulin M (IgM), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) increased with increasing dietary Thr levels up to 1.58-2.08% and thereafter tended to decrease. Dietary Thr regulated the gene expressions of Cu/Zn-SOD, Mn-SOD and CAT, GPx1, glutathione S-transferase mu (GST), nuclear factor erythroid 2-related factor 2 (Nrf2), heat shock protein-70 (Hsp70), tumor necrosis factor-alpha (TNF-α), apolipoprotein A-I (ApoA1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and fructose-bisphosphate aldolase B (ALDOB); while the gene expression of peroxiredoxin II (PrxII) was not significantly modified by graded Thr levels. These genes are involved in different functions including antioxidant, immune, and defense responses, energy metabolism and protein synthesis. Therefore, this study could provide a new molecular tool for studies in fish immunonutrition and shed light on the regulatory mechanisms that dietary Thr improved the antioxidant and immune capacities of fish.

Peroxiredoxin II Is an Antioxidant Enzyme That Negatively Regulates Collagen-stimulated Platelet Function

Collagen-induced platelet signaling is mediated by binding to the primary receptor glycoprotein VI (GPVI). Reactive oxygen species produced in response to collagen have been found to be responsible for the propagation of GPVI signaling pathways in platelets. Therefore, it has been suggested that antioxidant enzymes could down-regulate GPVI-stimulated platelet activation. Although the antioxidant enzyme peroxiredoxin II (PrxII) has emerged as having a role in negatively regulating signaling through various receptors by eliminating H2O2 generated upon receptor stimulation, the function of PrxII in collagen-stimulated platelets is not known. We tested the hypothesis that PrxII negatively regulates collagen-stimulated platelet activation. We analyzed PrxII-deficient murine platelets. PrxII deficiency enhanced GPVI-mediated platelet activation through the defective elimination of H2O2 and the impaired protection of SH2 domain-containing tyrosine phosphatase 2 (SHP-2) against oxidative inactivation, which resulted in increased tyrosine phosphorylation of key components for the GPVI signaling cascade, including Syk, Btk, and phospholipase Cγ2. Interestingly, PrxII-mediated antioxidative protection of SHP-2 appeared to occur in the lipid rafts. PrxII-deficient platelets exhibited increased adhesion and aggregation upon collagen stimulation. Furthermore, in vivo experiments demonstrated that PrxII deficiency facilitated platelet-dependent thrombus formation in injured carotid arteries. This study reveals that PrxII functions as a protective antioxidant enzyme against collagen-stimulated platelet activation and platelet-dependent thrombosis.

Peroxiredoxin II negatively regulates lipopolysaccharide-induced osteoclast formation and bone loss via JNK and STAT3.

Lipopolysaccharide (LPS) is considered a prominent pathogenic factor in inflammatory bone diseases. LPS challenge contributes to the production of reactive oxygen species (ROS) in diverse inflammatory diseases. However, its mechanism remains to be clarified in bone. Thus, we investigated the critical mechanism of ROS in LPS-induced osteoclastogenesis and bone loss. Antioxidant prevented LPS-induced osteoclast formation via inhibition of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and c-Fos expression in preosteoclasts. Moreover, LPS-induced osteoclast formation via ROS was attenuated by treatment with c-Jun N-terminal protein kinase (JNK) inhibitor. Interestingly, LPS also activated signal transducer and activator of transcription 3 (STAT3), which is suppressed by antioxidants. We found that knockdown of STAT3 or use of a STAT3 inhibitor resulted in a significant reduction in interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and nitric oxide (NO) production, followed by decreased osteoclast formation by LPS. Peroxiredoxin II (PrxII) is a member of the antioxidant enzyme family, and it plays a protective role against oxidative damage caused by ROS. In our study, ROS production and osteoclast formation by LPS was significantly enhanced in PrxII(-/-) cells. Moreover, JNK-mediated c-Fos and NFATc1 expression was promoted in PrxII(-/-) cells. Furthermore, STAT3 activation and accompanying IL-1β, IL-6, and NO production was also increased in PrxII(-/-) cells. Consistent with the in vitro result, PrxII-deficient mice showed increased osteoclast formation and bone loss by LPS challenge compared with wild-type mice. For the first time, we showed that LPS-induced ROS signaling is dependent on the coordinated mechanism of JNK and STAT3 during osteoclastogenesis, which is negatively regulated by PrxII. We suggest that PrxII could be useful in the development of a novel target for inflammatory bone loss.

Abstract 3953: Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II

Abstract In our previous study, we identified a protein target, Peroxiredoxin II (PrxII) to be overexpressed in radioresistant MCF+FIR3 breast cancer cells and found that its expression and function is associated with breast cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting the PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by siRNA that inhibits PrxII gene expression. Our results presented here show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to its targeted property to specific cancer related genes. Citation Format: Tieli Wang, Anthony Joseph Diaz, Jian-Jian Li, Yun Yen, Daniel Tamae. Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2014-3953

Enhanced radiation response in radioresistant MCF-7 cells by targeting peroxiredoxin II

In our previous study, we identified that a protein target, peroxiredoxin II (PrxII), is overexpressed in radioresistant MCF+FIR3 breast-cancer cells and found that its expression and function is associated with breast-cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast-cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by the siRNA that inhibits PrxII gene expression. Our results, presented here, show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells, and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to this technology’s property of targeting to specific cancer-related genes.

Peroxiredoxin II Is an Essential Antioxidant Enzyme that Prevents the Oxidative Inactivation of VEGF Receptor-2 in Vascular Endothelial Cells

Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H(2)O(2) level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrates a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.

Oxidative stress-dependent oligomeric status of erythrocyte peroxiredoxin II (PrxII) during storage under standard blood banking conditions

Although biochemical properties of 2-Cys peroxiredoxins have been extensively studied in various cell lines and organisms, redox-induced structural transitions of peroxiredoxin II (PrxII) in human erythrocytes certainly warrant further investigation. In this work, cytosol and membrane ghosts of both fresh erythrocytes (cells obtained just after blood collection) and 28-day stored erythrocytes were analyzed by proteomics tools. We demonstrated that in fresh red blood cells PrxII exhibits four different oligomeric states in cytosol, whereas no PrxII complexes are in the membrane. The highest molecular weight PrxII protein complex (440 kDa) was proven to derive from the association between tetrameric catalase (CAT, 232 kDa) and decameric PrxII, whereas oligomers at 140, 100 and 67 kDa resulted to be homo-polymeric complexes composed of variable copies of PrxII monomeric subunits. Interestingly, the 440 kDa complex contained both reduced and oxidized (disulphide-linked dimers) PrxII decamers. Upon oxidative stress (28-day storage), the PrxII oligomers at 100 kDa in the cytosol disappeared and the CAT–PrxII hetero-oligomeric complex at 440 kDa is converted to a higher molecular weight structure (480 kDa) due to the presence therein of cross-linked species of PrxII and hemoglobin. More interestingly, oxidized red cell membranes contained the CAT–PrxII complex detected in 0-day cytosol as a consequence of protein recruitments induced by oxidative stress, however it showed a greater percentage of PrxII dimers. Finally, since the adoption of distinct PrxII structures is known to be closely related to different functions, peroxidase activity assays were performed demonstrating a positive reaction for oligomers at 440 kDa (both in cytosol and membrane compartment) and at 140 kDa. Our results contribute to clarify structural and functional switching of peroxiredoxin II in erythrocytes, thus possibly opening new scenarios in the biological roles played by this protein in defense mechanisms against oxidative stress, especially with the reference to red cell storage lesions.

Heat‐shock protein‐27, ‐70 and peroxiredoxin–II show molecular chaperone function in sickle red cells: Evidence from transgenic sickle cell mouse model

Sickle cell disease (SCD) is an autosomal recessive genetic red cell disorder characterized by the production of a defective form of hemoglobin, hemoglobin-S, that is worldwide-distributed. The acute clinical manifestations of SCD are related to hemoglobin cyclic-polymerization and to the generation of rigid, dense red blood cells (RBCs). We studied RBCs membrane proteome from human sickle RBCs, fractioned according to density compared to normal RBCs. 2-DE followed by MS analysis was carried out. We identified 65 proteins differently expressed, divided into five major clusters according to their functions: (i) membrane-cytoskeleton proteins; (ii) metabolic enzymes; (iii) ubiquitin-proteasome-system; (iv) flotillins; (v) chaperones. HSP27, HSP70 and peroxiredoxin-II (Prx-II) showed the most relevant changes. They were differently recruited to sickle RBCs membrane in response to in vitro hypoxia. Potential markers were then validated in a transgenic-mouse model for SCD, the SAD mice, exposed to hypoxia mimicking acute SCD vaso-occlusive-crisis (VOCs); we found that HSP70 and HSP27 bound to RBCs membrane respectively after 12 h and 48 h of hypoxia, while Prx-II membrane binding was modulated during hypoxia. Our data indicate that HSP27 and HSP70 play a novel role as RBCs membrane protein protectors and as possibly new markers of severity of RBCs membrane damage during acute VOCs.

Peroxiredoxin II Regulates CD8+ T Cell Expansion and Survival During Acute and Chronic Viral Infections

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. We examined the role of the antioxidant enzyme, Peroxiredoxin II (PrxII), during T cell activation and throughout the immune response to acute and chronic viral infections. Following TCR stimulation, PrxII levels increased 50‐fold by 30 hours. Examination of differentiated T cells demonstrated that effector cells possessed lower PrxII levels than naïve or memory CD8+ T cells. Utilizing mice deficient in PrxII, we determined that ROI levels measured in T cells, either directly ex vivo or in response to stimulation, were higher than wild‐type mice. Deletion of PrxII also increased in vitro T cell proliferation, but decreased cell survival during division. Acute infection of PrxII−/− mice with LCMV Armstrong revealed an increased expansion of effector CD8+ T cells on day 8, but yielded no significant difference in memory CD8+ T cells by day 97. Additionally, no differences were observed in virus‐specific CD4+ T cells or antibody secreting cells at any time during infection. In contrast, chronic infection with LCMV Clone 13 resulted in death by day 13 post infection. Together, these results demonstrate that redox regulation of T cell activation through PrxII is critical during acute and chronic viral infections.Supported by ACS‐ #RSG‐04‐066‐01‐MBC to J.G.

Differential role of peroxiredoxin II (PrxII) on the expression of toll-like receptor 4 (TLR4) and B-cell activating factor (BAFF) in ovalbumin (OVA)-induced mouse asthma

Peroxiredoxin II (PrxII) is one of reactive oxygen species (ROS)-degrading enzyme. Here, we investigated the role of PrxII on toll-like receptor 4 (TLR4) and B-cell activating factor (BAFF) expression in ovalbumin (OVA)-induced mouse asthma. We used ROS-producing PrxII −/− mice of which cells up-regulate BAFF expression. As significant changes were detected in TLR4 mRNA with real-time quantitative RT-PCR analysis, TLR4 protein was decreased in PrxII −/− mouse splenocytes and peritoneal macrophages, compared to wild type cells. Airway hyper-responsiveness (AHR) was more severe in PrxII −/− mice than wild type mice, which was measured by the level of various parameters, number of eosinophils, IgE level, airway thickness, and mucous secretion. BAFF was detected in cells surrounding airways of OVA-induced mouse and it was highly augmented in PrxII −/− mice. BAFF promoter activity was also higher in PrxII −/− mouse embryonic fibroblast (MEF) than in wild type MEF. Collectively, results show that PrxII may have benefits in asthma through reducing ROS. It suggests that BAFF and TLR4 expressions are differentially regulated by PrxII and TLR4 protein level may not be crucial in OVA-induced asthma.

Bronchoalveolar Lavage Fluid Proteome in Bronchiolitis Obliterans Syndrome: Possible Role for Surfactant Protein A in Disease Onset

Bronchiolitis obliterans syndrome (BOS) affects long-term survival of lung transplant (Tx) recipients (LTRs), with no consistently effective treatment strategy. Identifying early markers of BOS is of paramount importance for improving graft survival. We used 2-dimensional gel electrophoresis and protein identification by mass spectrometry to compare the protein profile of bronchoalveolar lavage fluid (BALf) in two groups of LTRs: one composed of patients with BOS and the other composed of patients with good graft function at >5 years post-surgery (stable LTRs). Based on the hypothesis that only proteins of lung origin could represent reliable BOS markers, we also evaluated paired plasma samples. Proteins of interest were also assessed in the BALf of control subjects and results confirmed by dot- blot analysis. Among 11 differentially expressed proteins, we identified 2 locally produced factors: peroxiredoxin II (PRXII), exclusively expressed in BOS; and surfactant protein A (SP-A), expressed consistently less in BOS patients than in stable LTRs. PRXII expression was never observed in BALf from control subjects, whereas SP-A was present in higher amounts compared with stable LTRs and BOS patients. Finally, the time course of SP-A was studied in 5 LTRs who subsequently developed BOS. A reduction in BALf SP-A content was detectable early after Tx, preceding BOS onset in 4 of 5 patients. Our results suggest that testing SP-A levels in BALf could predict LTR patients who are at higher risk of BOS development.

Reactive oxygen species augment B-cell-activating factor expression

B-cell-activating factor (BAFF) plays a role in mature B-cell generation and maintenance. Lipopolysaccharide (LPS) activates toll-like receptor 4 (TLR4)-dependent signal transduction and induces ROS production. Here, we investigated BAFF production regulated by reactive oxygen species (ROS). BAFF expression was augmented by LPS stimulation and by serum deprivation that induced ROS production. BAFF expression was inhibited by treatment with various antioxidants including N-acetyl -L-cysteine (NAC). We also investigated BAFF expression in vivo using peroxiredoxin II (PrxII)-deficient mouse spleen cells. PrxII is a member of the antioxidant enzyme family that protects cells from oxidative damage. Constitutive production of endogenous ROS was detected in spleen cells lacking PrxII. Serum BAFF protein level and BAFF transcript expression in splenocytes were significantly higher in PrxII –/– mice than wildtype mice. A higher BAFF level is consistent with the higher total number of splenocytes and B220 +cells. Results were supported by NF-κB activation as judged by reduced IκBα degradation and increased nuclear translocation of p65/RelA with LPS stimulation, serum deprivation, and PrxII deletion. Data suggest that TLR4-mediated BAFF expression was increased by ROS and it was inhibited by PrxII controlling ROS production.

The Role of Peroxiredoxin II in Radiation-Resistant MCF-7 Breast Cancer Cells

Although several signaling pathways have been suggested to be involved in the cellular response to ionizing radiation, the molecular basis of tumor resistance to radiation remains elusive. We have developed a unique model system based upon the MCF-7 human breast cancer cell line that became resistant to radiation treatment (MCF+FIR30) after exposure to chronic ionizing radiation. By proteomics analysis, we found that peroxiredoxin II (PrxII), a member of a family of peroxidases, is up-regulated in the radiation-derived MCF+FIR3 cells but not in the MCF+FIS4 cells that are relatively sensitive to radiation. Both MCF+FIR3 and MCF+FIS4 cell lines are from MCF+FIR30 populations. Furthermore, the resistance to ionizing radiation can be partially reversed by silencing the expression of PrxII by PrxII/small interfering RNA treatment of MCF+FIR3 resistant cells, suggesting that PrxII is not the sole factor responsible for the resistant phenotype. The relevance of this mechanism was further confirmed by the increased radioresistance in PrxII-overexpressing MCF+FIS4 cells when compared with vector control cells. The up-regulation of the PrxII protein in radioresistant cancer cells suggested that human peroxiredoxin plays an important role in eliminating the generation of reactive oxygen species by ionizing radiation. The present finding, together with the observation that PrxII is also up-regulated in response to ionizing radiation in other cell systems, strengthens the hypothesis that the PrxII antioxidant protein is involved in the cellular response to ionizing radiation and functions to reduce the intracellular reactive oxygen species levels, resulting in increased resistance of breast cancer cells to ionizing radiation.

Peroxiredoxin II functions as a signal terminator for H2O2‐activated phospholipase D1

Phospholipase D1 (PLD1) is a signal-transduction regulated enzyme which regulates several cell intrinsic processes including activation of NAPDH oxidase, which elevates intracellular H2O2. Several proteins have been reported to interact with PLD1 in resting cells. We sought to identify proteins that interact with PLD1 after phorbol 12-myristate 13-acetate (PMA) stimulation. A novel interaction with peroxiredoxin II (PrxII), an enzyme that eliminates cellular H2O2, which is a known stimulator of PLD1, was identified by PLD1-affinity pull-down and MS. PMA stimulation was confirmed to promote physical interaction between PLD1 and PrxII and to cause PLD1 and PrxII to colocalize subcellularly. Functional significance of the interaction was suggested by the observation that over-expression of PrxII specifically reduces the response of PLD1 to stimulation by H2O2. These results indicate that PrxII may have a signal-terminating role for PLD1 by being recruited to sites containing activated PLD1 after cellular stimulation involving production of H2O2.

Identification of differentially expressed proteins during human urinary bladder cancer progression

Comparative proteome analysis was performed between RT4 (grade-1) and T24 (grade-3) bladder cancer cell lines, in an attempt to identify differentially expressed proteins during bladder cancer progression. Among those relatively abundant proteins, seven spots changed more than two-fold reproducibly and identified by peptide mass fingerprinting using mass spectrometry and database search. We found most extensive and reproducible down-regulation of NADP dependent isocitrate dehydrogenase cytoplasmic (IDPc) and peroxiredoxin-II (Prx-II), in poorly differentiated T24 compared to well-differentiated RT4 bladder cancer cell line. Subsequent Western blotting analysis of human biopsy samples from bladder cancer patient revealed significant loss of IDPc and Prx-II in more advance tumor samples, in agreement with data on cell lines. These results suggest that loss of IDPc and Prx-II during tumor development may involve in tumor progression and metastasis. However, additional investigations are needed on large number of human samples to further verify these findings.

Reactive Oxygen Species and RBC Membrane Integrity in Peroxyredoxin II Deficient Mice.

Red blood cells (RBCs) contain a complex set of enzymes and non-enzymatic scavengers as defense against reactive oxygen species (ROS). Peroxiredoxin II (PrxII), a member of a family of small proteins with strong antioxidant properties, is highly abundant in RBCs. PrxII is likely to play an essential role in ROS protection, as RBCs generate high levels of ROS due to their role in oxygen transport and the presence of redox active hemoglobin. Phosphatidylserine (PS) asymmetry in RBCs is maintained by the active transport of PS from outer to inner monolayer by the oxidant sensitive aminophospholipid translocase or flipase. PS exposure is found in RBCs where phospholipid scrambling is activated and the flipase is inhibited. In PrxII−/− mice, PrxII is absent from the RBC. These mice are anemic (Hct 41% vs. 46% in wild type mice (WT)), with increased reticulocyte count (4.7% vs. 2.0 % in WT), and a morphologically diverse RBC population. RBC indices after isovolumetric sphering showed a similar MCH (14.3 vs. 14.2), slightly increased MCV (49.2 fl vs. 45.5 fl) and slightly decreased MCHC (30.1 vs. 32.1) in PrxII−/− mice as compared to WT. In flow cytometric analysis, two distinct populations of RBC are found with either slight or significantly increased autofluorescence in the fluorescein channel (excitation 488 nm, emission 515 nm), indicative of oxidant damage. These two populations of low (LF) and high (HF) fluorescent cells comprise 70–80% and 20–30% of the total RBC population respectively. RBC from PrxII−/− and WT mice were biotinylated using EZ-Link Sulfo-NHS-Biotin (Pierce) allowing turnover studies of the LF and HF population. At set time points, the number of biotinylated cells was determined in small blood samples by flow cytometry using fluorescently labeled streptavidin. The data were mathematically fitted to 100–100*[1−(1/T)*t]exp(−kt), where t is the time point, T is the extinction time, and k the exponential rate of RBC removal. The data in the WT showed a linear removal rate (k=0), and a T of 40 days (R2=0.99). In PrxII−/−, an overall faster disappearance of biotinylated cells was noted, and the number of surviving (biotinylated) cells in the population followed an exponential pattern, consistent with random removal (k=0.08, R2= 0.98). At 20 days, 50% of biotinylated RBC were present in WT, but only 18% were found in PrxII−/− mice. In the non-biotinylated RBC, HF cells started to appear at day 13, indicating that autofluorescence is acquired in time. Using the fluorescent ROS membrane probe C11-BODIPY, our data indicate a higher level of ROS in the HF population. The HF population exhibited a lower flipase activity and increased phospholipid scrambling, as measured by labeling with annexin V. Together, our data indicate the importance of PrxII in the maintenance of RBC membrane integrity and suggest that oxidant induced PS exposure is in part responsible for shortened RBC survival in these mice. These findings indicate a role for oxidation in the exposure of PS on the RBC surface, which may clarify mechanisms in oxidant induced membrane alterations in hemoglobinopathies.

Proteomics of Membrane from Human Sickle and Normal Fractioned Red Cells Identifies Different Expression of Stress-Response Proteins.

Sickle cell disease (SCD) is characterized by the presence of sickle hemoglobin (HbS), which has the unique property of polymerizing when deoxygenated and thereby triggering erythrocyte sickling and dehydration. Repeated HbS polymerization is associated with generation of dense, distorted red cells characterized by an extensive membrane oxidative damage and abnormal activation of membrane cation transport pathways. The recently developed proteomic technologies represent a useful tool for identification of new functional pathways in sickle red cells. We studied the membrane proteoma of sickle red cells from homozygous patients (SS), not carrying alpha-thalassemia, not undergoing hydroxyurea treatment and not transfused at least three months before the study. The buffy-coat was removed and the red cells were separated into two fractions corresponding to the reticulocytes enriched fraction and to the dense red cell fraction (n= 8). Following osmotic lysis, the red cell ghost proteins were separated by one- dimensional or by two-dimensional gel electrophoresis (1-DE, 2-DE). Using the ImageMaster 2D Platinum (5.0 Amersham-Biosciences) software the 2-DE gels from control and sickle cells were compared, allowing us to identify spots differently expressed in sickle cells compared to normal cells. The selected spots were excised and trypsinized before mass spectrometric analysis and compared with the SWISS-PROT and NCBI non-redundant databases. In sickle reticulocytes enriched fraction compared to the control one, we selectively identified the following cystokeleton proteins: four spots for beta actin, two spots for myosin light chain and a train of four proteins identified as protein 4.1, one of which was phosphorylated on Tyr13, a protein similar to flotillin 2, carbonic anhydrase I and the following stress-response proteins: heat shock protein 70 (HSP70) protein 9B, HSPC108 and peroxiredoxin II (PrxII), which was phosphorylated in Thr142. In dense sickle red cell fraction compared to normal one we selectively identified a train of two spots for 4.1R, band 3 and aldolase and two spots identified as HSP70 protein 5.The identified proteins were then confirmed by western-blotting analysis.These data suggest that in sickle red cells HSP70, HSP108 function may be required by the extensive assistance in the refolding of misfolded proteins most likely related to abnormal sickle red cell oxidative environment as also supported by the presence of phosphorylated peroxiredoxin II, which has been recently proposed as regulator of redox-sensitive signaling in other cell types.

Reactive oxygen species induced by the deletion of peroxiredoxin II (PrxII) increases the number of thymocytes resulting in the enlargement of PrxII-null thymus.

In the thymus, CD4+ or CD8+ single-positive (SP) thymocytes develop and mature by positive and negative selection or undergo "death by neglect". CD4+ or CD8+ SP then circulate to other lymphoid tissues. We have investigated the role of reactive oxygen species (ROS) in thymocyte development using peroxiredoxin II (PrxII)-null mice. The level ofROS in PrxII-null thymocytes is higher than that in wild-type mice. Deletion of the PrxII gene leads to enlargement of the thymus in young (9 weeks) and old (64 weeks) mice. The increased number ofthymocytes in PrxII-null thymus is related to reduced hypodiploid cell formation. For mice on a normal diet, the ratio of SP to double-positive (DP) thymocytes in thymus of PrxII-null mice is lower than that in wild-type mice. After food restriction, which leads to increased ROS production, this ratio becomes much higher in PrxII-null thymus. The amount of apoptosis, induced by food restriction orby the injection of dexamethasone, is consistently lower in PrxII-null thymocytes than in wild-type thymocytes. In the presence of low serum concentrations, PrxII-deleted T cells proliferate more vigorously after stimulation with concanavalin A. Phytohemagglutinin- or OKT3-stimulated proliferation of human peripheral blood mononuclear cells is also higher in the presence of lower serum concentrations. Collectively, the results suggest for the first time that thymocyte maturations and proliferations are regulated by ROS levels induced by the deletion of PrxII gene in vivo.