The novel role of peroxiredoxin-2 in red cell membrane protein homeostasis and senescence.

Peroxiredoxin-2 (Prx2), a typical two-cysteine peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2(-/-) mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2(-/-) red cells and reduced Prx2(-/-) red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2(-/-) mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2(-/-) mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2(-/-) mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.

Altered expression of Peroxiredoxin and Thioredoxin in septic animal model / 결핵

BACKGROUND: In sepsis, excessive generation of reactive oxygen species plays key roles in the pathogenesis of acute lung injury. The serum antioxidants such as catalase and MnSOD are elevated in sepsis and considered as predictors of acute respiratory distress syndrome(ARDS) and prognostic factors of sepsis. Peroxiredoxin(Prx) has recently been known as an unique and major intracellular antioxidant. In this study, we evaluated the expression of Prx I and Prx II in mouse monocyte-macrophage cells(RAW267.7) after treatment of oxidative stress and endotoxin and measured the amount of Prx I, Prx II and thioredoxin(Trx) in peritoneal and bronchoalveolar lavage fluid of septic animal model. METHODS: Using immunoblot analysis with specific antibodies against Prx I, Prx II and Trx, we evaluated the distribution of Prx I and Prx II in human neutrophil, alveolar macrophage and red blood cell. We evaluated the expression of Prx I and Prx II in mouse monocyte-macrophage cells after treatment of 5 micro M menadione and 1 micro gram/ml lipopolysaccharide(LPS) and measured the amount of Prx I, Prx II and Trx in peritoneal lavage fluid of intraperitoneal septic animals(septic animal model induced with intraperitoneal 6 micro gram/kg LPS injection) and those in bronchoalveolar lavage fluid of intraperitoneal septic animals and intravenous septic animals(septic animal model induced with intravenous 5 micro gram/kg LPS injection) and compared with the severity of lung inflammation. RESULTS: The distribution of Prx I and Prx II were so different among human neutrophil, alveolar macrophage and red blood cell. The expression of Prx I in mouse monocyte-macrophage cells was increased after treatment of 5 micro M menadione and 1 micro gram/ml lipopolysaccharide but that of Prx II was not increased. The amount of Prx I, Prx II and Trx are increased in peritoneal lavage fluid of intraperitoneal septic animals but were not increased in bronchoalveolar lavage fluid of intraperitoneal and intravenous septic animals regardless of the severity of lung inflammation. CONCLUSION: As intracellular antioxidant, the expression of Prx I is increased in mouse monocyte-macrophage cells after treatment of oxidative stress and endotoxin. The amount of Prx I, Prx II and Trx are increased in local inflammatory site but not increased in injured lung of septic animal model.

Protective Role of Prx(Peroxiredoxin) I and II against H2O2-Induced Apoptosis of MCF7 Cell Lines / 한국유방암학회지

PURPOSE: Apoptosis is known to be induced either by direct oxidative damage from oxygen free radicals or hydrogen peroxide, or from their generation in cells by injurious agents. Peroxiredoxin plays an important role in eliminating peroxides generated during metabolism. The aim of this study is to elucidate the role of Prx (peroxiredoxin) enzymes during the cellular response to oxidative stress. METHODS: The presence of Prx isoforms was demonstrated by immunoblot analysis using Prx isoforms-specific antibodies, and RT-PCR using Prx isozyme coding sequences. Annexin V-FITO apoptosis detection method was used to measure the cell death following exposure to H2O2. RESULTS: Treatment of MCF7 cell lines with H2O2 resulted in the dose-dependent expression of Prx I and II. Observed decreases in the mRNA expressions of Prx I and II, analyzed by RT-PCR, correlated well with the results of immunoblot analysis. The treatment of normal breast cell line, MCF10A, with H2O2 resulted in rapid cell death, while the breast cancer cell line, MCF7, was resistant. In addition, we confirmed that Prx I and II transfected MCF10A cells were more prone to cell death than MCF10A transfected with vector alone, after H2O2 treatment. CONCLUSION: These findings suggest that Prx I and II have an important function as inhibitors of cell death during the cellular response to oxidative stress.

Distribution of Thioredoxin Peroxidases and Expression of Thioredoxin Peroxidases in Various Human Cancers / 대한내과학회지

OBJECTIVES: Thioredoxin peroxidase(TPx), which does not exhibit similar activity and amino acid sequence homology to conventional antioxidant enzymes has been purified from S cerevisiae and bovine brain. Natural killer enhancing factor-A(NKEF-A)/ proliferation associated gene(PAG), natural killer enhancing factor-B(NKEF-B)/TPx and MER5 which has sequence homology to yeast TPx has been recently characterized biochemically, Prosperi has reported that the level of PAG in HL-60 cells was increased after serum stimulation and decreased after differentiation induced by DMSO treatment. It is well known that thioredoxin, the electron donor to thioredoxin peroxidase, also implicated in cell proliferation via protein kinase C pathway. Disturbed balance of reactive oxygen species and antioxidant in tumor tissue could enhance the cancer promotion This study was designed to investigate the distribution of NKEF-A/FAG, NKEF-B/TPx and MER5 in various tissues, and the expression of NKEF-A/PAG, NKEF-B/TPx and MER5 in human cancers. METHODS: We used antibodies against the purified recombinant protein of NKEF-A/PAG and NKEF-B/TPx and C-terminus amino acids(SP-TASKEYFEKVHO) of MER5, We separated cytosole and mitochondria from rat liver and prepared crude extract from these. We prepared crude extract of various tissues from rat and cancer tissue from lung, stomach and breast and paired normal tissue. Immunoblot analysis of these crude extracts was performed. RESULTS: 1) NKEF-A/PAG and NKEF-B/TPx existed in cytosolic fraction as Cu, Zn-SOD and MER5 mainly exist mainly in mitochondrial fraction as Mn-SOD. Although the level of NKEF-AIPAG, NKEF-B/TPx and MER5 was different, all tissues exhibited NKEF-A/PAG, NKEF-B/TPx and MER5 immunoreactive bands. The adrenal gland had relatively strong band of MEK. 2) The expression of NKEF-A/PAG in HL-60 cell was increased after serum stimulation and decreased after cell differentiation induced by DMSO treatment. 3) The expression of NKEF-A/PAG was increased in lung and breast cancer tissues compaired to paired normal tissues but was not changed in stomach cancer tissues and the expression of NKEF-B/TPx and MER5 was not changed in lung, stomach and breast cancer tissues compaired to paired normal tissues. 4) The level of NKEF-A/PAG, NKEF-B/TPx and MER5 was not different among various lung cancer cell lines. CONCLUSION: The NKEF-A/PAG, NKRF-B/TPx and MER5 are present in the cytosol and mitochondria of various tissues. The NKEF-A/PAG, in particular, is associated with cell proliferation and differentiation and overexpressed in lung and breast cancer.