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Luis Eduardo Soares Netto - One of the best experts on this subject based on the ideXlab platform.
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crystallization and preliminary x ray analysis of a decameric form of cytosolic Thioredoxin Peroxidase 1 tsa1 c47s mutant from saccharomyces cerevisiae
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2007Co-Authors: Marcos Antonio De Oliveira, Luis Eduardo Soares Netto, Victor Genu, Karen Fulan Discola, Simone Vidigal Alves, Beatriz G GuimaraesAbstract:Saccharomyces cerevisiae cytosolic Thioredoxin Peroxidase 1 (cTPxI or Tsa1) is a bifunctional enzyme with protective roles in cellular defence against oxidative and thermal stress that exhibits both Peroxidase and chaperone activities. Protein overoxidation and/or high temperatures induce great changes in its quaternary structure and lead to its assembly into large complexes that possess chaperone activity. A recombinant mutant of Tsa1 from S. cerevisiae, with Cys47 substituted by serine, was overexpressed in Escherichia coli as a His6-tagged fusion protein and purified by nickel-affinity chromatography. Crystals were obtained from protein previously treated with 1,4-dithiothreitol by the hanging-drop vapour-diffusion method using PEG 3000 as precipitant and sodium fluoride as an additive. Diffraction data were collected to 2.8 A resolution using a synchrotron-radiation source. The crystal structure was solved by molecular-replacement methods and structure refinement is currently in progress.
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yeast oxidative stress response influences of cytosolic Thioredoxin Peroxidase i and of the mitochondrial functional state
FEBS Journal, 2006Co-Authors: Ana Paula Dias Demasi, Goncalo A G Pereira, Luis Eduardo Soares NettoAbstract:We investigated the changes in the oxidative stress response of yeast cells suffering mitochondrial dysfunction that could impair their viability. First, we demonstrated that cells with this dysfunction rely exclusively on cytosolic Thioredoxin Peroxidase I (cTPxI) and its reductant sulfiredoxin, among other antioxidant enzymes tested, to protect them against H2O2-induced death. This cTPxI-dependent protection could be related to its dual functions, as Peroxidase and as molecular chaperone, suggested by mixtures of low and high molecular weight oligomeric structures of cTPxI observed in cells challenged with H2O2. We found that cTPxI deficiency leads to increased basal sulfhydryl levels and transcriptional activation of most of the H2O2-responsive genes, interpreted as an attempt by the cells to improve their antioxidant defense. On the other hand, mitochondrial dysfunction, specifically the electron transport blockage, provoked a huge depletion of sulfhydryl groups after H2O2 treatment and reduced the H2O2-mediated activation of some genes otherwise observed, impairing cell defense and viability. The transcription factors Yap1 and Skn7 are crucial for the antioxidant response of cells under inhibited electron flow condition and probably act in the same pathway of cTPxI to protect cells affected by this disorder. Yap1 cellular distribution was not affected by cTpxI deficiency and by mitochondrial dysfunction, in spite of the observed expression alterations of several Yap1-target genes, indicating alternative mechanisms of Yap1 activation/deactivation. Therefore, we propose that cTPxI is specifically important in the protection of yeast with mitochondrial dysfunction due to its functional versatility as an antioxidant, chaperone and modulator of gene expression.
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cytosolic Thioredoxin Peroxidase i and ii are important defenses of yeast against organic hydroperoxide insult catalases and peroxiredoxins cooperate in the decomposition of h2o2 by yeast
Journal of Biological Chemistry, 2004Co-Authors: Daniela Cristina Munhoz, Luis Eduardo Soares NettoAbstract:The cytosolic Thioredoxin Peroxidase II (cTPxII/Tsa2p) from Saccharomyces cerevisiae shares 86% identity with the relatively well characterized cytosolic Thioredoxin Peroxidase I (cTPxI/Tsa1p). In contrast to cTPxI protein, cTPxII is not abundant and is highly inducible by peroxides. Here, we describe a unique phenotype for ΔcTPxII strain; these cells were highly sensitive to tert-butylhydroperoxide (TBHP) but presented resistance to H2O2 in fermentative and respiratory conditions. In contrast, ΔcTPxI strain was very sensitive to both TBHP and H2O2, whatever the carbon source present in the media. These differences in the response of mutant cells to the different kinds of peroxide insult could not be attributed to enzymatic properties of cTPxI and cTPxII since the recombinant proteins showed similar in vitro efficiencies (Kcat /Km) in the removals of both kinds of peroxide. This specific sensitivity of ΔcTPxII cells to TBHP could not be related to the expression pattern of TSA2 (cytosolic Thioredoxin Peroxidase II gene) either, since this gene is highly inducible by both H2O2 and TBHP when cells were grown in different conditions. Finally, peroxide-removing assays were performed and showed that catalase activity increased significantly only in ΔcTPxII cells, which appear to be related with the resistance of this strain to H2O2. Taken together, present data indicate that cTPxII and cTPxI are key components of the yeast defense system against organic peroxide insult. In regard to the stress induced by H2O2, catalases (peroxisomal and/or cytosolic) and cTPxII seemed to cooperate with cTPxI in the defense of yeast against this oxidant.
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regulation of mitochondrial Thioredoxin Peroxidase i expression by two different pathways one dependent on camp and the other on heme
Free Radical Biology and Medicine, 2002Co-Authors: Gisele Monteiro, Goncalo Amarante Guimaraes Pereira, Luis Eduardo Soares NettoAbstract:Abstract Mitochondrial isoform of Thioredoxin Peroxidase (mTPx I) is an antioxidant protein recently described in Saccharomyces cerevisiae. Here we characterized pathways that lead to mTPx I induction in two situations: growth in media containing low glucose concentrations and treatment with peroxides. The induction of mTPx I by growth on low glucose concentrations was dependent on cAMP and on the transcription factors Msn2p/Msn4p as demonstrated by northern blot experiments using yeast strains with deletion of MSN2 and MSN4 genes and also using a strain permeable to cAMP. mTPx I expression was also induced by peroxides in a time- and dose-dependent manner and varied with the carbon source present in the media. Deletion of HAP1 or inhibition of heme synthesis abolished induction of mTPx I by H2O2 on cells which were grown in media containing glucose, indicating that Hap1p is involved in the regulation of this process. mTPx I was induced by H2O2 on glycerol/ethanol-containing media, but we could not associate any transcription factor with this phenomenon. Finally, mTPx I also induced by t-butyl hydroperoxide in a Hap1p-independent manner. In conclusion, mTPx I expression is under a complex regulatory network, which involves, at least, two signaling pathways: one sensing the carbon source (which is signalized by cAMP) and the other sensing the intracellular redox state (which is signalized by heme).
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cytosolic Thioredoxin Peroxidase i is essential for the antioxidant defense of yeast with dysfunctional mitochondria
FEBS Letters, 2001Co-Authors: Ana Paula Dias Demasi, Goncalo A G Pereira, Luis Eduardo Soares NettoAbstract:The specific role of cytosolic Thioredoxin Peroxidase I (cTPx I), encoded by TSA1 (thiol-specific antioxidant), was investigated in the oxidative stress response of Saccharomyces cerevisiae. In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity. However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident. All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H2O2 in cells, which could be related to their elevated sensitivity to oxidative stress. In fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H2O2. In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory-deficient cells.
Shin-ichiro Kawazu - One of the best experts on this subject based on the ideXlab platform.
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field evaluation of recombinant antigen elisa in detecting zoonotic schistosome infection among water buffaloes in endemic municipalities in the philippines
Frontiers in Veterinary Science, 2020Co-Authors: Jose Ma. M. Angeles, Yasuyuki Goto, Masashi Kirinoki, Elena A Villacorte, Yuichi Chigusa, Kharleezelle J Moendeg, Pilarita T Rivera, Shin-ichiro KawazuAbstract:In this study, we investigated the use of recombinant antigens Thioredoxin Peroxidase-1 (rSjTPx-1) and tandem repeat rSj1TR in evaluating the antibody positivity rates of Schistosoma japonicum infection among water buffaloes from four endemic areas in the Philippines, two municipalities with high endemicity (Calatrava, Negros Occidental and Catarman, Northern Samar) and two municipalities nearing elimination with no cases of human schistosomiasis (Talibon and Trinidad, Bohol). These recombinant antigen ELISA assays were compared with other diagnostic tests including SEA-ELISA, FECT, and fecal-based PCR. Results showed that rSj1TR-ELISA has the highest agreement with PCR in all study areas. Furthermore, significant positivity rates among water buffaloes were seen in Talibon and Trinidad, indicating that water buffaloes are maintaining the schistosome parasites in transmission areas even in the absence of human infection. Hence, serological assay using a more sensitive and specific rSj1TR-ELISA can be used for animal surveillance to prevent emergence and re-emergence of human schistosomiasis.
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development of monoclonal antibodies against plasmodium falciparum Thioredoxin Peroxidase 1 and its possible application for malaria diagnosis
Experimental Parasitology, 2015Co-Authors: Hassan Hakimi, Jose Ma. M. Angeles, Noboru Inoue, Keisuke Suganuma, Satoru Kawai, Yasuyuki Goto, Shin-ichiro KawazuAbstract:Rapid diagnostic tests (RDTs) have been considered as an ideal alternative for light microscopy to detect malaria parasites especially in remote areas. The development and improvement of RDTs is an area of intensive research in the last decade. To date, few parasite proteins have been targeted in RDTs which are known to have certain deficiencies and made the researchers to look for other promising candidates to address this problem. Plasmodium falciparum Thioredoxin Peroxidase 1 (PfTPx-1) is abundantly expressed in the cytoplasm of the parasite and well conserved across Plasmodium species, making this antigen a promising target for malaria diagnosis. Several monoclonal antibodies (mAbs) were produced against PfTPx-1. The binding affinities of mAbs were measured. Several immunochromatographic tests (ICTs) were developed using different combination of mAbs. All mAbs showed promising affinities to be used for diagnosis. The sensitivities of ICTs were evaluated using recombinant PfTPx-1 whose results lead us to the preparation of 4 different ICTs. These tests showed positive reaction with P. falciparum in vitro culture supernatant indicating the release of PfTPx-1 during schizont rupture. Altogether, these findings suggest that PfTPx-1 is a promising biomarker to diagnose P. falciparum infection. However, the diagnostic performance of this antigen should be further validated using clinical samples.
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effect of Thioredoxin Peroxidase 1 gene disruption on the liver stages of the rodent malaria parasite plasmodium berghei
Parasitology International, 2015Co-Authors: Miho Usui, Hassan Hakimi, Jose Ma. M. Angeles, Noboru Inoue, Hirono Masudasuganuma, Shinya Fukumoto, Shin-ichiro KawazuAbstract:Phenotypic observation of Thioredoxin Peroxidase-1 (TPx-1) gene-disrupted Plasmodium berghei (TPx-1 KO) in the liver-stage was performed with an in vitro infection system in order to investigate defective liver-stage development in a mouse infection model. Indirect immunofluorescence microscopy assay with anti-circumsporozoite protein antibody revealed that in the liver schizont stage, TPx-1 KO parasite cells were significantly smaller than cells of the wild-type parent strain (WT). Indirect immunofluorescence microscopy assay with anti-merozoite surface protein-1 antibody, which was used to evaluate late schizont-stage development, indicated that TPx-1 KO schizont development was similar to WT strain development towards the merozoite-forming stage (mature schizont). However, fewer merozoites were produced in the mature TPx-1 KO schizont than in the mature WT schizont. Taken together, the results suggest that TPx-1 may be involved in merozoite formation during liver schizont development.
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transfection of babesia bovis by double selection with wr99210 and blasticidin s and its application for functional analysis of Thioredoxin Peroxidase 1
PLOS ONE, 2015Co-Authors: Masahito Asada, Hassan Hakimi, Kazuhide Yahata, Naoaki Yokoyama, Ikuo Igarashi, Osamu Kaneko, Carlos E Suarez, Shin-ichiro KawazuAbstract:Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd) or human dihydrofolate reductase (hdhfr). In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP) and human dihydrofolate reductase (hDHFR), was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP) and blasticidin-S deaminase (BSD). Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which Thioredoxin Peroxidase-1 (Bbtpx-1) gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO) parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.
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plasmodium knowlesi Thioredoxin Peroxidase 1 binds to nucleic acids and has rna chaperone activity
Parasitology Research, 2014Co-Authors: Hassan Hakimi, Masahito Asada, Jose Ma. M. Angeles, Noboru Inoue, Miho Usui, Hirono Masudasuganuma, Keisuke Suganuma, Satoru Kawai, Shin-ichiro KawazuAbstract:Malaria parasites are under oxidative attack throughout their life cycle in human body and mosquito vector. Therefore, Plasmodium antioxidant defenses are crucial for its survival and being considered as interesting target for antimalarial drug design. Plasmodium knowlesi has emerged recently from its simian host to human in Southeast Asia and has been recognized as the fifth Plasmodium species that can cause human malaria. In this study, we cloned and characterized Thioredoxin Peroxidase 1 from P. knowlesi (PkTPx-1). PkTPx-1 gene was cloned, and recombinant protein was produced by heterologous overexpression in Escherichia coli. The recombinant protein was used for evaluation of enzymatic activity and polyclonal antibody production. Using the recombinant PkTPx-1 protein, its antioxidant activity was confirmed in a mixed-function oxidation assay where PkTPx-1 prevented nicking of DNA by hydroxyl radicals. PkTPx-1 was able to bind to double-strand DNA and RNA and had RNA chaperone activity in a nucleic acid melting assay indicating new function of PkTPx-1 other than antioxidant activity. Using specific polyclonal antibodies, it was indicated that PkTPx-1 is expressed in the cytoplasm of the parasite. Altogether, these results suggest that PkTPx-1 not only protects the parasite from the adverse effects of reactive oxygen species but also has RNA chaperone activity.
Xingqi Guo - One of the best experts on this subject based on the ideXlab platform.
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a novel 1 cys Thioredoxin Peroxidase gene in apis cerana cerana characterization of acctpx4 and its role in oxidative stresses
Cell Stress & Chaperones, 2015Co-Authors: Yifeng Huaxia, Fang Wang, Yan Yan, Feng Liu, Hongfang Wang, Xingqi GuoAbstract:Thioredoxin Peroxidase (Tpx), also named peroxiredoxin (Prx), is an important Peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys Thioredoxin Peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H2O2, and HgCl2. The in vivo Peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H2O2 in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees.
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identification and characterisation of a novel 1 cys Thioredoxin Peroxidase gene acctpx5 from apis cerana cerana
Comparative Biochemistry and Physiology B, 2014Co-Authors: Yan Yan, Yifeng Huaxia, Pengbo Yao, Xiuling Wang, Yuanying Zhang, Xingqi GuoAbstract:Abstract Thioredoxin Peroxidases (Tpxs), members of the antioxidant protein family, play critical roles in resisting oxidative stress. In this work, a novel 1-Cys Thioredoxin Peroxidase gene was isolated from Apis cerana cerana and was named AccTpx5. The open reading frame (ORF) of AccTpx5 is 663 bp in length and encodes a 220-amino acid protein with a predicted molecular mass and isoelectric point of 24,921 kDa and 5.45, respectively. Promoter sequence analysis of AccTpx5 revealed the presence of putative transcription factor binding sites related to early development and stress responses. Additionally, real-time quantitative PCR (Q-PCR) analysis indicated that AccTpx5 was primarily present in some developmental stages, with the highest expression levels in the first-instar larvae. The expression level of AccTpx5 was up-regulated under various abiotic stresses, including 4 °C, 42 °C, HgCl2, H2O2, phoxim and acaricide treatments. Conversely, it was down-regulated by UV and pyriproxyfen treatments. Moreover, H2O2 concentration dramatically increased under a variety of stressful conditions. Finally, the purified recombinant AccTpx5 protein protected the supercoiled form of plasmid DNA from damage in the thiol-dependent mixed-function oxidation (MFO) system. These results suggest that AccTpx5 most likely plays an essential role in antioxidant defence.
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glutaredoxin 1 glutaredoxin 2 Thioredoxin 1 and Thioredoxin Peroxidase 3 play important roles in antioxidant defense in apis cerana cerana
Free Radical Biology and Medicine, 2014Co-Authors: Pengbo Yao, Yan Yan, Feng Liu, Yuanying Zhang, Xiaobo Chen, Xingqi GuoAbstract:Abstract Glutaredoxins (Grxs) and Thioredoxins (Trxs) play important roles in maintaining intracellular thiol-redox homeostasis by scavenging reactive oxygen species. However, few Grxs and Trxs have been functionally characterized in Apis cerana cerana. In this study, we identified three genes, AccGrx1, AccGrx2, and AccTrx1, and investigated their connection to antioxidant defense. AccGrx1 and AccGrx2 were mainly detected in dark-eyed pupae, whereas AccTrx1 was highly concentrated in 15-day postemergence adults. The expression levels of AccGrx1 and AccTrx1 were the highest in fat body and epidermis, respectively. However, the expression level of AccGrx2 was the highest in muscle, followed by the epidermis. AccGrx1, AccGrx2, and AccTrx1 were induced by 4, 16, and 42 °C; H 2 O 2 ; and pesticide (acaricide, paraquat, cyhalothrin, and phoxime) treatments and repressed by UV light. AccGrx1 and AccGrx2 were upregulated by HgCl 2 treatment, whereas AccTrx1 was downregulated. We investigated the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 in A. cerana cerana and surprisingly found that knockdown of the these four genes enhanced the enzymatic activities of CAT and POD; the metabolite contents of hydrogen peroxide, carbonyls, and ascorbate; and the ratios of GSH/GSSG and NADP + /NADPH. In addition, we also analyzed the transcripts of other antioxidant genes and found that some were upregulated and others were downregulated, revealing that the upregulated genes may be involved in compensating for the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1. Taken together, these results suggest that AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 may play critical roles in antioxidant defense.
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molecular cloning expression and oxidative stress response of a mitochondrial Thioredoxin Peroxidase gene acctpx 3 from apis cerana cerana
Journal of Insect Physiology, 2013Co-Authors: Pengbo Yao, Fei Meng, Xiuling Wang, Xingqi GuoAbstract:Abstract Thioredoxin Peroxidase (Tpxs) plays an important role in maintaining redox homeostasis and in protecting organisms from the accumulation of toxic reactive oxygen species (ROS). Here, we isolated a mitochondrial Thioredoxin Peroxidase gene from Apis cerana cerana , AccTpx-3 . The open reading frame (ORF) of AccTpx-3 is 729 bp in length and encodes a predicted protein of 242 amino acids, 27.084 kDa and an isoelectric point of 8.70. Furthermore, the 980 bp 5′ flanking region was cloned, and the transcription factor binding sites were predicted. A quantitative RT-PCR (Q-PCR) analysis indicated that AccTpx-3 was expressed higher in muscle than other tissues, with its highest expression occurring on the fourth day of the larval stage, followed by the fifteenth day of the adult stage. Moreover, the expression of the AccTpx-3 transcript was upregulated by such abiotic stresses as 4 °C, 42 °C, H 2 O 2 , cyhalothrin, acaricide and phoxime treatments. In contrast, AccTpx-3 transcription was downregulated by other abiotic stresses, including 16 °C, 25 °C, ultraviolet light and HgCl 2 . Recombinant AccTpx-3 protein acted as a potent antioxidant that resisted paraquat-induced oxidative stress and protected DNA from oxidative damage. Taken together, these results suggest that the AccTpx-3 protein is an antioxidant enzyme that may protect organisms from oxidative stress.
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molecular cloning and characterization of a Thioredoxin Peroxidase gene from apis cerana cerana
Insect Molecular Biology, 2011Co-Authors: Mingjiang Kang, Fei Meng, Xingqi GuoAbstract:Thioredoxin Peroxidases (Tpxs) play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS) and regulating intracellular signal transduction. In the present study, we cloned the full cDNA of Tpx1 encoding a 195-amino acid protein from Apis cerana cerana (Acc). Based on the genomic DNA sequence, a 1442-bp 5'-flanking region was obtained, and the putative transcription factor binding sites were predicted. Quantitative PCR analysis showed that AccTpx1 was highly expressed in thorax and that the AccTpx1 transcript reached its highest level in two-week-old adult worker honeybees. Moreover, expression of the AccTpx1 transcript was increased by various abiotic stresses, such as ultraviolet light, HgCl(2) , and insecticide treatments. In addition, the recombinant AccTpx1 protein exhibited antioxidant activity; it removed hydrogen peroxide and protected DNA. These results suggest that AccTpx1 plays an important role in protecting honeybees from oxidative injury and may act in extending the lifespan of them.
Sue Goo Rhee - One of the best experts on this subject based on the ideXlab platform.
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catalases and Thioredoxin Peroxidase protect saccharomyces cerevisiae against ca2 induced mitochondrial membrane permeabilization and cell death
FEBS Letters, 2000Co-Authors: Alicia J Kowaltowski, Anibal E. Vercesi, Sue Goo Rhee, Luis Eduardo Soares NettoAbstract:The involvement of reactive oxygen species in Ca2+-induced mitochondrial membrane permeabilization and cell viability was studied using yeast cells in which the Thioredoxin Peroxidase (TPx) gene was disrupted and/or catalase was inhibited by 3-amino-1,2,4-triazole (ATZ) treatment. Wild-type Saccharomyces cerevisiae cells were very resistant to Ca2+ and inorganic phosphate or t-butyl hydroperoxide-induced mitochondrial membrane permeabilization, but suffered an immediate decrease in mitochondrial membrane potential when treated with Ca2+ and the dithiol binding reagent phenylarsine oxide. In contrast, S. cerevisiae spheroblasts lacking the TPx gene and/or treated with ATZ suffered a decrease in mitochondrial membrane potential, generated higher amounts of hydrogen peroxide and had decreased viability under these conditions. In all cases, the decrease in mitochondrial membrane potential could be inhibited by ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid, dithiothreitol or ADP, but not by cyclosporin A. We conclude that TPx and catalase act together, maintaining cell viability and protecting S. cerevisiae mitochondria against Ca2+-promoted membrane permeabilization, which presents similar characteristics to mammalian permeability transition.
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catalases and Thioredoxin Peroxidase protect saccharomyces cerevisiae against ca2 induced mitochondrial membrane permeabilization and cell death
FEBS Letters, 2000Co-Authors: Alicia J Kowaltowski, Anibal E. Vercesi, Sue Goo Rhee, Luis Eduardo Soares NettoAbstract:The involvement of reactive oxygen species in Ca(2+)-induced mitochondrial membrane permeabilization and cell viability was studied using yeast cells in which the Thioredoxin Peroxidase (TPx) gene was disrupted and/or catalase was inhibited by 3-amino-1,2, 4-triazole (ATZ) treatment. Wild-type Saccharomyces cerevisiae cells were very resistant to Ca(2+) and inorganic phosphate or t-butyl hydroperoxide-induced mitochondrial membrane permeabilization, but suffered an immediate decrease in mitochondrial membrane potential when treated with Ca(2+) and the dithiol binding reagent phenylarsine oxide. In contrast, S. cerevisiae spheroblasts lacking the TPx gene and/or treated with ATZ suffered a decrease in mitochondrial membrane potential, generated higher amounts of hydrogen peroxide and had decreased viability under these conditions. In all cases, the decrease in mitochondrial membrane potential could be inhibited by ethylene glycol-bis(beta-aminoethyl ether) N,N, N',N'-tetraacetic acid, dithiothreitol or ADP, but not by cyclosporin A. We conclude that TPx and catalase act together, maintaining cell viability and protecting S. cerevisiae mitochondria against Ca(2+)-promoted membrane permeabilization, which presents similar characteristics to mammalian permeability transition.
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purification and characterization of a second type Thioredoxin Peroxidase type ii tpx from saccharomyces cerevisiae
Biochemistry, 1999Co-Authors: Jin Sook Jeong, Sue Goo Rhee, Soo Jin Kwon, Sang Won Kang, Kanghwa KimAbstract:A yeast Peroxidase that reduces H2O2 and alkyl hydroperoxides with the use of reducing equivalents provided by Thioredoxin was identified previously and named Thioredoxin Peroxidase (TPx) [Chae, H. Z., Chung, S. J., and Rhee, S. G. (1994) J. Biol. Chem. 269, 27670−27678]. A second type Thioredoxin-dependent Peroxidase, named type II TPx, has now been purified from yeast, and several peptide sequences have been obtained. Using those sequences, the corresponding cDNA has been identified from the GenBank database. Comparison of the predicted sequence of 176 amino acids of type II TPx with that of the 195 residues of TPx, now renamed type I TPx, revealed no substantial homology except for a short segment preceding Cys62 of type II TPx. Kinetic characterization of the reactions catalyzed by type I and II TPxs revealed that type I preferentially reduces H2O2 rather than alkyl hydroperoxides, whereas type II shows the reverse specificity. Type II TPx contains three cysteine residues at positions 31, 62, and 120....
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regulatory role for a novel human Thioredoxin Peroxidase in nf κb activation
Journal of Biological Chemistry, 1997Co-Authors: Dongyan Jin, Sue Goo Rhee, Ho Zoon Chae, Kuanteh JeangAbstract:Reduction-oxidation (redox) plays a critical role in NF-kappaB activation. Diverse stimuli appear to utilize reactive oxygen species (e.g. hydrogen peroxide) as common effectors for activating NF-kappaB. Antioxidants govern intracellular redox status, and many such molecules can reduce H2O2. However, functionally, it does appear that different antioxidants are variously selective for redox regulation of certain transcription factors such as NF-kappaB. For NF-kappaB, Thioredoxin has been described to be a more potent antioxidant than either glutathione or N-acetylcysteine. Thioredoxin Peroxidase is the immediate enzyme that links reduction of H2O2 to Thioredoxin. Several putative human Thioredoxin Peroxidases have been identified using recursive sequence searches/alignments with yeast or prokaryotic enzymes. None has been characterized in detail for intracellular function(s). Here, we describe a new human Thioredoxin Peroxidase, antioxidant enzyme AOE372, identified by virtue of its protein-protein interaction with the product of a proliferation association gene, pag, which is also a thiol-specific antioxidant. In human cells, AOE372 defines a redox pathway that specifically regulates NF-kappaB activity via a modulation of IkappaB-alpha phosphorylation in the cytoplasm. We show that AOE372 activity is regulated through either homo- or heterodimerization with other thiol Peroxidases, implicating subunit assortment as a mechanism for regulating antioxidant specificities. AOE372 function suggests Thioredoxin Peroxidase as an immediate regulator of H2O2-mediated activation of NF-kappaB.
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Thioredoxin Peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of bcl 2
Journal of Biological Chemistry, 1997Co-Authors: Ping Zhang, Sue Goo Rhee, Bin Liu, San Won Kang, Min Seok Seo, Lina M ObeidAbstract:Thioredoxin Peroxidase (TPx) is a member of a newly discovered family of proteins that are conserved from yeast to mammals and to which natural killer enhancing factor belongs. These proteins are antioxidants that function as Peroxidases only when coupled to a sulfhydryl reducing system. The physiological function of TPx in cells is not yet known. Here we demonstrate that when the human TPx II, a member of this family, is stably overexpressed in Molt-4 leukemia cells, it protects from apoptosis induced by serum deprivation, ceramide, or etoposide. TPx II, like Bcl-2, is able to inhibit release of cytochrome c from mitochondria to cytosol, and it inhibits lipid peroxidation in cells. TPx II, unlike Bcl-2, could prevent hydrogen peroxide accumulation in cells, suggesting that it functions upstream of Bcl-2 in the protection from apoptosis and may be implicated as an endogenous regulator of apoptosis.
Jose Ma. M. Angeles - One of the best experts on this subject based on the ideXlab platform.
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field evaluation of recombinant antigen elisa in detecting zoonotic schistosome infection among water buffaloes in endemic municipalities in the philippines
Frontiers in Veterinary Science, 2020Co-Authors: Jose Ma. M. Angeles, Yasuyuki Goto, Masashi Kirinoki, Elena A Villacorte, Yuichi Chigusa, Kharleezelle J Moendeg, Pilarita T Rivera, Shin-ichiro KawazuAbstract:In this study, we investigated the use of recombinant antigens Thioredoxin Peroxidase-1 (rSjTPx-1) and tandem repeat rSj1TR in evaluating the antibody positivity rates of Schistosoma japonicum infection among water buffaloes from four endemic areas in the Philippines, two municipalities with high endemicity (Calatrava, Negros Occidental and Catarman, Northern Samar) and two municipalities nearing elimination with no cases of human schistosomiasis (Talibon and Trinidad, Bohol). These recombinant antigen ELISA assays were compared with other diagnostic tests including SEA-ELISA, FECT, and fecal-based PCR. Results showed that rSj1TR-ELISA has the highest agreement with PCR in all study areas. Furthermore, significant positivity rates among water buffaloes were seen in Talibon and Trinidad, indicating that water buffaloes are maintaining the schistosome parasites in transmission areas even in the absence of human infection. Hence, serological assay using a more sensitive and specific rSj1TR-ELISA can be used for animal surveillance to prevent emergence and re-emergence of human schistosomiasis.
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evaluation of schistosoma japonicum Thioredoxin Peroxidase 1 as a potential circulating antigen target for the diagnosis of asian schistosomiasis
Journal of Veterinary Medical Science, 2017Co-Authors: Adrian Miki C Macalanda, Jose Ma. M. Angeles, Noboru Inoue, Masashi Kirinoki, Yuichi Chigusa, Kharleezelle J Moendeg, Anh Tm Dang, Luna Higuchi, Xuenan Xuan, Lydia LeonardoAbstract:Asian schistosomiasis caused by Schistosoma japonicum is a serious zoonotic disease endemic in China, the Philippines and parts of Indonesia. Mass drug administration in endemic areas resulted to decline in disease severity and intensity. The low intensity of infection limits the use of current parasitological methods for schistosomiasis diagnosis. Detection of parasite circulating antigens might provide more informative result as it may indicate the true status of infection. In this study, S. japonicum Thioredoxin Peroxidase-1 (SjTPx-1) a 22 kDa secreted antioxidant enzyme expressed throughout the life stages of the parasite was evaluated for its potential use as a biomarker for schistosomiasis japonica infection. Rabbit polyclonal antibody and mouse monoclonal antibodies (mAbs) were raised against the recombinant SjTPx-1 (rSjTPx-1). The antibodies produced against the recombinant antigen was confirmed to detect the native SjTPx-1 in crude adult worm lysate. Likewise, the specific binding of mAbs to parasite TPx-1 and not to mammalian peroxiredoxin-1 orthologues was also confirmed. The double antibody sandwich ELISA developed in this study was able to detect at least 1 ng/ml of rSjTPx-1. In addition, this method was able to detect the antigen from all serum samples of experimentally infected rabbit and mice. The diagnostic potential of SjTPx-1 in human clinical samples was also evaluated, in which 4 out of 10 stool-confirmed serum samples had detectable levels of the antigen. The results suggest that SjTPx-1 can be a potential biomarker for Asian zoonotic schistosomiasis.
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development of monoclonal antibodies against plasmodium falciparum Thioredoxin Peroxidase 1 and its possible application for malaria diagnosis
Experimental Parasitology, 2015Co-Authors: Hassan Hakimi, Jose Ma. M. Angeles, Noboru Inoue, Keisuke Suganuma, Satoru Kawai, Yasuyuki Goto, Shin-ichiro KawazuAbstract:Rapid diagnostic tests (RDTs) have been considered as an ideal alternative for light microscopy to detect malaria parasites especially in remote areas. The development and improvement of RDTs is an area of intensive research in the last decade. To date, few parasite proteins have been targeted in RDTs which are known to have certain deficiencies and made the researchers to look for other promising candidates to address this problem. Plasmodium falciparum Thioredoxin Peroxidase 1 (PfTPx-1) is abundantly expressed in the cytoplasm of the parasite and well conserved across Plasmodium species, making this antigen a promising target for malaria diagnosis. Several monoclonal antibodies (mAbs) were produced against PfTPx-1. The binding affinities of mAbs were measured. Several immunochromatographic tests (ICTs) were developed using different combination of mAbs. All mAbs showed promising affinities to be used for diagnosis. The sensitivities of ICTs were evaluated using recombinant PfTPx-1 whose results lead us to the preparation of 4 different ICTs. These tests showed positive reaction with P. falciparum in vitro culture supernatant indicating the release of PfTPx-1 during schizont rupture. Altogether, these findings suggest that PfTPx-1 is a promising biomarker to diagnose P. falciparum infection. However, the diagnostic performance of this antigen should be further validated using clinical samples.
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effect of Thioredoxin Peroxidase 1 gene disruption on the liver stages of the rodent malaria parasite plasmodium berghei
Parasitology International, 2015Co-Authors: Miho Usui, Hassan Hakimi, Jose Ma. M. Angeles, Noboru Inoue, Hirono Masudasuganuma, Shinya Fukumoto, Shin-ichiro KawazuAbstract:Phenotypic observation of Thioredoxin Peroxidase-1 (TPx-1) gene-disrupted Plasmodium berghei (TPx-1 KO) in the liver-stage was performed with an in vitro infection system in order to investigate defective liver-stage development in a mouse infection model. Indirect immunofluorescence microscopy assay with anti-circumsporozoite protein antibody revealed that in the liver schizont stage, TPx-1 KO parasite cells were significantly smaller than cells of the wild-type parent strain (WT). Indirect immunofluorescence microscopy assay with anti-merozoite surface protein-1 antibody, which was used to evaluate late schizont-stage development, indicated that TPx-1 KO schizont development was similar to WT strain development towards the merozoite-forming stage (mature schizont). However, fewer merozoites were produced in the mature TPx-1 KO schizont than in the mature WT schizont. Taken together, the results suggest that TPx-1 may be involved in merozoite formation during liver schizont development.
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plasmodium knowlesi Thioredoxin Peroxidase 1 binds to nucleic acids and has rna chaperone activity
Parasitology Research, 2014Co-Authors: Hassan Hakimi, Masahito Asada, Jose Ma. M. Angeles, Noboru Inoue, Miho Usui, Hirono Masudasuganuma, Keisuke Suganuma, Satoru Kawai, Shin-ichiro KawazuAbstract:Malaria parasites are under oxidative attack throughout their life cycle in human body and mosquito vector. Therefore, Plasmodium antioxidant defenses are crucial for its survival and being considered as interesting target for antimalarial drug design. Plasmodium knowlesi has emerged recently from its simian host to human in Southeast Asia and has been recognized as the fifth Plasmodium species that can cause human malaria. In this study, we cloned and characterized Thioredoxin Peroxidase 1 from P. knowlesi (PkTPx-1). PkTPx-1 gene was cloned, and recombinant protein was produced by heterologous overexpression in Escherichia coli. The recombinant protein was used for evaluation of enzymatic activity and polyclonal antibody production. Using the recombinant PkTPx-1 protein, its antioxidant activity was confirmed in a mixed-function oxidation assay where PkTPx-1 prevented nicking of DNA by hydroxyl radicals. PkTPx-1 was able to bind to double-strand DNA and RNA and had RNA chaperone activity in a nucleic acid melting assay indicating new function of PkTPx-1 other than antioxidant activity. Using specific polyclonal antibodies, it was indicated that PkTPx-1 is expressed in the cytoplasm of the parasite. Altogether, these results suggest that PkTPx-1 not only protects the parasite from the adverse effects of reactive oxygen species but also has RNA chaperone activity.