Thiol-specific Antioxidant Research Articles

Prdx Overexpression in Tumor Tissue of Breast Cancer Patients

Peroxiredoxin (Prdx) proteins are evolutionarily conserved thiol-specific antioxidant enzymes that reduce various cellular peroxides, protecting cells from oxidative damage. Prdxs also have been demonstrated to play an important role in regulating redox-sensitive cell signaling in a number of cell processes.

Identification and molecular characterization of peroxiredoxin 6 from Japanese eel (Anguilla japonica) revealing its potent antioxidant properties and putative immune relevancy.

Peroxiredoxins (Prdx) are thiol specific antioxidant enzymes that play a pivotal role in cellular oxidative stress by reducing toxic peroxide compounds into nontoxic products. In this study, we identified and characterized a peroxiredoxin 6 counterpart from Japanese eel (Anguilla japonica) (AjPrdx6) at molecular, transcriptional and protein level. The identified full-length coding sequence of AjPrdx6 (669bp) coded for a polypeptide of 223 aa residues (24.9kDa). Deduced protein of AjPrdx6 showed analogy to characteristic structural features of 1-cysteine peroxiredoxin sub-family. According to the topology of the generated phylogenetic reconstruction AjPrdx6 showed closest evolutionary relationship with Salmo salar. As detected by Quantitative real time PCR (qPCR), AjPrdx6 mRNA was constitutively expressed in all the tissues examined. Upon the immune challenges with Edwardsiella tarda, lipopolysaccharides and polyinosinic:polycytidylic acid, expression of AjPrdx6 mRNA transcripts were significantly induced. The general functional properties of Prdx6 were confirmed using purified recombinant AjPrdx6 protein by deciphering its potent protective effects on cultured vero cells (kidney epithelial cell from an African green monkey) against H2O2-induced oxidative stress and protection against oxidative DNA damage elicited by mixed function oxidative (MFO) system. Altogether, our findings suggest that AjPrdx6 is a potent antioxidant protein in Japanese eels and its putative immune relevancy in pathogen stress mounted by live-bacteria or pathogen associated molecular patterns (PAMPs).

Abstract 1245: Peroxiredoxin induction in breast cancer

Abstract Cancer cells possess elevated levels of reactive oxygen species (ROS) that can enhance genomic instability and alter redox signaling. Aberrant antioxidant expression has also been recognized in many cancers, suggesting an adaptive response to the cancerous state. Breast cancers express significantly elevated levels of Peroxiredoxins (Prdxs), a family of thiol-specific antioxidant proteins involved in regulating cell growth and proliferation in many cell types. We previously reported overexpression of many Prdx proteins in the MCF-7 breast cancer line, as well as their importance in doxorubicin-resistance in this line. In the present study, we analyzed Prdx levels in several human breast tumor samples, and further investigated possible mechanisms of Prdx regulation in MCF-7 cells. Comparison of breast tumor and adjacent normal breast tissue from almost 20 patients showed that most patients exhibited elevated levels of several Prdx proteins in the tumor tissue. To begin to understand possible regulators of Prdxs in breast cancer, we focused on transcriptional regulators known to be aberrantly expressed in breast cancer, and also implicated in antioxidant regulation. Sequence analysis revealed potential binding sites for NF-κB, Nrf2, and AP-1 in several peroxiredoxin promoters. We found high levels of NF-κB in MCF-7 cells, as compared to normal breast epithelial cells. We further showed that a chemo-resistant MCF-7 culture generated from 14 days of doxorubicin treatment exhibited markedly decreased NF-κB expression. We then used both siRNA and chemical inhibition of NF-κB to examine the effect on Prdx regulation in these cells and found gene-specific effects. Similar studies are currently underway for Nrf2 and AP-1. This investigation will help to elucidate the role of peroxiredoxins in breast cancer cell survival, and the mechanism by which these cells alter Prdx levels to avoid death and achieve chemoresistance. Citation Format: Valerie Buurma, Brendan Freeman, Akosua Adzenyah, Gregory Pettit, Felicitas Heinen, Jillian Muhlbauer, Shelley A. Phelan. Peroxiredoxin induction in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1245. doi:10.1158/1538-7445.AM2015-1245

Yeast 2-Cys peroxiredoxin Tsa1 protects cells from DNA damage-induced reactive oxygen species through peroxidase activity

Peroxiredoxins (Prxs) belong to a family of thiol-specific peroxidases that scavenge reactive oxygen species (ROS). Yeast Prx thiol-specific antioxidant (Tsa1) protects cells from oxidative stress and maintains genome stability. In this study, we investigated the role of Tsa1 in the response to DNA damage. We observed that Tsa1 among all five Prxs of yeast is the most responsive in cell viability and mutation rate assays. The Δtsa1 mutant is hypersensitive to DNA-damaging agents such as hydroxyurea, methyl methane sulfonate, and camptothecin. The Δtsa1 cells lead to reduced cell viability and cell growth after DNA damage. The Δtsa1-expressing wild-type Tsa1 rescues the sensitivity of Δtsa1 cells to DNA damage, but not in Δtsa1 cells carrying the active-site mutant Tsa1-C47S. We found that the level of ROS in Δtsa1 cells was increased more than in wild-type cells by DNA damage. Tsa1 completely restored the production of DNA damage-induced ROS in Δtsa1 cells, but not Tsa1-C47S. Finally, DNA damage-induced ROS resulted in a switch of Tsa1 from a low-molecular-weight to a high-molecular-weight complex. Taken together, these findings suggest that Tsa1 protects yeast cells from DNA damage by regulating the homeostasis of ROS through peroxidase activity.

Induction of Peroxiredoxins in Breast Cancer

Cancer cells possess elevated levels of reactive oxygen species (ROS) that can enhance genomic instability and alter redox signaling. Aberrant antioxidant expression has also been recognized in many cancers, suggesting an adaptive response to the cancerous state. Breast cancers express significantly elevated levels of Peroxiredoxins (Prdxs), a family of thiol‐specific antioxidant proteins involved in regulating cell growth and proliferation in many cell types. We previously reported overexpression of many Prdx proteins in the MCF‐7 breast cancer line, as well as their importance in doxorubicin‐resistance in this line. In the present study, we analyzed Prdx levels in several human breast tumor samples, and further investigated possible mechanisms of Prdx regulation in MCF‐7 cells. Comparison of breast tumor and adjacent normal breast tissue from almost 20 patients showed that most patients exhibited elevated levels of several Prdx proteins in the tumor tissue. Sequence analysis revealed potential binding sites for NF‐κB, Nrf2, and AP‐1 in several peroxiredoxin promoters. We found high levels of NF‐κB in MCF‐7 cells, as compared to normal breast epithelial cells. We further showed that a chemo‐resistant MCF‐7 culture generated from 14 days of doxorubicin treatment exhibited markedly decreased NF‐ κB expression. We then used both siRNA and chemical inhibition of NF‐κB to examine the effect on Prdx regulation in these cells and found gene‐specific effects. This investigation will help to elucidate the role of peroxiredoxins in breast cancer cell survival, and the mechanism by which these cells alter Prdx levels to avoid death and achieve chemoresistance.

Overexpression and activities of 1-Cys peroxiredoxin from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode.

Peroxiredoxins (Prxs) are enzymatic antioxidants widely distributed in biological kingdoms, which constitute a family of heme-free peroxidases that reduce alkyl hydroperoxides and hydrogen peroxide. In this paper, an open reading frame (ORF) of 639 bp, which encoded a protein of 213 amino acid residues, was cloned from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode. Amino acid sequence alignment showed that the encoded protein shared 99, 97, and 97 % identity with the thiol-specific antioxidant protein LsfA of P. fluorescens Q2-87, the peroxiredoxin of Pseudomonas sp. GM17 and 1-Cys peroxiredoxin of P. fluorescens Pf 0-1, respectively. The ORF was cloned into expressing vector pET-15b and introduced into Escherichia coli BL21 (DE3). Overexpression of a 27-kDa protein was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on a Ni(2+) matrix column. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that part of the recombinant appeared in dimer form. Bioassay results showed that purified recombinant protein had both peroxidase and thioredoxin activity. Furthermore, E. coli expressing the ORF showed tolerance to hydrogen peroxide stress, which indicated that the gene might help P. fluorescens GcM5-1A resist hydrogen peroxide generated by host pines after pine wood nematode associated with this bacterium infected pine trees.

Abstract 2262: Elevated peroxiredoxin expression in breast cancer and its protective role in doxorubicin-resistance

Abstract Peroxiredoxin (Prdx) proteins are thiol-specific antioxidants that protect cells from oxidative stress in various normal and disease states. There are six different Prdx proteins expressed in mammals, each possessing a characteristic tissue and subcellular distribution. Recent studies have revealed elevated Prdx levels in many cancers, suggesting a protective role for these proteins in cancer cell survival. In the present study we analyzed Prdx levels in several human breast tumor samples, and also investigated the function of Prdx proteins in the MCF-7 breast cancer cell line. Analysis of breast tumor and adjacent normal breast tissue from almost 20 patients showed elevated levels of most Prdx proteins in tumor tissue from the majority of patients. To address the role of Prdxs in breast cancer, we used transient siRNA transfections of MCF-7 cells and examined resulting cell viability in the absence and presence of doxorubicin treatment. Our data show that individual suppression of four of six of the Prdx proteins leads to increased cell death, as well as increased sensitivity to doxorubicin-induced toxicity. Finally, we show that clonal selection of doxorubicin-resistant MCF-7 cells by two-week culture in 0.1uM doxorubicin resulted in a marked elevation in the expression of several Prdx proteins. Similar studies are currently underway in the highly metastatic MDA cell line, and possible upstream regulators of Prdx overexpression in these lines are being investigated. Together, these data show a strong protective role for Peroxiredoxins in breast cancer cell survival, and suggest that Prdx overexpression in breast cancer may play a role in chemotherapy-resistance. Citation Format: Jillian Muhlbauer, Gregg Perlmutter, Caitlin McDonald, Harry Cintineo, Caterina Aiello, Shelley A. Phelan. Elevated peroxiredoxin expression in breast cancer and its protective role in doxorubicin-resistance. [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 2262. doi:10.1158/1538-7445.AM2014-2262

The role of Montanide ISA 70 as an adjuvant in immune responses against Leishmania major induced by thiol-specific antioxidant-based protein vaccine.

Leishmaniasis is a parasitic disease caused by several species of the genus Leishmania. Montanide ISA 70 is an adjuvant composed of a natural metabolizable oil and a very refined emulsifier from the manide monooleate family. The TSA (thiol-specific antioxidant) is a important antigen of Leishmania major. The purpose of this work was protein-vaccine efficacy as an protection and excellent candidate in the presence Montanide. The expression of recombinant protein was confirmed with SDS (sodium dodecyl sulfate) page and Western bloting. 48 BALB/c mice were divided into four groups (TSA/Freund,TSA/Alum+BCG, TSA/Montanide and PBS groups) and immunized with 20μg of vaccine subcutaneously three times intervals on days 0, 14 and 28. The mice were challenged with parasite 21days after final immunization. The lymphocyte proliferation was evaluated with Brdu method. Cytokines and also total antibody and subclasses were evaluated with ELISA method. The vaccine formulated with the recombinant TSA protein with Montanide induced lymphocytes proliferation cytokines and total antibody and subclasses as compared with the control group.

Survey Protein Vaccine Formulated with Montanide ISA 70 Effects following Immunization and after Challenge with Leishmania major

Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which, in the infected host are obliging intracellular parasite . Montanide ISA 70 is an adjuvant composed of a natural metabolizable oil and a highly refined emulsifier from the manide monooleate family. The TSA (thiol-specific antioxidant) is a considerable antigen of Leishmania major. The purpose of this work was Protein-vaccine efficacy in the presence Montanide comparing to absence of it. The expression of recombinant protein was confirmed with SDS (sodium dodecyl sulfate) page and Western bloting . 48 BALB/c mice were divided into four groups [TSA/ Freund ,TSA/Alum+BCG , TSA/ Montanide and PBS groups] and immunized with 20 mg of vaccine subcutaneously three times intervals on days 0, 14 and 28. The mice were challenged with parasite 21 days after final immunization . The lymphocyte proliferation was evaluated with Brdu method. Cytokines was evaluated with ELISA method. The vaccine formulated with the recombinant TSA protein with Montanide induced lymphocytes proliferation , cytokines as compared with the control group. Base on results, current candidate vaccine has potency for futher studies.

Peroxiredoxin proteins protect MCF-7 breast cancer cells from doxorubicin-induced toxicity.

Peroxiredoxin (Prdx) proteins are thiol-specific antioxidants that protect cells from oxidative stress in many normal and disease states. There are six Prdx proteins expressed in mammals, each with a characteristic tissue expression, subcellular distribution and substrate specificity. Recent studies have revealed elevated Prdx levels in many cancers, suggesting a protective role for these proteins in cancer cell survival. The present study is the first to investigate the function of all six Prdx proteins in the MCF-7 breast cancer cell line. We show that these cells have both higher resistance to doxorubicin-induced toxicity and significantly elevated Prdx levels, compared to the non-cancer MCF-10A cells. Using transient siRNA transfections, we show that Prdx3 suppression leads to decreased MCF-7 cell survival in the absence of doxorubicin. We further demonstrate that individual suppression of four of six of the Prdx proteins leads to increased doxorubicin-induced toxicity by apoptosis. Finally, we show that clonal selection of a doxorubicin-resistant MCF-7 subline by 2-week culture in 0.1 µM doxorubicin resulted in a marked elevation in the expression of several Prdx proteins. Together, these data reveal a protective function for peroxiredoxins in MCF-7 cell survival, and suggest that Prdx overexpression in breast cancer may play a role in doxorubicin-resistance in these, and possibly other, breast cancer cells. This study is the first to investigate the function of the entire Prdx family in a breast cancer cell line.

Th1 Platform Immune Responses Against Leishmania major Induced by Thiol-Specific Antioxidant-Based DNA Vaccines.

Background:The Thiol-specific antioxidant (TSA) is an antigen of Leishmania major which is believed to be the most promising molecule as a vaccine candidate against leishmaniasis.Objectives:In this study, we investigated the protective efficacy of TSA-based DNA vaccine against L. major infection.Materials and Methods:Recombinant plasmid construction TSA (pcTSA) was prepared and transfected into eukaryotic cells and expression was confirmed with western blot and RT-PCR. The mice were assigned to six different groups and DNA immunization was performed with 100 µg intramuscular recombinant plasmid with a two-week interval. Cytokines and lymphocyte proliferation assay, antibody responses and determination of parasite burden were performed following immunization and the challenging infection with L. major.Results:The antibody and IFN-γ titers were higher in pcTSA + AlPO4 group the immunized mice with pcTSA alone, but there was no statistically significant difference between the two groups. Additionally the IL-4 titer was not statistically different between the groups following immunization and challenge. After infection with L. major promastigotes, the immunized mice with pcTSA and the one immunized with both pcTSA + AlPO4 presented a considerable reduction in diameter of lesion but there was no statistical difference between the two groups. The immunized mice had significantly lower parasite loads. No significant differences were observed between the two vaccinated groups. However the highest reduction in parasite burden was observed in the group immunized with pcDNA + AlPO4. No significant differences were observed in survival rate of the immunized mice after the challenge with L. major.Conclusions:In conclusion, TSA-based DNA vaccine induced Th1 platform immune response and aluminum phosphate could improve the efficacy of these vaccines with induction of humoral and cellular immune responses against L. major infection. There were no significant differences observed between pcTSA and pcTSA + AlPO4 groups.

Attenuation of hydrogen peroxide-mediated oxidative stress by Brassica juncea annexin-3 counteracts thiol-specific antioxidant (TSA1) deficiency in Saccharomyces cerevisiae

Brassica juncea annexin-3 (BjAnn3) was functionally characterized for its ability to modulate H2O2-mediated oxidative stress in Saccharomyces cerevisiae. BjAnn3 showed a significant protective role in cellular-defense against oxidative stress and partially alleviated inhibition of mitochondrial respiration in presence of exogenously applied H2O2. Heterologous expression of BjAnn3 protected membranes from oxidative stress-mediated damage and positively regulated antioxidant gene expression for ROS detoxification. We conclude that, BjAnn3 partially counteracts the effects of thioredoxin peroxidase 1 (TSA1) deficiency and aids in cellular-protection across kingdoms. Despite partial compensation of TSA1 by BjAnn3 in cell-viability tests, the over-complementation in ROS-related features suggests the existence of both redundant (e.g. ROS detoxification) and distinct features (e.g. membrane protection versus proximity-based redox regulator) of both proteins.

Peroxiredoxin Overexpression in MCF-7 Breast Cancer Cells and Regulation by Cell Proliferation and Oxidative Stress

Peroxiredoxins are thiol-specific antioxidant proteins that protect cells from ROS-induced cell death and are elevated in several cancers. We found that five of the six mammalian peroxiredoxins are overexpressed in MCF-7 breast cancer cells at the mRNA and protein levels, compared to noncancerous MCF-10A cells. Inhibition of MCF-7 proliferation reduced the levels of several peroxiredoxins. In contrast, all six proteins were strongly and transiently induced in MCF-7 cells by H2O2. These data suggest that coordinate overexpression of peroxiredoxins may be an important cancer cell adaptation, and that these proteins can be regulated by cell proliferation and oxidative stress.

Abstract 2908: Function and regulation of peroxiredoxin proteins in doxorubicin-treated MCF-7 breast cancer cells.

Abstract The Peroxiredoxin (Prdx) family of genes encodes thiol-specific antioxidant proteins that protect cells from oxidative stress. Mammalian cells express six different Prdx proteins, and these proteins play a role in cell signaling, proliferation and apoptosis. Recent studies have shown that Prdx levels are elevated in many cancers, suggesting that Prdx upregulation may be an advantageous adaptation to the cancerous state. In the present study we investigated the expression and function of different Prdx proteins in untreated and doxorubicin-treated MCF-7 breast cancer cells, as well as noncancerous MCF-10A cells. We used real-time PCR and western blotting to examine Prdx expression in both cell lines, and also measured Prdx protein expression in doxorubicin-treated MCF-7 cells. In addition, we suppressed Prdx expression using transiently transfected siRNA and measured cell proliferation and apoptosis. Our results show that MCF-7 cells are significantly more resistant to cell death induced by the chemotherapy agent doxorubicin. We also found that five of the six Prdxs are overexpressed in MCF-7 cells at the mRNA and protein levels, and that serum deprivation reduced Prdx expression. Treatment of MCF-7 cells with doxorubicin altered expression of specific Prdxs, demonstrating a specific effect on these antioxidants. Transient transfection of MCF-7 cells with Prdx6 siRNA resulted in a marked reduction in Prdx6 suppression, with no change in other Prdx proteins. Suppression of Prdx6 in these cells led to reduced cell proliferation and increased susceptibility to doxorubicin-induced death. Similar studies are currently underway for the other Prdx proteins. Together, these findings suggest that Prdx overexpression in MCF-7 cells may be cytoprotective and that Prdxs may contribute to doxorubicin-resistance in these and other breast cancer cells. Citation Format: Caitlin McDonald, Gregg Perlmutter, Kekoa Taparra, Jillian Muhlbauer, Julie Passarelli, Shelley A. Phelan. Function and regulation of peroxiredoxin proteins in doxorubicin-treated MCF-7 breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2908. doi:10.1158/1538-7445.AM2013-2908

항산화반응을 유발하는 물질의 검색에 적용할 수 있는 형질전환 마우스 생산을 위한 새로운 Prx 6/Luc 벡터시스템의 제조 및 폐암세포주에서 반응성 확인

Peroxiredoxin 6 (Prx 6) is a member of the thiol-specific antioxidant protein family, which may play a role in protection against oxidative stress and in regulating phospholipid turnover. The aim of this study was to determine whether a human Prx 6/Luc vector was stably expressed and responded to antioxidants in a lung cell line (NCI-H460). To achieve this, the luciferase signal, hPrx 6 mRNA expression, and superoxide dismutase (SOD) activity were measured in transfectants with a hPrx 6/Luc plasmid after treatment with four antioxidant extracts, including Korea white ginseng (KWG), Korea red ginseng (KRG), Liriope platyphylla (LP), and red Liriope platyphylla (RLP). First, the hPrx 6/Luc plasmid was successfully constructed with DNA fragments of human Prx 6 promoter, amplified by PCR using genomic DNA isolated from NCI-H460 cells, and cloned into the pTransLucent reporter vector. The orientation and sequencing of the hPrx 6/Luc plasmid were identified with restriction enzyme and automatic sequencing. A luciferase assay revealed significant enhancement of luciferase activity in the four treatment groups compared with a vehicle-treated group, although the ratio of the increase was different within each group. The KRG- and LP-treated groups showed higher activity than the KWG- and RLP-treated groups. Furthermore, the luciferase activity against RLP occurred roughly in a dose-dependent manner. However, the level of endogenous hPrx 6 mRNA did not change in any group treated with the four extracts. The SOD activity was in agreement with the luciferase activity. Therefore, these results indicate that the hPrx 6/Luc vector system may successfully express and respond to antioxidant compounds in NCI-H460 cells. The data also suggest that the Prx 6/Luc vector system may be effectively applied in screening the response of hPrx 6 to antioxidant compounds in transgenic mice.

The N-Terminal β-Sheet of Peroxiredoxin 4 in the Large Yellow Croaker Pseudosciaena crocea Is Involved in Its Biological Functions

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that exhibit peroxidase and peroxynitrite reductase activities involved in the reduction of reactive oxygen species. The peroxiredoxin Prx4 from the large yellow croaker Pseudosciaena crocea is a typical 2-Cys Prx with an N-terminal signal peptide. We solved the crystal structure of Prx4 at 1.90 Å and revealed an N-terminal antiparallel β-sheet that contributes to the dimer interface. Deletion of this β-sheet decreased the in vitro peroxidase activity to about 50% of the wild-type. In vivo assays further demonstrated that removal of this β-sheet led to some impairment in the ability of Prx4 to negatively regulate nuclear factor-κB (NF-κB) activity and to perform its role in anti-bacterial immunity. These results provide new insights into the structure and function relationship of a peroxiredoxin from bony fish.

Systemic delivery of thiol-specific antioxidant hPRDX6 gene by AAV8 inhibits atherogenesis in LDLR KO mice on HCD

Systemic delivery of thiol-specific antioxidant hPRDX6 gene by AAV8 inhibits atherogenesis in LDLR KO mice on HCD

Expression of salt-induced 2-Cys peroxiredoxin from Oryza sativa increases stress tolerance and fermentation capacity in genetically engineered yeast Saccharomyces cerevisiae

Peroxiredoxins (Prxs), also termed thioredoxin peroxidases (TPXs), are a family of thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative chloroplastic 2-Cys thioredoxin peroxidase (OsTPX) was identified by proteome analysis from leaf tissue samples of rice (Oryza sativa) seedlings exposed to 0.1 M NaCl for 3 days. To investigate the relationship between the OsTPX gene and the stress response, OsTPX was cloned into the yeast expression vector p426GPD under the control of the glyceraldehyde-3-phosphate dehydrogenase (GPD1) promoter, and the construct was transformed into Saccharomyces cerevisiae cells. OsTPX expression was confirmed by semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses. OsTPX contained two highly conserved cysteine residues (Cys114 and Cys236) and an active site region (FTFVCPT), and it is structurally very similar to human 2-Cys Prx. Heterologous OsTPX expression increased the ability of the transgenic yeast cells to adapt and recover from reactive oxygen species (ROS)-induced oxidative stresses, such as a reduction of cellular hydroperoxide levels in the presence of hydrogen peroxide and menadione, by improving redox homeostasis. OsTPX expression also conferred enhanced tolerance to tert-butylhydroperoxide, heat shock, and high ethanol concentrations. Furthermore, high OsTPX expression improved the fermentation capacity of the yeast during glucose-based batch fermentation at a high temperature (40 °C) and at the general cultivation temperature (30 °C). The alcohol yield in OsTPX-expressing transgenic yeast increased by approximately 29 % (0.14 g g(-1)) and 21 % (0.12 g g(-1)) during fermentation at 40 and 30 °C, respectively, compared to the wild-type yeast. Accordingly, OsTPX-expressing transgenic yeast showed prolonged cell survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsTPX expression increases acquired tolerance to ROS-induced oxidative stress by improving cellular redox homeostasis and improves fermentation capacity due to improved cell survival during fermentation, especially at a high temperature.

Enhancement of immune response induced by DNA vaccine cocktail expressing complete LACK and TSA genes against Leishmania major

Leishmaniasis is an important disease in humans. Leishmania homologue of receptor for Activated C Kinase (LACK) and thiol specific antioxidant (TSA) as immuno-dominant antigens of Leishmania major are considered the most promising molecules for a DNA vaccine. We constructed a DNA cocktail, containing plasmids encoding LACK and TSA genes of Leishmania major and evaluated the immune response and survival rate in BALB/c mice. IgG and Interferon gamma values were noticeably increased in the immunized group with DNA cocktail vaccine, which were significantly higher than those in the single-gene vaccinated and control groups (p < 0.05) following the immunization and after challenging with Leishmania major. Interleukin 4 values were decreased in all immunized groups, but only in DNA vaccine cocktail and single-gene vaccination with pc-LACK there were statistical differences with control groups (p > 0.05). The immunized mice with the cocktail DNA vaccine presented a considerable reduction in diameter of lesion compared to other groups and a significant difference was observed (p < 0.05) in this regard. The survival time of the immunized mice with the cocktail DNA vaccine was significantly higher than that in the other groups (p < 0.05) after their being challenged with Leishmania major. The findings of this study indicated that the cocktail DNA vaccine increased the cellular response and survival rate and induced protection against infection with Leishmania in the mice.

Disulfide Biochemistry in 2-Cys Peroxiredoxin: Requirement of Glu50 and Arg146 for the Reduction of Yeast Tsa1 by Thioredoxin

2-Cys peroxiredoxin (Prx) enzymes are ubiquitously distributed peroxidases that make use of a peroxidatic cysteine (Cys(P)) to decompose hydroperoxides. A disulfide bond is generated as a consequence of the partial unfolding of the α-helix that contains Cys(P). Therefore, during its catalytic cycle, 2-Cys Prx alternates between two states, locally unfolded and fully folded. Tsa1 (thiol-specific antioxidant protein 1 from yeast) is by far the most abundant Cys-based peroxidase in Saccharomyces cerevisiae. In this work, we present the crystallographic structure at 2.8Å resolution of Tsa1(C47S) in the decameric form [(α(2))(5)] with a DTT molecule bound to the active site, representing one of the few available reports of a 2-Cys Prx (AhpC-Prx1 subfamily) (AhpC, alkyl hydroperoxide reductase subunit C) structure that incorporates a ligand. The analysis of the Tsa1(C47S) structure indicated that Glu50 and Arg146 participate in the stabilization of the Cys(P) α-helix. As a consequence, we raised the hypothesis that Glu50 and Arg146 might be relevant to the Cys(P) reactivity. Therefore, Tsa1(E50A) and Tsa1(R146Q) mutants were generated and were still able to decompose hydrogen peroxide, presenting a second-order rate constant in the range of 10(6)M(-1)s(-1). Remarkably, although Tsa1(E50A) and Tsa1(R146Q) were efficiently reduced by the low-molecular-weight reductant DTT, these mutants displayed only marginal thioredoxin (Trx)-dependent peroxidase activity, indicating that Glu50 and Arg146 are important for the Tsa1-Trx interaction. These results may impact the comprehension of downstream events of signaling pathways that are triggered by the oxidation of critical Cys residues, such as Trx.