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Sample records for 2-cys peroxiredoxins prxs

  1. Microbial 2-Cys Peroxiredoxins: Insights into Their Complex Physiological Roles

    PubMed Central

    Toledano, Michel B.; Huang, Bo

    2016-01-01

    The peroxiredoxins (Prxs) constitute a very large and highly conserved family of thiol-based peroxidases that has been discovered only very recently. We consider here these enzymes through the angle of their discovery, and of some features of their molecular and physiological functions, focusing on complex phenotypes of the gene mutations of the 2-Cys Prxs subtype in yeast. As scavengers of the low levels of H2O2 and as H2O2 receptors and transducers, 2-Cys Prxs have been highly instrumental to understand the biological impact of H2O2, and in particular its signaling function. 2-Cys Prxs can also become potent chaperone holdases, and unveiling the in vivo relevance of this function, which is still not established, should further increase our knowledge of the biological impact and toxicity of H2O2. The diverse molecular functions of 2-Cys Prx explain the often-hard task of relating them to peroxiredoxin genes phenotypes, which underscores the pleiotropic physiological role of these enzymes and complex biologic impact of H2O2. PMID:26813659

  2. Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms

    PubMed Central

    Tairum, Carlos A.; Santos, Melina Cardoso; Breyer, Carlos A.; Geyer, R. Ryan; Nieves, Cecilia J.; Portillo-Ledesma, Stephanie; Ferrer-Sueta, Gerardo; Toledo, José Carlos; Toyama, Marcos H.; Augusto, Ohara; Netto, Luis E. S.; de Oliveira, Marcos A.

    2016-01-01

    Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states. PMID:27629822

  3. Overoxidation of chloroplast 2-Cys peroxiredoxins: balancing toxic and signaling activities of hydrogen peroxide.

    PubMed

    Puerto-Galán, Leonor; Pérez-Ruiz, Juan M; Ferrández, Julia; Cano, Beatriz; Naranjo, Belén; Nájera, Victoria A; González, Maricruz; Lindahl, Anna M; Cejudo, Francisco J

    2013-01-01

    Photosynthesis, the primary source of biomass and oxygen into the biosphere, involves the transport of electrons in the presence of oxygen and, therefore, chloroplasts constitute an important source of reactive oxygen species, including hydrogen peroxide. If accumulated at high level, hydrogen peroxide may exert a toxic effect; however, it is as well an important second messenger. In order to balance the toxic and signaling activities of hydrogen peroxide its level has to be tightly controlled. To this end, chloroplasts are equipped with different antioxidant systems such as 2-Cys peroxiredoxins (2-Cys Prxs), thiol-based peroxidases able to reduce hydrogen and organic peroxides. At high peroxide concentrations the peroxidase function of 2-Cys Prxs may become inactivated through a process of overoxidation. This inactivation has been proposed to explain the signaling function of hydrogen peroxide in eukaryotes, whereas in prokaryotes, the 2-Cys Prxs of which were considered to be insensitive to overoxidation, the signaling activity of hydrogen peroxide is less relevant. Here we discuss the current knowledge about the mechanisms controlling 2-Cys Prx overoxidation in chloroplasts, organelles with an important signaling function in plants. Given the prokaryotic origin of chloroplasts, we discuss the occurrence of 2-Cys Prx overoxidation in cyanobacteria with the aim of identifying similarities between chloroplasts and their ancestors regarding their response to hydrogen peroxide.

  4. The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts.

    PubMed

    Puerto-Galán, Leonor; Pérez-Ruiz, Juan M; Guinea, Manuel; Cejudo, Francisco Javier

    2015-05-01

    Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock.

  5. Molecular recognition in the interaction of chloroplast 2-Cys peroxiredoxin with NADPH-thioredoxin reductase C (NTRC) and thioredoxin x.

    PubMed

    Bernal-Bayard, Pilar; Ojeda, Valle; Hervás, Manuel; Cejudo, Francisco J; Navarro, José A; Velázquez-Campoy, Adrián; Pérez-Ruiz, Juan M

    2014-11-28

    In addition to the standard NADPH thioredoxin reductases (NTRs), plants hold a plastidic NTR (NTRC), with a thioredoxin module fused at the C-terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs). The interaction of NTRC and chloroplastic thioredoxin x with 2-Cys Prxs has been confirmed in vivo, by bimolecular fluorescence complementation (BiFC) assays, and in vitro, by isothermal titration calorimetry (ITC) experiments. In comparison with thioredoxin x, NTRC interacts with 2-Cys Prx with higher affinity, both the thioredoxin and NTR domains of NTRC contributing significantly to this interaction, as demonstrated by using the NTR and thioredoxin modules of the enzyme expressed separately. The presence of the thioredoxin domain seems to prevent the interaction of NTRC with thioredoxin x.

  6. Characterization of one typical 2-Cys peroxiredoxin gene of Taenia solium and Taenia crassiceps.

    PubMed

    Vaca-Paniagua, Felipe; Parra-Unda, Ricardo; Landa, Abraham

    2009-09-01

    The Taenia genus is capable of living for long periods within its hosts. Reports have shown that this successful establishment is related to its efficient defense mechanisms against host immune response and its high tolerance to oxidative stress. In this work, we describe the genomic sequences of one Taenia solium and Taenia crassiceps typical 2-Cys peroxiredoxins (Ts2-CysPrx, Tc2-CysPrx) genes, which are 94% identical in primary sequence with the typical 2-Cys Prxs catalytic motifs. Both genes have the same genomic architecture, showing a TATA box and Initiator (Inr) sequence in their proximal promoter, two exons split by a 67-bp type III intron and one unique transcription start site located inside the Inr. We show that T. crassiceps cysticerci are highly tolerant to H(2)O(2) presenting a lethal concentration 50 of 3.0 mM and demonstrate that the typical Tc2-CysPrx gene is not induced by H(2)O(2), showing a behavior of an antioxidant housekeeping gene. This study describes for first time the gene structure of a typical 2-Cys Prx in the Taenia genus.

  7. Evolution and function of the Mycoplasma hyopneumoniae peroxiredoxin, a 2-Cys-like enzyme with a single Cys residue.

    PubMed

    Gonchoroski, Taylor; Virginio, Veridiana G; Thompson, Claudia E; Paes, Jéssica A; Machado, Cláudio X; Ferreira, Henrique B

    2017-04-01

    The minimal genome of the mollicute Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia, encodes a limited repertoire of antioxidant enzymes that include a single and atypical peroxiredoxin (MhPrx), whose evolution and function were studied here. MhPrx has only one catalytic cysteine, in contrast with some of its possible ancestors (2-Cys peroxiredoxins), which have two. Although it is more similar to 2-Cys orthologs, MhPrx can still function with a single peroxidatic cysteine (CysP), using non-thiolic electron donors to reduce it. Therefore, MhPrx could be a representative of a possible group of 2-Cys peroxiredoxins, which have lost the resolving cysteine (CysR) residue without losing their catalytic properties. To further investigate MhPrx evolution, we performed a comprehensive phylogenetic analysis in the context of several bacterial families, including Prxs belonging to Tpx and AhpE families, shedding light on the evolutionary history of Mycoplasmataceae Prxs and giving support to the hypothesis of a relatively recent loss of the CysR within this family. Moreover, mutational analyses provided insights into MhPrx function with one, two, or without catalytic cysteines. While removal of the MhPrx putative CysP caused complete activity loss, confirming its catalytic role, the introduction of a second cysteine in a site correspondent to that of the CysR of a 2-Cys orthologue, as in the MhPrx supposed ancestral form, was compatible with enzyme activity. Overall, our phylogenetic and mutational studies support that MhPrx recently diverged from a 2-Cys Prx ancestor and pave the way for future studies addressing structural, functional, and evolutive aspects of peroxiredoxin subfamilies in Mollicutes and other bacteria.

  8. Expression, purification and characterization of an atypical 2-Cys peroxiredoxin from the silkworm, Bombyx mori.

    PubMed

    Zhang, L; Lu, Z

    2015-04-01

    Peroxiredoxins (Prxs) play important roles in protecting organisms against damage caused by reactive oxygen species (ROS). In this study, we cloned a cDNA of Bombyx mori peroxiredoxin 5 (BmPrx5), which contained a 565-bp open reading frame for a 188-residue protein. Sequence analysis indicated that BmPrx5 belongs to the atypical 2-Cys peroxiredoxin family. Recombinant BmPrx5 purified from Escherichia coli showed antioxidant activity that removes H2 O2 and protects DNA from oxidative damage. Quantitative real-time PCR showed that the level of BmPrx5 mRNA in haemocytes increased early and decreased by 24 h after injection of H2 O2 whereas, in the fat body, the transcript level decreased at 6 h and increased at 12 h. Pseudomonas aeruginosa and Staphylococcus aureus infection resulted in higher levels of H2 O2 in the haemolymph and of BmPrx5 mRNA in haemocytes at 8 h postinfection. These data suggest that BmPrx5 acts as an antioxidant enzyme to protect the silkworm from oxidative damage induced by bacterial infection. Further study is needed to elucidate the exact role of BmPrx5 in the silkworm immune system.

  9. The C-type Arabidopsis thioredoxin reductase ANTR-C acts as an electron donor to 2-Cys peroxiredoxins in chloroplasts

    SciTech Connect

    Moon, Jeong Chan; Jang, Ho Hee; Chae, Ho Byoung; Lee, Jung Ro; Lee, Sun Yong; Jung, Young Jun; Shin, Mi Rim; Lim, Hye Song |; Chung, Woo Sik |; Yun, Dae-Jin |; Lee, Kyun Oh; Lee, Sang Yeol . E-mail: sylee@gsnu.ac.kr

    2006-09-22

    2-Cys peroxiredoxins (Prxs) play important roles in the antioxidative defense systems of plant chloroplasts. In order to determine the interaction partner for these proteins in Arabidopsis, we used a yeast two-hybrid screening procedure with a C175S-mutant of Arabidopsis 2-Cys Prx-A as bait. A cDNA encoding an NADPH-dependent thioredoxin reductase (NTR) isotype C was identified and designated ANTR-C. We demonstrated that this protein effected efficient transfer of electrons from NADPH to the 2-Cys Prxs of chloroplasts. Interaction between 2-Cys Prx-A and ANTR-C was confirmed by a pull-down experiment. ANTR-C contained N-terminal TR and C-terminal Trx domains. It exhibited both TR and Trx activities and co-localized with 2-Cys Prx-A in chloroplasts. These results suggest that ANTR-C functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TR/Trx system in chloroplasts.

  10. Engineering of 2-Cys Peroxiredoxin for Enhanced Stress-Tolerance

    PubMed Central

    An, Byung Chull; Lee, Seung Sik; Lee, Jae Taek; Hong, Sung Hyun; Wi, Seung Gon; Chung, Byung Yeoup

    2011-01-01

    A typical 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) that alternatively acts as a peroxidase or a molecular chaperone in Pseudomonas putida KT2440 was previously characterized. The dual functions of PpPrx are regulated by the existence of an additional Cys112 between the active Cys51 and Cys171 residues. In the present study, additional Cys residues (Cys31, Cys112, and Cys192) were added to PpPrx variants to improve their enzymatic function. The optimal position of the additional Cys residues for the dual functionality was assessed. The peroxidase activities of the S31C and Y192C mutants were increased 3- to 4-fold compared to the wild-type, while the chaperone activity was maintained at > 66% of PpPrx. To investigate whether optimization of the dual functions could enhance stress-tolerance in vivo, a complementation study was performed. The S31C and Y192C mutants showed a much greater tolerance than other variants under a complex condition of heat and oxidative stresses. The optimized dual functions of PpPrx could be adapted for use in bioengineering systems and industries, such as to develop organisms that are more resistant to extreme environments. PMID:21773675

  11. A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection

    PubMed Central

    Peterson, Tina M.L.; Luckhart, Shirley

    2008-01-01

    Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster (S2) and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito. PMID:16540402

  12. A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection.

    PubMed

    Peterson, Tina M L; Luckhart, Shirley

    2006-03-15

    Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster S2 and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito.

  13. Multilevel regulation of 2-Cys peroxiredoxin reaction cycle by S-nitrosylation.

    PubMed

    Engelman, Rotem; Weisman-Shomer, Pnina; Ziv, Tamar; Xu, Jianqiang; Arnér, Elias S J; Benhar, Moran

    2013-04-19

    S-nitrosothiols (SNOs), formed by nitric oxide (NO)-mediated S-nitrosylation, and hydrogen peroxide (H2O2), a prominent reactive oxygen species, are implicated in diverse physiological and pathological processes. Recent research has shown that the cellular action and metabolism of SNOs and H2O2 involve overlapping, thiol-based mechanisms, but how these reactive species may affect each other's fate and function is not well understood. In this study we investigated how NO/SNO may affect the redox cycle of mammalian peroxiredoxin-1 (Prx1), a representative of the 2-Cys Prxs, a group of thioredoxin (Trx)-dependent peroxidases. We found that, both in a cell-free system and in cells, NO/SNO donors such as S-nitrosocysteine and S-nitrosoglutathione readily induced the S-nitrosylation of Prx1, causing structural and functional alterations. In particular, nitrosylation promoted disulfide formation involving the pair of catalytic cysteines (Cys-52 and Cys-173) and disrupted the oligomeric structure of Prx1, leading to loss of peroxidase activity. A highly potent inhibition of the peroxidase catalytic reaction by NO/SNO was seen in assays employing the coupled Prx-Trx system. In this setting, S-nitrosocysteine (10 μM) effectively blocked the Trx-mediated regeneration of oxidized Prx1. This effect appeared to be due to both competition between S-nitrosocysteine and Prx1 for the Trx system and direct modulation by S-nitrosocysteine of Trx reductase activity. Our findings that NO/SNO target both Prx and Trx reductase may have implications for understanding the impact of nitrosylation on cellular redox homeostasis.

  14. Physiological relevance of plant 2-Cys peroxiredoxin overoxidation level and oligomerization status.

    PubMed

    Cerveau, Delphine; Ouahrani, Djelloul; Marok, Mohamed Amine; Blanchard, Laurence; Rey, Pascal

    2016-01-01

    Peroxiredoxins are ubiquitous thioredoxin-dependent peroxidases presumed to display, upon environmental constraints, a chaperone function resulting from a redox-dependent conformational switch. In this work, using biochemical and genetic approaches, we aimed to unravel the factors regulating the redox status and the conformation of the plastidial 2-Cys peroxiredoxin (2-Cys PRX) in plants. In Arabidopsis, we show that in optimal growth conditions, the overoxidation level mainly depends on the availability of thioredoxin-related electron donors, but not on sulfiredoxin, the enzyme reducing the 2-Cys PRX overoxidized form. We also observed that upon various physiological temperature, osmotic and light stress conditions, the overoxidation level and oligomerization status of 2-Cys PRX can moderately vary depending on the constraint type. Further, no major change was noticed regarding protein conformation in water-stressed Arabidopsis, barley and potato plants, whereas species-dependent up- and down-variations in overoxidation were observed. In contrast, both 2-Cys PRX overoxidation and oligomerization were strongly induced during a severe oxidative stress generated by methyl viologen. From these data, revealing that the oligomerization status of plant 2-Cys PRX does not exhibit important variation and is not tightly linked to the protein redox status upon physiologically relevant environmental constraints, the possible in planta functions of 2-Cys PRX are discussed.

  15. Characterization of plant sulfiredoxin and role of sulphinic form of 2-Cys peroxiredoxin

    PubMed Central

    Iglesias-Baena, Iván; Barranco-Medina, Sergio; Lázaro-Payo, Alfonso; López-Jaramillo, Francisco Javier; Sevilla, Francisca; Lázaro, Juan-José

    2010-01-01

    The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO2H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO2H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function. PMID:20176891

  16. How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily*

    PubMed Central

    Morais, Mariana A. B.; Giuseppe, Priscila O.; Souza, Tatiana A. C. B.; Alegria, Thiago G. P.; Oliveira, Marcos A.; Netto, Luis E. S.; Murakami, Mario T.

    2015-01-01

    2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His113) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp76 from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress. PMID:25666622

  17. Biochemical characterization of Toxoplasma gondii 1-Cys peroxiredoxin 2 with mechanistic similarities to typical 2-Cys Prx.

    PubMed

    Deponte, Marcel; Becker, Katja

    2005-03-01

    TgPrx2 represents a recently discovered cytosolic 1-Cys peroxiredoxin (Prx) from the intracellular parasite Toxoplasma gondii. Over-expression of the respective gene confers protection against H(2)O(2), suggesting that the protein possesses peroxidase activity. According to the current nomenclature eukaryotic typical and atypical 2-Cys Prx contain a second conserved resolving cysteine residue whereas 1-Cys Prx work on the basis of a monothiol mechanism. Only a few 1-Cys peroxiredoxins have been biochemically characterized to date. Here we describe the mechanistic characterization of TgPrx2 in vitro, including site directed mutagenesis studies, gel filtration chromatography, and molecular modeling. TgPrx2 has general antioxidant properties as indicated by its ability to protect glutamine synthetase against a dithiothreitol Fe(3+)-catalyzed oxidation system. However, TgPrx2 does not reduce H(2)O(2) nor tert-butyl hydroperoxide at the expense of glutaredoxin, thioredoxin or glutathione. Cys(47) was identified as the active site cysteine residue. Most interestingly, Cys(47) was found to form an intermolecular disulfide with Cys(209) from the C-terminal domain of a second subunit which acts as the resolving cysteine. This is a mechanism analogous to typical peroxiredoxins. In contrast to the latter, however, dimeric TgPrx2 does not oligomerize to decamers but is able to form tetramers and hexamers which are non-covalently associated. To our knowledge, TgPrx2 is the first eukaryotic 'so called' 1-Cys peroxiredoxin shown to act on the basis of a 2-Cys mechanism. Our data indicate that mechanistic studies are essential for classifying peroxiredoxins.

  18. Calcium and Magnesium Ions Modulate the Oligomeric State and Function of Mitochondrial 2-Cys Peroxiredoxins in Leishmania Parasites.

    PubMed

    Morais, Mariana A B; Giuseppe, Priscila O; Souza, Tatiana A C B; Castro, Helena; Honorato, Rodrigo V; Oliveira, Paulo S L; Netto, Luis E S; Tomas, Ana M; Murakami, Mario T

    2017-03-14

    Leishmania parasites have evolved a number of strategies to cope with the harsh environmental changes during mammalian infection. One of these mechanisms involves the functional gain that allowed mitochondrial 2-Cys peroxiredoxins to act as molecular chaperones when forming decamers. This function was demonstrated to be critical for the parasite infectivity in mammals and its activation was considered to be controlled exclusively by the enzyme redox state under physiological conditions. Herein, we revealed that magnesium and calcium ions play a major role in modulating the ability of these enzymes to act as molecular chaperones, surpassing the redox effect. These ions are directly involved in the mitochondrial metabolism and now also integrate a novel mechanism to stabilize the decameric form of 2-Cys peroxiredoxins in Leishmania mitochondrion. Moreover, we demonstrated that a constitutively dimeric Prx1m mutant impairs Leishmania's survival under heat stress, supporting the central role of chaperone function of Prx1m for Leishmania parasites during the transition from insect to mammalian hosts.

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

    PubMed

    Kim, Il-Sup; Kim, Young-Saeng; Yoon, Ho-Sung

    2013-04-01

    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

  20. Methyl jasmonate promotes the transient reduction of the levels of 2-Cys peroxiredoxin in Ricinus communis plants.

    PubMed

    dos Santos Soares, Alexandra Martins; de Souza, Thiago Freitas; de Souza Domingues, Sarah Jane; Jacinto, Tânia; Tavares Machado, Olga Lima

    2004-06-01

    Jasmonates are signaling molecules that play a key role in the regulation of metabolic processes, reproduction and defense against insects and pathogens. This study investigated the effects of methyl jasmonate on the protein pattern of Ricinus communis plants and the activity of guaiacol peroxidase, an antioxidant enzyme. Methyl jasmonate treatment caused a transient reduction in guaiacol peroxidase activity. A similar response was observed for the levels of 2-Cys peroxiredoxin protein. Moreover, the levels of the small and large chains of Rubisco were also reduced. The transient reduction of the levels and activity of antioxidant enzymes could account for the increase in the levels of H2O2, an important signaling molecule in plant defense.

  1. Interactions between 2-Cys peroxiredoxins and ascorbate in autophagosome formation during the heat stress response in Solanum lycopersicum.

    PubMed

    Cheng, Fei; Yin, Ling-Ling; Zhou, Jie; Xia, Xiao-Jian; Shi, Kai; Yu, Jing-Quan; Zhou, Yan-Hong; Foyer, Christine Helen

    2016-03-01

    2-Cys peroxiredoxins (2-CPs) function in the removal of hydrogen peroxide and lipid peroxides but their precise roles in the induction of autophagy have not been characterized. Here we show that heat stress, which is known to induce oxidative stress, leads to the simultaneous accumulation of transcripts encoding 2-CPs and autophagy proteins, as well as autophagosomes, in tomato (Solanum lycopersicum) plants. Virus-induced gene silencing of the tomato peroxiredoxin genes 2-CP1, 2-CP2, and 2-CP1/2 resulted in an increased sensitivity of tomato plants to heat stress. Silencing 2-CP2 or 2-CP1/2 increased the levels of transcripts associated with ascorbate biosynthesis but had no effect on the glutathione pool in the absence of stress. However, the heat-induced accumulation of transcripts associated with the water-water cycle was compromised by the loss of 2-CP1/2 functions. The transcript levels of autophagy-related genes ATG5 and ATG7 were higher in plants with impaired 2-CP1/2 functions, and the formation of autophagosomes increased, together with an accumulation of oxidized and insoluble proteins. Silencing of ATG5 or ATG7 increased the levels of 2-CP transcripts and protein but decreased heat stress tolerance. These results demonstrate that 2-CPs fulfil a pivotal role in heat stress tolerance in tomato, via interactions with ascorbate-dependent pathways and autophagy.

  2. Hydrogen peroxide-mediated inactivation of two chloroplastic peroxidases, ascorbate peroxidase and 2-cys peroxiredoxin.

    PubMed

    Kitajima, Sakihito

    2008-01-01

    Reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide, are generated by the photosystems because photoexcited electrons are often generated in excess of requirements for CO2 fixation and used for reducing molecular oxygen, even under normal environmental conditions. Moreover, ROS generation is increased in chloroplasts if plants are subjected to stresses, such as drought, high salinity and chilling. Chloroplast-localized isoforms of ascorbate peroxidase and possibly peroxiredoxins assume the principal role of scavenging hydrogen peroxide. However, in vitro studies revealed that both types of peroxidases are easily damaged by hydrogen peroxide and lose their catalytic activities. This is one contributing factor for cellular damage that occurs under severe oxidative stress. In this review, I describe mechanisms of hydrogen peroxide-mediated inactivation of these two enzymes and discuss a reason why they became susceptible to damage by hydrogen peroxide.

  3. A 2-Cys peroxiredoxin in response to oxidative stress in the pine wood nematode, Bursaphelenchus xylophilus.

    PubMed

    Li, Zhen; Zhang, Qingwen; Zhou, Xuguo

    2016-06-07

    The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease that has devastated pine forests in Asia. Parasitic nematodes are known to have evolved antioxidant stress responses that defend against host plant defenses. In this study, the infestation of whitebark pine, Pinus bungean, with B. xylophilus led to a significant increase in plant hydrogen peroxide (H2O2) and salicylic acid levels. Correspondingly, the expression of an antioxidative enzyme, 2-Cysteine peroxiredoxin (BxPrx), was elevated in B. xylophilus following the H2O2 treatments. Recombinant BxPrx, a thermal stabile and pH tolerant enzyme, exhibited high level of antioxidant activity against H2O2, suggesting that it is capable of protecting cells from free radical attacks. Immunohistochemical localization study showed that BxPrx was broadly expressed across different tissues and could be secreted outside the nematode. Finally, the number of BxPrx homologs in both dauer-like and fungi-feeding B. xylophilus were comparable based on bioinformatics analysis of existing EST libraries, indicating a potential role of BxPrx in both propagative and dispersal nematodes. These combined results suggest that BxPrx is a key genetic factor facilitating the infestation and distribution of B. xylophilus within pine hosts, and consequently the spread of pine wilt disease.

  4. A 2-Cys peroxiredoxin in response to oxidative stress in the pine wood nematode, Bursaphelenchus xylophilus

    PubMed Central

    Li, Zhen; Zhang, Qingwen; Zhou, Xuguo

    2016-01-01

    The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease that has devastated pine forests in Asia. Parasitic nematodes are known to have evolved antioxidant stress responses that defend against host plant defenses. In this study, the infestation of whitebark pine, Pinus bungean, with B. xylophilus led to a significant increase in plant hydrogen peroxide (H2O2) and salicylic acid levels. Correspondingly, the expression of an antioxidative enzyme, 2-Cysteine peroxiredoxin (BxPrx), was elevated in B. xylophilus following the H2O2 treatments. Recombinant BxPrx, a thermal stabile and pH tolerant enzyme, exhibited high level of antioxidant activity against H2O2, suggesting that it is capable of protecting cells from free radical attacks. Immunohistochemical localization study showed that BxPrx was broadly expressed across different tissues and could be secreted outside the nematode. Finally, the number of BxPrx homologs in both dauer-like and fungi-feeding B. xylophilus were comparable based on bioinformatics analysis of existing EST libraries, indicating a potential role of BxPrx in both propagative and dispersal nematodes. These combined results suggest that BxPrx is a key genetic factor facilitating the infestation and distribution of B. xylophilus within pine hosts, and consequently the spread of pine wilt disease. PMID:27271000

  5. Functional complementation in yeast reveals a protective role of chloroplast 2-Cys peroxiredoxin against reactive nitrogen species.

    PubMed

    Sakamoto, Atsushi; Tsukamoto, Shigefumi; Yamamoto, Hiroshi; Ueda-Hashimoto, Manami; Takahashi, Misa; Suzuki, Hitomi; Morikawa, Hiromichi

    2003-03-01

    The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism.

  6. 2-Cys peroxiredoxin responds to low temperature and other cues in Caragana jubata, a plant species of cold desert of Himalaya.

    PubMed

    Bhardwaj, Pardeep Kumar; Mala, Deep; Kumar, Sanjay

    2014-05-01

    A 2-Cys peroxiredoxin cDNA (CjPrx) was isolated and characterized from Caragana jubata, a temperate/alpine plant species of high altitude cold desert of Himalaya and Eurasia. The cDNA obtained was 1,064 bp long consisting of an open reading frame of 789 bp encoding 262 amino acids. The calculated molecular mass of the mature protein was 28.88 kDa and pI was 5.84. Deduced amino acid sequence of CjPrx shared a high degree homology with 2-CysPrx proteins from other plants. CjPrx had both the PRX_type 2-Cys domain and thioredoxin-like superfamily domains. CjPrx contained 26.72% α-helices, 6.87% β-turns, 20.61% extended strands and 45.80% random coils, and was a hydrophilic protein. Expression of CjPrx was modulated by low temperature, methyl jasmonate (MJ), salicylic acid and drought stress, but no significant change was observed in response to abscisic acid treatment. Among all the treatments, a strong up-regulation of CjPrx was observed in response to MJ treatment.

  7. Cloning and Characterization of a 2-Cys Peroxiredoxin in the Pine Wood Nematode, Bursaphelenchus xylophilus, a Putative Genetic Factor Facilitating the Infestation

    PubMed Central

    Li, Zhen; Liu, Xiaoxia; Chu, Yanna; Wang, Yan; Zhang, Qingwen; Zhou, Xuguo

    2011-01-01

    The pine wood nematode, Bursaphelenchus xylophilus, is an invasive plant parasitic nematode and a worldwide quarantine pest. An indigenous species in North America and the causal agent of pine wilt disease, B. xylophilus has devastated pine production in Southeastern Asia including Japan, China, and Korea since its initial introduction in the early 1900s. The reactive oxygen species (ROS) is the first line of defense utilized by host plants against parasites, while nematodes, counteractively, employ antioxidants to facilitate their infection. Peroxiredoxins (Prxs) are a large class of antioxidants recently found in a wide variety of organisms. In this report, a gene encoding a novel 2-cysteine peroxiredoxin protein in B. xylophilus was cloned and characterized. The 2-cysteine peroxiredoxin in B. xylophilus (herein refers to as “BxPrx”) is highly conserved in comparison to 2-cysteine peroxiredoxins (Prx2s) in other nematodes, which have two conserved cysteine amino acids (Cp and Cr), a threonine-cysteine-arginine catalytic triad, and two signature motifs (GGLG and YF) sensitive to hydrogen peroxide. In silico assembly of BxPrx tertiary structure reveals the spatial configuration of these conserved domains and the simulated BxPrx 3-dimensional structure is congruent with its presumed redox functions. Although no signal peptide was identified, BxPrx was abundantly expressed and secreted under the B. xylophilus cuticle. Upon further analysis of this leader-less peptide, a single transmembrane α-helix composed of 23 consecutive hydrophobic amino acids was found in the primary structure of BxPrx. This transmembrane region and/or readily available ATP binding cassette transporters may facilitate the transport of non-classical BxPrx across the cell membrane. Recombinant BxPrx showed peroxidase activity in vitro reducing hydrogen peroxide using glutathione as the electron donor. The combined results from gene discovery, protein expression and distribution profiling

  8. Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins

    PubMed Central

    COX, Andrew G.; Pearson, Andree G.; Pullar, Juliet M.; Jönsson, Thomas J.; Lowther, W. Todd; Winterbourn, Christine C.; Hampton, Mark B.

    2013-01-01

    The Prxs (peroxiredoxins) are a family of cysteine-dependent peroxidases that decompose hydrogen peroxide. Prxs become hyperoxidized when a sulfenic acid formed during the catalytic cycle reacts with hydrogen peroxide. In the present study, Western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidized at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 μM in the mitochondrial matrix of Jurkat T-lymphoma cells. Although the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs, other structural features must contribute to the innate resilience of Prx 3 to hyperoxidation. PMID:19356151

  9. Glutathionylation of Pea Chloroplast 2-Cys Prx and Mitochondrial Prx IIF Affects Their Structure and Peroxidase Activity and Sulfiredoxin Deglutathionylates Only the 2-Cys Prx.

    PubMed

    Calderón, Aingeru; Lázaro-Payo, Alfonso; Iglesias-Baena, Iván; Camejo, Daymi; Lázaro, Juan J; Sevilla, Francisca; Jiménez, Ana

    2017-01-01

    Together with thioredoxins (Trxs), plant peroxiredoxins (Prxs), and sulfiredoxins (Srxs) are involved in antioxidant defense and redox signaling, while their regulation by post-translational modifications (PTMs) is increasingly regarded as a key component for the transduction of the bioactivity of reactive oxygen and nitrogen species. Among these PTMs, S-glutathionylation is considered a protective mechanism against overoxidation, it also modulates protein activity and allows signaling. This study explores the glutathionylation of recombinant chloroplastic 2-Cys Prx and mitochondrial Prx IIF from Pisum sativum. Glutathionylation of the decameric form of 2-Cys Prx produced a change in the elution volume after FPLC chromatography and converted it to its dimeric glutathionylated form, while Prx IIF in its reduced dimeric form was glutathionylated without changing its oligomeric state. Mass spectrometry demonstrated that oxidized glutathione (GSSG) can glutathionylate resolving cysteine (Cys(174)), but not the peroxidatic equivalent (Cys(52)), in 2-Cys Prx. In contrast, GSSG was able to glutathionylate both peroxidatic (Cys(59)) and resolving (Cys(84)) cysteine in Prx IIF. Glutathionylation was seen to be dependent on the GSH/GSSG ratio, although the exact effect on the 2-Cys Prx and Prx IIF proteins differed. However, the glutathionylation provoked a similar decrease in the peroxidase activity of both peroxiredoxins. Despite growing evidence of the importance of post-translational modifications, little is known about the enzymatic systems that specifically regulate the reversal of this modification. In the present work, sulfiredoxin from P. sativum was seen to be able to deglutathionylate pea 2-Cys Prx but not pea Prx IIF. Redox changes during plant development and the response to stress influence glutathionylation/deglutathionylation processes, which may represent an important event through the modulation of peroxiredoxin and sulfiredoxin proteins.

  10. Glutathionylation of Pea Chloroplast 2-Cys Prx and Mitochondrial Prx IIF Affects Their Structure and Peroxidase Activity and Sulfiredoxin Deglutathionylates Only the 2-Cys Prx

    PubMed Central

    Calderón, Aingeru; Lázaro-Payo, Alfonso; Iglesias-Baena, Iván; Camejo, Daymi; Lázaro, Juan J.; Sevilla, Francisca; Jiménez, Ana

    2017-01-01

    Together with thioredoxins (Trxs), plant peroxiredoxins (Prxs), and sulfiredoxins (Srxs) are involved in antioxidant defense and redox signaling, while their regulation by post-translational modifications (PTMs) is increasingly regarded as a key component for the transduction of the bioactivity of reactive oxygen and nitrogen species. Among these PTMs, S-glutathionylation is considered a protective mechanism against overoxidation, it also modulates protein activity and allows signaling. This study explores the glutathionylation of recombinant chloroplastic 2-Cys Prx and mitochondrial Prx IIF from Pisum sativum. Glutathionylation of the decameric form of 2-Cys Prx produced a change in the elution volume after FPLC chromatography and converted it to its dimeric glutathionylated form, while Prx IIF in its reduced dimeric form was glutathionylated without changing its oligomeric state. Mass spectrometry demonstrated that oxidized glutathione (GSSG) can glutathionylate resolving cysteine (Cys174), but not the peroxidatic equivalent (Cys52), in 2-Cys Prx. In contrast, GSSG was able to glutathionylate both peroxidatic (Cys59) and resolving (Cys84) cysteine in Prx IIF. Glutathionylation was seen to be dependent on the GSH/GSSG ratio, although the exact effect on the 2-Cys Prx and Prx IIF proteins differed. However, the glutathionylation provoked a similar decrease in the peroxidase activity of both peroxiredoxins. Despite growing evidence of the importance of post-translational modifications, little is known about the enzymatic systems that specifically regulate the reversal of this modification. In the present work, sulfiredoxin from P. sativum was seen to be able to deglutathionylate pea 2-Cys Prx but not pea Prx IIF. Redox changes during plant development and the response to stress influence glutathionylation/deglutathionylation processes, which may represent an important event through the modulation of peroxiredoxin and sulfiredoxin proteins. PMID:28197170

  11. The crystal structure of the C45S mutant of annelid Arenicola marina peroxiredoxin 6 supports its assignment to the mechanistically typical 2-Cys subfamily without any formation of toroid-shaped decamers

    PubMed Central

    Smeets, Aude; Loumaye, Eléonore; Clippe, André; Rees, Jean-François; Knoops, Bernard; Declercq, Jean-Paul

    2008-01-01

    The peroxiredoxins (PRDXs) define a superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high-molecular-weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 Å resolution. Although A. marina PRDX6 was cloned during the search of annelid homologs of mammalian 1-Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2-Cys PRDX subfamily. The protein is composed of two distinct domains: a C-terminal domain and an N-terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild-type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid-shaped decamers formed by dimer association and observed in most typical 2-Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2-Cys PRDXs without any formation of toroid-shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide-dependent cell signaling rather than a molecular chaperone. PMID:18359859

  12. 2-Cysteine Peroxiredoxins and Thylakoid Ascorbate Peroxidase Create a Water-Water Cycle That Is Essential to Protect the Photosynthetic Apparatus under High Light Stress Conditions1

    PubMed Central

    Awad, Jasmin; Stotz, Henrik U.; Fekete, Agnes; Krischke, Markus; Engert, Cornelia; Havaux, Michel; Berger, Susanne; Mueller, Martin J.

    2015-01-01

    Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H2O2)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H2O2, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H2O2- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX. PMID:25667319

  13. The fission yeast Schizosaccharomyces pombe as a model to understand how peroxiredoxins influence cell responses to hydrogen peroxide.

    PubMed

    Veal, Elizabeth A; Tomalin, Lewis E; Morgan, Brian A; Day, Alison M

    2014-08-01

    As a more selectively reactive oxygen species, H2O2 (hydrogen peroxide) has been co-opted as a signalling molecule, but high levels can still lead to lethal amounts of cell damage. 2-Cys Prxs (peroxiredoxins) are ubiquitous thioredoxin peroxidases which utilize reversibly oxidized catalytic cysteine residues to reduce peroxides. As such, Prxs potentially make an important contribution to the repertoire of cell defences against oxidative damage. Although the abundance of eukaryotic 2-Cys Prxs suggests an important role in maintaining cell redox, the surprising sensitivity of their thioredoxin peroxidase activity to inactivation by H2O2 has raised questions as to their role as an oxidative stress defence. Indeed, work in model yeast has led the way in revealing that Prxs do much more than simply remove peroxides and have even uncovered circumstances where their thioredoxin peroxidase activity is detrimental. In the present paper, we focus on what we have learned from studies in the fission yeast Schizosaccharomyces pombe about the different roles of 2-Cys Prxs in responses to H2O2 and discuss the general implications of these findings for other systems.

  14. Comparative analysis of cyanobacterial and plant peroxiredoxins and their electron donors: peroxidase activity and susceptibility to overoxidation.

    PubMed

    Lindahl, Marika; Cejudo, Francisco Javier

    2013-01-01

    Peroxiredoxins (Prxs) are peroxidases that use thiol-based catalytic mechanisms implying redox-active cysteines. The different Prx families have homologs in all photosynthetic organisms, including plants, algae, and cyanobacteria. However, recent studies show that the physiological reduction systems that provide Prxs with reducing equivalents to sustain their activities differ considerably between cyanobacterial strains. Thus, for example, the filamentous cyanobacterium Anabaena sp. PCC 7120 is similar to the chloroplast in that it possesses an abundant 2-Cys Prx, which receives electrons from the NADPH-dependent thioredoxin reductase C (NTRC). In contrast, the unicellular cyanobacterium Synechocystis sp. PCC 6803, which lacks NTRC, has little 2-Cys Prx but high amounts of PrxII and 1-Cys Prx. The characterization of cyanobacterial Prxs and their electron donors relies on straightforward enzymatic assays and tools to study the physiological relevance of these systems. Here, we present methods to measure peroxidase activities in vitro and peroxide decomposition in vivo. Several approaches to detect overoxidation of the active site cysteine in cyanobacterial 2-Cys Prxs are also described.

  15. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes

    PubMed Central

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene. PMID:26529408

  16. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    PubMed

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

  17. Expression Analysis of Four Peroxiredoxin Genes from Tamarix hispida in Response to Different Abiotic Stresses and Exogenous Abscisic Acid (ABA)

    PubMed Central

    Gao, Caiqiu; Zhang, Kaimin; Yang, Guiyan; Wang, Yucheng

    2012-01-01

    Peroxiredoxins (Prxs) are a recently discovered family of antioxidant enzymes that catalyze the reduction of peroxides and alkyl peroxides. In this study, four Prx genes (named as ThPrxII, ThPrxIIE, ThPrxIIF, and Th2CysPrx) were cloned from Tamarix hispida. Their expression profiles in response to stimulus of NaCl, NaHCO3, PEG, CdCl2 and abscisic acid (ABA) in roots, stems and leaves of T. hispida were investigated using real-time RT-PCR. The results showed that the four ThPrxs were all expressed in roots, stems and leaves. Furthermore, the transcript levels of ThPrxIIE and ThPrxII were the lowest and the highest, respectively, in all tissue types. All the ThPrx genes were induced by both NaCl and NaHCO3 and reached their highest expression levels at the onset of stress in roots. Under PEG and CdCl2 stress, the expression patterns of these ThPrxs showed temporal and spatial specificity. The expressions of the ThPrxs were all differentially regulated by ABA, indicating that they are all involved in the ABA signaling pathway. These findings reveal a complex regulation of Prxs that is dependent on the type of Prx, tissue, and the signaling molecule. The divergence of the stress-dependent transcriptional regulation of the ThPrx gene family in T. hispida may provide an essential basis for the elucidation of Prx function in future work. PMID:22489180

  18. Expression analysis of four peroxiredoxin genes from Tamarix hispida in response to different abiotic stresses and Exogenous Abscisic Acid (ABA).

    PubMed

    Gao, Caiqiu; Zhang, Kaimin; Yang, Guiyan; Wang, Yucheng

    2012-01-01

    Peroxiredoxins (Prxs) are a recently discovered family of antioxidant enzymes that catalyze the reduction of peroxides and alkyl peroxides. In this study, four Prx genes (named as ThPrxII, ThPrxIIE, ThPrxIIF, and Th2CysPrx) were cloned from Tamarix hispida. Their expression profiles in response to stimulus of NaCl, NaHCO(3), PEG, CdCl(2) and abscisic acid (ABA) in roots, stems and leaves of T. hispida were investigated using real-time RT-PCR. The results showed that the four ThPrxs were all expressed in roots, stems and leaves. Furthermore, the transcript levels of ThPrxIIE and ThPrxII were the lowest and the highest, respectively, in all tissue types. All the ThPrx genes were induced by both NaCl and NaHCO(3) and reached their highest expression levels at the onset of stress in roots. Under PEG and CdCl(2) stress, the expression patterns of these ThPrxs showed temporal and spatial specificity. The expressions of the ThPrxs were all differentially regulated by ABA, indicating that they are all involved in the ABA signaling pathway. These findings reveal a complex regulation of Prxs that is dependent on the type of Prx, tissue, and the signaling molecule. The divergence of the stress-dependent transcriptional regulation of the ThPrx gene family in T. hispida may provide an essential basis for the elucidation of Prx function in future work.

  19. Peroxiredoxins in Parasites

    PubMed Central

    Gretes, Michael C.; Poole, Leslie B.

    2012-01-01

    Abstract Significance: Parasite survival and virulence relies on effective defenses against reactive oxygen and nitrogen species produced by the host immune system. Peroxiredoxins (Prxs) are ubiquitous enzymes now thought to be central to such defenses and, as such, have potential value as drug targets and vaccine antigens. Recent Advances: Plasmodial and kinetoplastid Prx systems are the most extensively studied, yet remain inadequately understood. For many other parasites our knowledge is even less well developed. Through parasite genome sequencing efforts, however, the key players are being discovered and characterized. Here we describe what is known about the biochemistry, regulation, and cell biology of Prxs in parasitic protozoa, helminths, and fungi. At least one Prx is found in each parasite with a sequenced genome, and a notable theme is the common patterns of expression, localization, and functionality among sequence-similar Prxs in related species. Critical Issues: The nomenclature of Prxs from parasites is in a state of disarray, causing confusion and making comparative inferences difficult. Here we introduce a systematic Prx naming convention that is consistent between organisms and informative about structural and evolutionary relationships. Future Directions: The new nomenclature should stimulate the crossfertilization of ideas among parasitologists and with the broader redox research community. The diverse parasite developmental stages and host environments present complex systems in which to explore the variety of roles played by Prxs, with a view toward parlaying what is learned into novel therapies and vaccines that are urgently needed. Antioxid. Redox Signal. 17, 608–633. PMID:22098136

  20. Cryo-electron microscopy structure of human peroxiredoxin-3 filament reveals the assembly of a putative chaperone.

    PubMed

    Radjainia, Mazdak; Venugopal, Hariprasad; Desfosses, Ambroise; Phillips, Amy J; Yewdall, N Amy; Hampton, Mark B; Gerrard, Juliet A; Mitra, Alok K

    2015-05-05

    Peroxiredoxins (Prxs) are a ubiquitous class of thiol-dependent peroxidases that play an important role in the protection and response of cells to oxidative stress. The catalytic unit of typical 2-Cys Prxs are homodimers, which can self-associate to form complex assemblies that are hypothesized to have signaling and chaperone activity. Mitochondrial Prx3 forms dodecameric toroids, which can further stack to form filaments, the so-called high-molecular-weight (HMW) form that has putative holdase activity. We used single-particle analysis and helical processing of electron cryomicroscopy images of human Prx3 filaments induced by low pH to generate a ∼7-Å resolution 3D structure of the HMW form, the first such structure for a 2-Cys Prx. The pseudo-atomic model reveals interactions that promote the stacking of the toroids and shows that unlike previously reported data, the structure can accommodate a partially folded C terminus. The HMW filament lumen displays hydrophobic patches, which we hypothesize bestow holdase activity.

  1. Fish Peroxiredoxins and Their Role in Immunity

    PubMed Central

    Valero, Yulema; Martínez-Morcillo, Francisco J.; Esteban, M. Ángeles; Chaves-Pozo, Elena; Cuesta, Alberto

    2015-01-01

    Peroxiredoxins (Prxs) are a family of antioxidant enzymes that protect cells from oxidative damage. In addition, Prxs may act as modulators of inflammation, protect against cell death and tumour progression, and facilitate tissue repair after damage. The most studied roles of Prx1 and Prx2 are immunological. Here we present a review on the effects of some immunostimulant treatments and bacterial, viral, or parasitic infections on the expression of fish Prxs at the gene and/or protein level, and point to their important role in immunity. The Prxs show antioxidant activity as well as a protective effect against infection. Some preliminary data are presented about the role of fish Prx1 and Prx2 in virus resistance although further studies are needed before the role of fish Prx in immunity can be definitively defined. PMID:26633533

  2. Functional characterization of peroxiredoxins from the human protozoan parasite Giardia intestinalis.

    PubMed

    Mastronicola, Daniela; Falabella, Micol; Testa, Fabrizio; Pucillo, Leopoldo Paolo; Teixeira, Miguel; Sarti, Paolo; Saraiva, Lígia M; Giuffrè, Alessandro

    2014-01-01

    The microaerophilic protozoan parasite Giardia intestinalis, causative of one of the most common human intestinal diseases worldwide, infects the mucosa of the proximal small intestine, where it has to cope with O2 and nitric oxide (NO). Elucidating the antioxidant defense system of this pathogen lacking catalase and other conventional antioxidant enzymes is thus important to unveil novel potential drug targets. Enzymes metabolizing O2, NO and superoxide anion (O2 (-•)) have been recently reported for Giardia, but it is yet unknown how the parasite copes with H2O2 and peroxynitrite (ONOO(-)). Giardia encodes two yet uncharacterized 2-cys peroxiredoxins (Prxs), GiPrx1a and GiPrx1b. Peroxiredoxins are peroxidases implicated in virulence and drug resistance in several parasitic protozoa, able to protect from nitroxidative stress and repair oxidatively damaged molecules. GiPrx1a and a truncated form of GiPrx1b (deltaGiPrx1b) were expressed in Escherichia coli, purified and functionally characterized. Both Prxs effectively metabolize H2O2 and alkyl-hydroperoxides (cumyl- and tert-butyl-hydroperoxide) in the presence of NADPH and E. coli thioredoxin reductase/thioredoxin as the reducing system. Stopped-flow experiments show that both proteins in the reduced state react with ONOO(-) rapidly (k = 4×10(5) M(-1) s(-1) and 2×10(5) M(-1) s(-1) at 4°C, for GiPrx1a and deltaGiPrx1b, respectively). Consistent with a protective role against oxidative stress, expression of GiPrx1a (but not deltaGiPrx1b) is induced in parasitic cells exposed to air O2 for 24 h. Based on these results, GiPrx1a and deltaGiPrx1b are suggested to play an important role in the antioxidant defense of Giardia, possibly contributing to pathogenesis.

  3. In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation.

    PubMed

    Noichri, Y; Palais, G; Ruby, V; D'Autreaux, B; Delaunay-Moisan, A; Nyström, T; Molin, M; Toledano, M B

    2015-12-01

    2-Cys Prxs are H2O2-specific antioxidants that become inactivated by enzyme hyperoxidation at elevated H2O2 levels. Although hyperoxidation restricts the antioxidant physiological role of these enzymes, it also allows the enzyme to become an efficient chaperone holdase. The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation. How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking. To begin addressing the link between enzyme hyperoxidation and HMW structures formation, we have evaluated the in vivo 2-Cys Prxs quaternary structure changes induced by H2O2 by size exclusion chromatography (SEC) on crude lysates, using wild type (Wt) untagged and Myc-tagged S. cerevisiae 2-Cys Prx Tsa1 and derivative Tsa1 mutants or genetic conditions known to inactivate peroxidase or chaperone activity or altering the enzyme sensitivity to hyperoxidation. Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.

  4. Tuning of Peroxiredoxin Catalysis for Various Physiological Roles

    PubMed Central

    2015-01-01

    Peroxiredoxins (Prxs) make up an ancient family of enzymes that are the predominant peroxidases for nearly all organisms and play essential roles in reducing hydrogen peroxide, organic hydroperoxides, and peroxynitrite. Even between distantly related organisms, the core protein fold and key catalytic residues related to its cysteine-based catalytic mechanism have been retained. Given that these enzymes appeared early in biology, Prxs have experienced more than 1 billion years of optimization for specific ecological niches. Although their basic enzymatic function remains the same, Prxs have diversified and are involved in roles such as protecting DNA against mutation, defending pathogens against host immune responses, suppressing tumor formation, and—for eukaryotes—helping regulate peroxide signaling via hyperoxidation of their catalytic Cys residues. Here, we review the current understanding of the physiological roles of Prxs by analyzing knockout and knockdown studies from ∼25 different species. We also review what is known about the structural basis for the sensitivity of some eukaryotic Prxs to inactivation by hyperoxidation. In considering the physiological relevance of hyperoxidation, we explore the distribution across species of sulfiredoxin (Srx), the enzyme responsible for rescuing hyperoxidized Prxs. We unexpectedly find that among eukaryotes appearing to have a “sensitive” Prx isoform, some do not contain Srx. Also, as Prxs are suggested to be promising targets for drug design, we discuss the rationale behind recently proposed strategies for their selective inhibition. PMID:25403613

  5. The conformational bases for the two functionalities of 2-cysteine peroxiredoxins as peroxidase and chaperone

    PubMed Central

    Dietz, Karl-Josef

    2013-01-01

    2-Cysteine peroxiredoxins (2-CysPrxs) are ubiquitous and highly abundant proteins that serve multiple functions as peroxidases, chaperones, and thiol oxidases and in redox-dependent cell signalling. The chloroplast protein plays a role in seedling development and protection of the photosynthetic apparatus. This study aimed to unequivocally link conformation and function. To this end, a set of non-tagged site-directed mutagenized At2-CysPrx variants was engineered, which mimicked the conformational states and their specific functions: hyperoxidized form (C54D), reduced form (C54S, C176S), oxidized form (C54DC176K), phosphorylated form (T92D), reduced ability for oligomerization by interfering with the dimer–dimer interface (F84R) and a C-terminally truncated form [ΔC (–20 aa)]. These variants were fully or partly fixed in their quaternary structure and function, respectively, and were analysed for their conformational state and peroxidase and chaperone activity, as well as for their sensitivity to hyperoxidation. The presence of a His6-tag strongly influenced the properties of the protein. The ΔC variant became insensitive to hyperoxidation, while T92D and F84R became more sensitive. The C54D variant revealed the highest chaperone activity. The highest peroxidase activity was observed for the F84R and ΔC variants. Efficient interaction with NADP-dependent thioredoxin reductase C depended on the presence of Cys residues and the C-terminal tail. The results suggest that the structural flexibility is important for the switch between peroxidase and chaperone function and that evolution has conserved the functional switch instead of maximizing a single function. These variants are ideal tools for future conformation-specific studies in vivo and in vitro. PMID:23828546

  6. Human Peroxiredoxins 1 and 2 and Their Interacting Protein Partners; Through Structure Toward Functions of Biological Complexes.

    PubMed

    Bertoldi, Mariarita

    2016-01-01

    Since their discovery in the mid-nineties, peroxiredoxins have drawn much attention and the number of papers publications on different Prxs has been multiplied. The rise in interest in this topic is probably due, at least in part, to the large and further increasing functions attributed to the members of this family of ubiquitous proteins, including many redox and non-redox physiological functions. This review presents a Since their discovery in the mid-nineties, peroxiredoxins have drawn much attention and the number of publications on different Prxs has been multiplied. The rise in interest in this topic is probably due, at least in part, to the large and further increasing functions attributed to the members of this family of ubiquitous proteins, including many redox and non-redox physiological functions. This review presents a literature survey of the protein partners of the human Peroxiredoxin-1 and Peroxiredoxin- 2 of the Peroxiredoxin 1 subfamily, the most abundant class. Three sequence motifs, or combinations thereof, were found in the protein partners, namely, CXXC, PXXP, and LXXLL. These findings are discussed in light of i) protein partner localization, function and biological pathways and ii) the peroxiredoxins regions important for partner interaction, as revealed by the Peroxiredoxin-1-Sulfiredoxin-1 complex structure. The outcome of these analyses is expected to unravel some common molecular bases underlying peroxiredoxins propensity to bind a partner, as well as to propose a functional role for this interaction that could help to widen the biological role of this important class of enzymes.

  7. Peroxiredoxin I and II in Human Eyes

    PubMed Central

    Klebe, Sonja; Callahan, Thomas

    2014-01-01

    Peroxiredoxin I and II are both 2-Cys members of the peroxiredoxin family of antioxidant enzymes and inactivate hydrogen peroxide. On western blotting, both enzymes appeared as 22-kD proteins and were present in the sclera, retina and iris. Immunohistochemistry showed strong cytoplasmic labeling in the basal cells of the corneal epithelial layer and the corneoscleral limbus. The melanocytes within the stroma of the iris and the anterior epithelial cells of the lens also showed strong cytoplasmic labeling. The fibrous structure of the stroma and the posterior surface of the ciliary body were also labeled. There was also strong labeling for both enzymes in the photoreceptors and the inner and outer plexiform layers of the retina. There was increased labeling of peroxiredoxin I and II in pterygium. In normal conjunctiva and cornea, only the basal cell layer showed labeling for peroxiredoxin I and II, whereas, in pterygia, there was strong cytoplasmic labeling in most cells involving the full thickness of the epithelium. Co-localization of the DNA oxidation product 8-hydroxy-2’-deoxyguanosine antibody with the nuclear dye 4’,6’-diamidino-2-phenylindole dihydrochloride indicated that the majority of the oxidative damage was cytoplasmic; this suggested that the mitochondrial DNA was most affected by the UV radiation in this condition. PMID:24152995

  8. Structural and functional analysis of native peroxiredoxin 2 in human red blood cells.

    PubMed

    Ogasawara, Yuki; Ohminato, Takuya; Nakamura, Yusuke; Ishii, Kazuyuki

    2012-07-01

    Peroxiredoxin 2, a typical 2-Cys peroxiredoxin, is the third most abundant protein in erythrocytes. It is understood that the physiologically functional state of peroxiredoxin 2 is the monomer, and that its role in scavenging low levels of H(2)O(2) results in the formation of disulfide-linked dimers, which are reversibly reduced to monomers by the thioredoxin-thioredoxin reductase system. Additionally, peroxiredoxins are highly susceptible to sulfinic acid formation through reactions with various peroxides. This overoxidized form, which is thought to convert peroxiredoxins into molecular chaperones and to be accompanied by a transition to polymeric forms, can be reversed by sulfiredoxins. However, physiological conformational changes and the antioxidant role of erythrocyte peroxiredoxin 2 are still unclear because there is low sulfiredoxin and thioredoxin-thioredoxin reductase activity in erythrocytes. In this study, we examined the structural and redox states of peroxiredoxin 2 in fresh hemolysates and estimated the activities of native and overoxidized peroxiredoxin 2 purified from red blood cells to clear the physiological roles of peroxiredoxin 2 in erythrocyte. Our findings demonstrate that native peroxiredoxin 2 exists as high molecular weight (>160 kDa) oligomers and that decamers or higher order molecular weight oligomers (260-460 kDa) have peroxidase activity. We further showed that peroxiredoxin 2 oligomers, which were predominantly composed of monomers in the reduced form, exert a chaperone activity equal to that of overoxidized peroxiredoxin 2 polymers. These results provide the novel insight that redox-active peroxiredoxin 2 functions in human red blood cells as high molecular weight oligomers that possess peroxidase and chaperone activities.

  9. Peroxiredoxins: Guardians Against Oxidative Stress and Modulators of Peroxide Signaling

    PubMed Central

    Perkins, Arden; Nelson, Kimberly J.; Parsonage, Derek; Poole, Leslie B.; Karplus, P. Andrew

    2015-01-01

    Peroxiredoxins (Prxs) are a ubiquitous family of cysteine-dependent peroxidase enzymes that play dominant roles in regulating peroxide levels within cells. These enzymes, often present at high levels and capable of rapidly clearing peroxides, display a remarkable array of variations in their oligomeric states and susceptibility to regulation by hyperoxidative inactivation and other post-translational modifications. Key conserved residues within the active site promote catalysis by stabilizing the transition state required for transferring the terminal oxygen of hydroperoxides to the active site (peroxidatic) cysteine residue. Extensive investigations continue to expand our understanding of the scope of their importance as well as the structures and forces at play within these critical defense and regulatory enzymes. PMID:26067716

  10. Peroxiredoxins: guardians against oxidative stress and modulators of peroxide signaling.

    PubMed

    Perkins, Arden; Nelson, Kimberly J; Parsonage, Derek; Poole, Leslie B; Karplus, P Andrew

    2015-08-01

    Peroxiredoxins (Prxs) are a ubiquitous family of cysteine-dependent peroxidase enzymes that play dominant roles in regulating peroxide levels within cells. These enzymes, often present at high levels and capable of rapidly clearing peroxides, display a remarkable array of variations in their oligomeric states and susceptibility to regulation by hyperoxidative inactivation and other post-translational modifications. Key conserved residues within the active site promote catalysis by stabilizing the transition state required for transferring the terminal oxygen of hydroperoxides to the active site (peroxidatic) cysteine residue. Extensive investigations continue to expand our understanding of the scope of their importance as well as the structures and forces at play within these critical defense and regulatory enzymes.

  11. Identification and functional analysis of peroxiredoxin isoforms in Euglena gracilis.

    PubMed

    Tamaki, Shun; Maruta, Takanori; Sawa, Yoshihiro; Shigeoka, Shigeru; Ishikawa, Takahiro

    2014-01-01

    Euglena gracilis lacks catalase and contains ascorbate peroxidase (APX) which is localized exclusively in the cytosol. Other enzymes that scavenge reactive oxygen species (ROS) in Euglena have not yet been identified; therefore, ROS metabolism, especially in organelles, remains unclear in Euglena. The full-length cDNAs of four Euglena peroxiredoxins (EgPrxs) were isolated in this study. EgPrx1 and -4 were predicted to be localized in the cytosol, and EgPrx2 and -3 in plastids and mitochondria, respectively. The catalytic efficiencies of recombinant EgPrxs were similar to those of plant thiol-peroxidases, but were markedly lower than those of APX from Euglena. However, transcript levels of EgPrx1, -2, and -3 were markedly higher than those of APX. The growth rate of Euglena cells, in which the expression of EgPrx1 and -4 was suppressed by gene silencing, was markedly reduced under normal conditions, indicating physiological significance of Prx proteins.

  12. The Extraordinary Catalytic Ability of Peroxiredoxins: a Combined Experimental and QM/MM Study on the Fast Thiol Oxidation Step

    PubMed Central

    Zeida, Ari; Reyes, Anibal M.; Lebrero, Mariano C. G.; Radi, Rafael; Trujillo, Madia; Estrina, Darío A.

    2014-01-01

    Peroxiredoxins (Prxs) catalyze the reduction of peroxides, a process of key relevance in a variety of cellular processes. The first step in the catalytic cycle of all Prxs is the oxidation of a cysteine residue to sulfenic acid, which occurs 103-107 times faster than in free cysteine. We present an experimental kinetics and hybrid QM/MM investigation to explore the reaction of Prxs with H2O2 using alkyl hydroperoxide reductase E from Mycobacterium tuberculosis as a Prx model. We report for the first time the reaction thermodynamical activation parameters of H2O2 reduction by a Prx, which show that the protein lowers significantly the activation enthalpy, with an unfavourable entropic effect, compared to the uncatalyzed reaction. The QM/MM simulations show that the remarkable catalytic effects responsible for the fast H2O2 reduction in Prxs are mainly due to an active-site arrangement, which establishes a complex hydrogen bond network activating both reactive species. PMID:25045760

  13. Differences in Proinflammatory Property of Six Subtypes of Peroxiredoxins and Anti-Inflammatory Effect of Ligustilide in Macrophages

    PubMed Central

    Zhao, Li-Xue; Du, Jun-Rong; Zhou, Hong-Jing; Liu, Dong-Ling; Gu, Man-Xia; Long, Fang-Yi

    2016-01-01

    Background Peroxiredoxins (Prxs) are proposed to function as damage-associated molecular patterns (DAMPs) and contribute to post-ischemic neuroinflammation and brain injury by activating Toll-like receptor (TLR) 4 at the acute and subacute phases after ischemic stroke. However, there are few studies concerning the inflammatory profiles of six distinct subtypes of Prxs (Prx1–Prx6). Our previous study demonstrated that the protective effect of ligustilide (LIG) against cerebral ischemia was associated with inhibition of neuroinflammatory response and Prx/TLR4 signaling in rats. Herein, the present study explored the inflammatory members of Prxs and the effect of LIG on their inflammatory responses in macrophages. Methodology/Principal Findings The murine RAW264.7 macrophages were treated with each of exogenous recombinant Prxs at a range of 1 to 50 nM for 24 h. The WST-1 test showed that Prx3 exhibited a significant cytotoxicity, whereas the rest five Prxs did not affect cellular viability. The quantitative measurements with spectrometry or ELISA indicated that three subtypes, Prx1, Prx2 and Prx4, increased production of proinflammatory mediators, including nitric oxide (NO) metabolites, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in a concentration-dependent manner. Immunostaining demonstrated that 20 nM Prx1, Prx2 or Prx4 significantly increased expression of TLR4 and iNOS and nuclear translocation of NF-κB p65. However, Prx5 and Prx6 showed no poinflammatory effect in macrophages. Remarkably, LIG treatment effectively inhibited the inflammatory response induced by Prx1, Prx2 and Prx4. Conclusion Three members of Prxs, Prx1, Prx2 and Prx4, are inflammatory DAMPs that induce TLR4 activation and inflammatory response in macrophages, which is effectively inhibited by LIG. These results suggest that inflammatory Prxs-activated macrophages may provide a novel cellular model for screening the potential inhibitors of DAMPs-associated inflammatory

  14. Transition steps in peroxide reduction and a molecular switch for peroxide robustness of prokaryotic peroxiredoxins

    PubMed Central

    Kamariah, Neelagandan; Sek, Mun Foong; Eisenhaber, Birgit; Eisenhaber, Frank; Grüber, Gerhard

    2016-01-01

    In addition to their antioxidant function, the eukaryotic peroxiredoxins (Prxs) facilitate peroxide-mediated signaling by undergoing controlled inactivation by peroxide-driven over-oxidation. In general, the bacterial enzyme lacks this controlled inactivation mechanism, making it more resistant to high H2O2 concentrations. During peroxide reduction, the active site alternates between reduced, fully folded (FF), and oxidized, locally unfolded (LU) conformations. Here we present novel insights into the divergence of bacterial and human Prxs in robustness and sensitivity to inactivation, respectively. Structural details provide new insights into sub-steps during the catalysis of peroxide reduction, enabling the transition from an FF to a LU conformation. Complementary to mutational and enzymatic results, these data unravel the essential role of the C-terminal tail of bacterial Prxs to act as a molecular switch, mediating the transition from an FF to a LU state. In addition, we propose that the C-terminal tail has influence on the propensity of the disulphide bond formation, indicating that as a consequence on the robustness and sensitivity to over-oxidation. Finally, a physical linkage between the catalytic site, the C-terminal tail and the oligomer interface is described. PMID:27892488

  15. Crystal Structure of Reduced and of Oxidized Peroxiredoxin IV Enzyme Reveals a Stable Oxidized Decamer and a Non-disulfide-bonded Intermediate in the Catalytic Cycle*

    PubMed Central

    Cao, Zhenbo; Tavender, Timothy J.; Roszak, Aleksander W.; Cogdell, Richard J.; Bulleid, Neil J.

    2011-01-01

    Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue. PMID:21994946

  16. Evidence for the formation of a covalent thiosulfinate intermediate with peroxiredoxin in the catalytic mechanism of sulfiredoxin.

    PubMed

    Roussel, Xavier; Béchade, Guillaume; Kriznik, Alexandre; Van Dorsselaer, Alain; Sanglier-Cianferani, Sarah; Branlant, Guy; Rahuel-Clermont, Sophie

    2008-08-15

    The typical 2-Cys peroxiredoxins are thiol-peroxidases involved in the physiology of hydrogen peroxide not only as a toxic but also as a signaling molecule. Coordination of these functions depends on the sulfinylation of the catalytic Cys, a modification reversed by ATP-dependent sulfiredoxin, which specifically reduces the sulfinic acid group of overoxidized 2-Cys peroxiredoxins into a sulfenic acid. Sulfiredoxin was originally proposed to operate by covalent catalysis, with formation of a peroxiredoxin-sulfiredoxin intermediate linked by a thiosulfinate bond between the catalytic Cys of both partners, a hypothesis rejected by a study of the human enzyme. To settle the argument, we investigated the catalytic mechanism of Saccharomyces cerevisiae sulfiredoxin, by the characterization of the nature and kinetics of formation of the protein species formed between sulfiredoxin and its substrate in the presence of ATP, using mutants of the non-essential Cys residues of both proteins. We observed the formation of a dithiothreitol-reducible peroxiredoxin-sulfiredoxin species using SDS-PAGE and Western blot analysis, and its mass was shown to correspond to a thiosulfinate complex by high resolution mass spectrometry coupled to liquid chromatography. We next measured indirectly and directly a rate constant of formation of the thiosulfinate species of approximately 2 min(-1), for both wild-type and mutant sulfiredoxins, at least equal to the steady-state rate constant of the reaction, with a stoichiometry of 1:1 relative to peroxiredoxin. Taken altogether, our results strongly argue in favor of the formation of a covalent thiosulfinate peroxiredoxin-sulfiredoxin species as an intermediate on the catalytic pathway.

  17. Utilizing Natural and Engineered Peroxiredoxins As Intracellular Peroxide Reporters.

    PubMed

    Van Laer, Koen; Dick, Tobias P

    2016-01-01

    It is increasingly apparent that nature evolved peroxiredoxins not only as H2O2 scavengers but also as highly sensitive H2O2 sensors and signal transducers. Here we ask whether the H2O2 sensing role of Prx can be exploited to develop probes that allow to monitor intracellular H2O2 levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular H2O2 concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of H2O2 levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redox-sensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for H2O2 probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular H2O2 homeostasis and Prx signaling.

  18. Peroxiredoxin I and II in human eyes: cellular distribution and association with pterygium and DNA damage.

    PubMed

    Klebe, Sonja; Callahan, Thomas; Power, John H T

    2014-01-01

    Peroxiredoxin I and II are both 2-Cys members of the peroxiredoxin family of antioxidant enzymes and inactivate hydrogen peroxide. On western blotting, both enzymes appeared as 22-kD proteins and were present in the sclera, retina and iris. Immunohistochemistry showed strong cytoplasmic labeling in the basal cells of the corneal epithelial layer and the corneoscleral limbus. The melanocytes within the stroma of the iris and the anterior epithelial cells of the lens also showed strong cytoplasmic labeling. The fibrous structure of the stroma and the posterior surface of the ciliary body were also labeled. There was also strong labeling for both enzymes in the photoreceptors and the inner and outer plexiform layers of the retina. There was increased labeling of peroxiredoxin I and II in pterygium. In normal conjunctiva and cornea, only the basal cell layer showed labeling for peroxiredoxin I and II, whereas, in pterygia, there was strong cytoplasmic labeling in most cells involving the full thickness of the epithelium. Co-localization of the DNA oxidation product 8-hydroxy-2'-deoxyguanosine antibody with the nuclear dye 4',6'-diamidino-2-phenylindole dihydrochloride indicated that the majority of the oxidative damage was cytoplasmic; this suggested that the mitochondrial DNA was most affected by the UV radiation in this condition.

  19. Characterization and expression analysis of peroxiredoxin family genes from the silkworm Bombyx mori in response to phoxim and chlorpyrifos.

    PubMed

    Shi, Gui-Qin; Zhang, Ze; Jia, Kun-Lun; Zhang, Kun; An, Dong-Xu; Wang, Gang; Zhang, Bao-Long; Yin, He-Nan

    2014-09-01

    The organophosphorus pesticide poisoning of the silkworm Bombyx mori is one of the major events causing serious damage to sericulture. Some antioxidant enzymes play roles in regulating generation of reactive oxygen species (ROS) by pesticides including phoxim and chlorpyrifos, but relatively little is known about their effects on the silkworm peroxiredoxin family genes. Here, five peroxiredoxin (Prx) genes have been identified in silkworm genome, and Prx genes of silkworm and mammalian homologs have apparent ortholog relationship. Based on the genomic DNA sequence, putative 5'-flanking region of five BmPrxs were obtained and the transcription factor binding sites were predicted. Their expression profiles exposed to different concentrations of phoxim and chlorpyrifos for 24 h, 48 h and 72 h in midgut of silkworm were investigated using quantitative RT-PCR (qRT-PCR). The results showed that five BmPrxs and dual oxidase (BmDUOX) gene were all expressed in midgut of silkworm. After feeding with 0.375 mg/L and 0.75 mg/L phoxim, the transcription levels of BmPrx3 and BmPrx5 that can be located in mitochondria reached their peak levels at an early time point (24h). However, the transcription levels of BmPrx4 and BmPrx6 that can be addressed to secrete from the cell and cytosol, respectively, reached their peak levels at a later time point (72 h). Similar to expose to phoxim, the transcription levels of BmPrx3 and BmPrx5 that can be located in mitochondria reached their peak levels at an early time point (24 h) under chlorpyrifos stress. However, the transcription levels of BmPrx4 and BmPrx6 that can be addressed to secrete from the cell and cytosol, respectively, reached their peak levels at a later time point (72 h) under chlorpyrifos stress. These results revealed that BmPrxs that can be located in mitochondria were able to protect cells even more efficiently than cytosolic from an oxidative stress caused by OP. In addition, BmDUOX was also induced by phomix and

  20. Molecular Basis of Hydroperoxide Specificity in Peroxiredoxins: The Case of AhpE from Mycobacterium tuberculosis.

    PubMed

    Zeida, Ari; Reyes, Aníbal M; Lichtig, Pablo; Hugo, Martín; Vazquez, Diego S; Santos, Javier; González Flecha, F Luis; Radi, Rafael; Estrin, Dario A; Trujillo, Madia

    2015-12-15

    Peroxiredoxins (Prxs) constitute a ubiquitous family of Cys-dependent peroxidases that play essential roles in reducing hydrogen peroxide, peroxynitrite, and organic hydroperoxides in almost all organisms. Members of the Prx subfamilies show differential oxidizing substrate specificities that await explanations at a molecular level. Among them, alkyl hydroperoxide reductases E (AhpE) is a novel subfamily comprising Mycobacterium tuberculosis AhpE and AhpE-like proteins expressed in some bacteria and archaea. We previously reported that MtAhpE reacts ∼10(4) times faster with an arachidonic acid derived hydroperoxide than with hydrogen peroxide, and suggested that this surprisingly high reactivity was related to the presence of a hydrophobic groove at the dimer interface evidenced in the crystallography structure of the enzyme. In this contribution we experimentally confirmed the existence of an exposed hydrophobic patch in MtAhpE. We found that fatty acid hydroperoxide reduction by the enzyme showed positive activation entropy that importantly contributed to catalysis. Computational dynamics indicated that interactions of fatty acid-derived hydroperoxides with the enzyme properly accommodated them inside the active site and modifies enzyme's dynamics. The computed reaction free energy profile obtained via QM/MM simulations is consistent with a greater reactivity in comparison with hydrogen peroxide. This study represents new insights on the understanding of the molecular basis that determines oxidizing substrate selectivity in the peroxiredoxin family, which has not been investigated at an atomic level so far.

  1. Dopamine DRD2/Cys311 is not associated with chronic schizophrenia

    SciTech Connect

    Crawford, F.; Hoyne, J.; Cai, Xingang

    1996-09-20

    A mutation in the DRD2 receptor gene has been reported in association with schizophrenia in Japanese and Caucasian populations. The variation, Ser to Cys at codon 311, occurs in the third intracellular loop of the receptor and is therefore putatively functional. We report the results of screening US Caucasian schizophrenic and nonschizophrenic populations. We detected the occurrence of the DRD2 Cys311 variant in both schizophrenics and controls. Our data demonstrates no significant difference between the frequency of Cys311 in Caucasian schizophrenic and non-schizophrenic populations, indicating no association with schizophrenia. 8 refs., 1 fig., 1 tab.

  2. An Atlas of Peroxiredoxins Created Using an Active Site Profile-Based Approach to Functionally Relevant Clustering of Proteins

    PubMed Central

    Babbitt, Patricia C.; Ferrin, Thomas E.

    2017-01-01

    Peroxiredoxins (Prxs or Prdxs) are a large protein superfamily of antioxidant enzymes that rapidly detoxify damaging peroxides and/or affect signal transduction and, thus, have roles in proliferation, differentiation, and apoptosis. Prx superfamily members are widespread across phylogeny and multiple methods have been developed to classify them. Here we present an updated atlas of the Prx superfamily identified using a novel method called MISST (Multi-level Iterative Sequence Searching Technique). MISST is an iterative search process developed to be both agglomerative, to add sequences containing similar functional site features, and divisive, to split groups when functional site features suggest distinct functionally-relevant clusters. Superfamily members need not be identified initially—MISST begins with a minimal representative set of known structures and searches GenBank iteratively. Further, the method’s novelty lies in the manner in which isofunctional groups are selected; rather than use a single or shifting threshold to identify clusters, the groups are deemed isofunctional when they pass a self-identification criterion, such that the group identifies itself and nothing else in a search of GenBank. The method was preliminarily validated on the Prxs, as the Prxs presented challenges of both agglomeration and division. For example, previous sequence analysis clustered the Prx functional families Prx1 and Prx6 into one group. Subsequent expert analysis clearly identified Prx6 as a distinct functionally relevant group. The MISST process distinguishes these two closely related, though functionally distinct, families. Through MISST search iterations, over 38,000 Prx sequences were identified, which the method divided into six isofunctional clusters, consistent with previous expert analysis. The results represent the most complete computational functional analysis of proteins comprising the Prx superfamily. The feasibility of this novel method is demonstrated

  3. The yeast Tsa1 peroxiredoxin is a ribosome-associated antioxidant.

    PubMed

    Trotter, Eleanor W; Rand, Jonathan D; Vickerstaff, Jill; Grant, Chris M

    2008-05-15

    The yeast Tsa1 peroxiredoxin, like other 2-Cys peroxiredoxins, has dual activities as a peroxidase and as a molecular chaperone. Its peroxidase function predominates in lower-molecular-mass forms, whereas a super-chaperone form predominates in high-molecular-mass complexes. Loss of TSA1 results in aggregation of ribosomal proteins, indicating that Tsa1 functions to maintain the integrity of the translation apparatus. In the present study we report that Tsa1 functions as an antioxidant on actively translating ribosomes. Its peroxidase activity is required for ribosomal function, since mutation of the peroxidatic cysteine residue, which inactivates peroxidase but not chaperone activity, results in sensitivity to translation inhibitors. The peroxidatic cysteine residue is also required for a shift from ribosomes to its high-molecular-mass form in response to peroxide stress. Thus Tsa1 appears to function predominantly as an antioxidant in protecting both the cytosol and actively translating ribosomes against endogenous ROS (reactive oxygen species), but shifts towards its chaperone function in response to oxidative stress conditions. Analysis of the distribution of Tsa1 in thioredoxin system mutants revealed that the ribosome-associated form of Tsa1 is increased in mutants lacking thioredoxin reductase (trr1) and thioredoxins (trx1 trx2) in parallel with the general increase in total Tsa1 levels which is observed in these mutants. In the present study we show that deregulation of Tsa1 in the trr1 mutant specifically promotes translation defects including hypersensitivity to translation inhibitors, increased translational error-rates and ribosomal protein aggregation. These results have important implications for the role of peroxiredoxins in stress and growth control, since peroxiredoxins are likely to be deregulated in a similar manner during many different disease states.

  4. Dissecting Peroxiredoxin Catalysis: Separating Binding, Peroxidation, and Resolution for a Bacterial AhpC

    PubMed Central

    Parsonage, Derek; Nelson, Kimberly J.; Ferrer-Sueta, Gerardo; Alley, Samantha; Karplus, P. Andrew; Furdui, Cristina M.; Poole, Leslie B.

    2015-01-01

    Peroxiredoxins make up a ubiquitous family of cysteine-dependent peroxidases that reduce hydroperoxide or peroxynitrite substrates through formation of a cysteine sulfenic acid (R-SOH) at the active site. In the 2-Cys peroxiredoxins, a second (resolving) cysteine reacts with the sulfenic acid to form a disulfide bond. For all peroxiredoxins, structural rearrangements in the vicinity of the active site cysteine(s) are necessary to allow disulfide bond formation and subsequent reductive recycling. In this study, we evaluated the rate constants for individual steps in the catalytic cycle of Salmonella typhimurium AhpC. Conserved Trp residues situated close to both peroxidatic and resolving cysteines in AhpC give rise to large changes in fluorescence during the catalytic cycle. For recycling, AhpF very efficiently reduces the AhpC disulfide, with a single discernible step and a rate constant of 2.3 × 107 M−1 s−1. Peroxide reduction was more complex and could be modeled as three steps, beginning with a reversible binding of H2O2 to the enzyme (k1 = 1.36 × 108 M−1 s−1, and k−1 = 53 s−1), followed by rapid sulfenic acid generation (620 s−1) and then rate-limiting disulfide bond formation (75 s−1). Using bulkier hydroperoxide substrates with higher Km values, we found that different efficiencies (kcat/Km) for turnover of AhpC with these substrates are primarily caused by their slower rates of binding. Our findings indicate that this bacterial peroxiredoxin exhibits rates for both reducing and oxidizing parts of the catalytic cycle that are among the fastest observed so far for this diverse family of enzymes. PMID:25633283

  5. Peroxiredoxins and NADPH-dependent thioredoxin systems in the model legume Lotus japonicus.

    PubMed

    Tovar-Méndez, Alejandro; Matamoros, Manuel A; Bustos-Sanmamed, Pilar; Dietz, Karl-Josef; Cejudo, Francisco Javier; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

    2011-07-01

    Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules.

  6. Cloning and functional characterisation of a peroxiredoxin 1 (NKEF A) cDNA from Atlantic salmon (Salmo salar) and its expression in fish infected with Neoparamoeba perurans.

    PubMed

    Loo, Grace H; Sutton, Drew L; Schuller, Kathryn A

    2012-06-01

    Peroxiredoxin 1 (Prx 1), also known as natural killer enhancing factor A (NKEF A), has been implicated in the immune response of both mammals and fish. Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a significant problem for the Atlantic salmon (Salmo salar L.) aquaculture industry based in Tasmania, Australia. Here we have cloned and functionally characterized a Prx 1 open reading frame (ORF) from Atlantic salmon liver and shown that Prx 1 gene expression was down-regulated in the gills of Atlantic salmon displaying symptoms of AGD. The Prx 1 ORF encoded all of the residues and motifs characteristic of typical 2-Cys Prx proteins from eukaryotes and the recombinant protein expressed in Escherichia coli catalyzed thioredoxin (Trx)-dependent reduction of H(2)O(2), cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH) with K(m) values of 122, 77 and 91 μM, respectively, confirming that it was a genuine 2-Cys Prx. The recombinant protein also displayed a double displacement reaction mechanism and a catalytic efficiency (k(cat)/K(m)) with H(2)O(2) of 1.5 × 10(5) M(-1) s(-1) which was consistent with previous reports for the 2-Cys Prx family of proteins. This is the first time that a Prx 1 protein has been functionally characterized from any fish species and it paves the way for further investigation of this important 2-Cys Prx family member in fish.

  7. Genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins with a Zn(II)2Cys6 domain, Cys-X2-Cys-X6-Cys-X5-12-Cys-X2-Cys-X6-9-Cys (hereafter, referred to as the C6 domain), form a subclass of zinc finger proteins found exclusively in fungi and yeast. Genome sequence databases of Saccharomyces cerevisiae and Candida albicans have provided an overvie...

  8. Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys Peroxide Sensor▿

    PubMed Central

    Soonsanga, Sumarin; Lee, Jin-Won; Helmann, John D.

    2008-01-01

    OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols. PMID:18586944

  9. Functional characterisation of the peroxiredoxin gene family members of Synechococcus elongatus PCC 7942.

    PubMed

    Stork, Tina; Laxa, Miriam; Dietz, Marina S; Dietz, Karl-Josef

    2009-02-01

    The genome of Synechococcus elongatus PCC 7942 encodes six peroxiredoxins (Prx). Single genes are present each for a 1-Cys Prx and a 2-Cys Prx, while four genes code for PrxQ-like proteins (prxQ-A1, -A2, -A3 and B). Their transcript accumulation varies with growth conditions in a gene-specific manner (Stork et al. in J Exp Bot 56:3193-3206, 2005). To address their functional properties, members of the prx gene family were produced as recombinant proteins and analysed for their peroxide detoxification capacity and quaternary structure by size exclusion chromatography. Independent of the reduction state, the 2-Cys Prx separated as oligomer, the 1-Cys Prx as dimer and the PrxQ-A1 as monomer. PrxQ-A2 was inactive in our assays, 1-Cys Prx activity was unaffected by addition of TrxA, while all others were stimulated to a variable extent by addition of E. coli thioredoxin. Sensitivity towards cumene hydroperoxide treatment of E. coli BL21 cells expressing the cyanobacterial PrxQ-A1 to A3 proteins was greatly reduced, while expression of the other Prx had no effect. The study shows differentiation of Prx functions in S. elongatus PCC 7942 which is discussed in relation to potential roles in site- and stress-specific defence.

  10. A peroxiredoxin, PRDX-2, is required for insulin secretion and insulin/IIS-dependent regulation of stress resistance and longevity

    PubMed Central

    Oláhová, Monika; Veal, Elizabeth A

    2015-01-01

    Peroxiredoxins (Prx) are abundant thiol peroxidases with a conserved anti-ageing role. In contrast to most animals, the nematode worm, Caenorhabditis elegans, encodes a single cytosolic 2-Cys Prx, PRDX-2, rendering it an excellent model for examining how peroxiredoxins affect animal physiology and ageing. Our previous work revealed that, although PRDX-2 protects against the toxicity of peroxides, enigmatically, prdx-2-mutant animals are hyper-resistant to other forms of oxidative stress. Here, we have investigated the basis for this increased resistance. Mammalian FOXO and Nrf2 transcription factors directly promote the expression of a range of detoxification enzymes. We show that the FOXO orthologue, DAF-16, and the Nrf2 orthologue, SKN-1, are required for the increased stress resistance of prdx-2-mutant worms. Our data suggest that PRDX-2 is required for normal levels of insulin secretion and hence the inhibition of DAF-16 and SKN-1 by insulin/IGF-1-like signalling (IIS) under nutrient-rich conditions. Intriguingly, loss of PRDX-2 increases DAF-16 and SKN-1 activities sufficiently to increase arsenite resistance without initiating other IIS-inhibited processes. Together, these data suggest that loss of peroxiredoxin function may increase stress resistance by reducing insulin secretion, but that further changes in insulin signalling are required for the reprogramming of development and fat metabolism. In addition, we reveal that the temperature-dependent prolongevity function of PRDX-2 is required for the extended lifespan associated with several pathways, including further reductions in IIS. PMID:25808059

  11. The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2.

    PubMed

    Manta, Bruno; Hugo, Martín; Ortiz, Cecilia; Ferrer-Sueta, Gerardo; Trujillo, Madia; Denicola, Ana

    2009-04-15

    Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K(M) of 2.4 microM for human thioredoxin and a very low K(M) for H2O2 (0.7 microM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H2O2 or peroxynitrite, were determined by competition kinetics, k(2) = 1.0 x 10(8) and 1.4 x 10(7) M(-1) s(-1) at 25 degrees C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K(d) < 10(-23) M(4) which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.

  12. Crystal structure of mammalian selenocysteine-dependent iodothyronine deiodinase suggests a peroxiredoxin-like catalytic mechanism

    PubMed Central

    Schweizer, Ulrich; Schlicker, Christine; Braun, Doreen; Köhrle, Josef; Steegborn, Clemens

    2014-01-01

    Local levels of active thyroid hormone (3,3′,5-triiodothyronine) are controlled by the action of activating and inactivating iodothyronine deiodinase enzymes. Deiodinases are selenocysteine-dependent membrane proteins catalyzing the reductive elimination of iodide from iodothyronines through a poorly understood mechanism. We solved the crystal structure of the catalytic domain of mouse deiodinase 3 (Dio3), which reveals a close structural similarity to atypical 2-Cys peroxiredoxin(s) (Prx). The structure suggests a route for proton transfer to the substrate during deiodination and a Prx-related mechanism for subsequent recycling of the transiently oxidized enzyme. The proposed mechanism is supported by biochemical experiments and is consistent with the effects of mutations of conserved amino acids on Dio3 activity. Thioredoxin and glutaredoxin reduce the oxidized Dio3 at physiological concentrations, and dimerization appears to activate the enzyme by displacing an autoinhibitory loop from the iodothyronine binding site. Deiodinases apparently evolved from the ubiquitous Prx scaffold, and their structure and catalytic mechanism reconcile a plethora of partly conflicting data reported for these enzymes. PMID:25002520

  13. Discovering Antioxidant Molecules in the Archaea Domain: Peroxiredoxin Bcp1 from Sulfolobus solfataricus Protects H9c2 Cardiomyoblasts from Oxidative Stress

    PubMed Central

    Sarcinelli, Carmen; Pizzo, Elio

    2016-01-01

    Peroxiredoxins (Prxs) are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps) of the hyperthermophilic archaeon Sulfolobus solfataricus that belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measured in vitro under physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR)/thioredoxin (yTrx) reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internalization and verify the effect of Bcp1 on H9c2 rat cardiomyoblasts subjected to oxidative stress. The results reveal that TAT-Bcp1 is not cytotoxic and inhibits H2O2-induced apoptosis in H9c2 cells by reducing the H2O2 content inside these cells. PMID:27752237

  14. Adenanthin targets peroxiredoxin I/II to kill hepatocellular carcinoma cells.

    PubMed

    Hou, J-K; Huang, Y; He, W; Yan, Z-W; Fan, L; Liu, M-H; Xiao, W-L; Sun, H-D; Chen, G-Q

    2014-09-04

    Adenanthin, a natural diterpenoid isolated from the leaves of Isodon adenanthus, has recently been reported to induce leukemic cell differentiation by targeting peroxiredoxins (Prx) I and II. On the other hand, increasing lines of evidence propose that these Prx proteins would become potential targets to screen drugs for the prevention and treatment of solid tumors. Therefore, it is of significance to explore the potential activities of adenanthin on solid tumor cells. Here, we demonstrate that Prx I protein is essential for the survival of hepatocellular carcinoma (HCC) cells, and adenanthin can kill these malignant liver cells in vitro and xenografts. We also show that the cell death-inducing activity of adenanthin on HCC cells is mediated by the increased reactive oxygen species (ROS) levels. Furthermore, the silencing of Prx I or Prx II significantly enhances the cytotoxic activity of adenanthin on HCC, whereas the ectopic expression of Prx I and Prx II but not their mutants of adenanthin-bound cysteines can rescue adenanthin-induced cytotoxicity in Prxs-silenced HCC cells. Taken together, our results propose that adenanthin targets Prx I/II to kill HCC cells and its therapeutic significance warrants to be further explored in HCC patients.

  15. Adenanthin targets peroxiredoxin I/II to kill hepatocellular carcinoma cells

    PubMed Central

    Hou, J-K; Huang, Y; He, W; Yan, Z-W; Fan, L; Liu, M-H; Xiao, W-L; Sun, H-D; Chen, G-Q

    2014-01-01

    Adenanthin, a natural diterpenoid isolated from the leaves of Isodon adenanthus, has recently been reported to induce leukemic cell differentiation by targeting peroxiredoxins (Prx) I and II. On the other hand, increasing lines of evidence propose that these Prx proteins would become potential targets to screen drugs for the prevention and treatment of solid tumors. Therefore, it is of significance to explore the potential activities of adenanthin on solid tumor cells. Here, we demonstrate that Prx I protein is essential for the survival of hepatocellular carcinoma (HCC) cells, and adenanthin can kill these malignant liver cells in vitro and xenografts. We also show that the cell death-inducing activity of adenanthin on HCC cells is mediated by the increased reactive oxygen species (ROS) levels. Furthermore, the silencing of Prx I or Prx II significantly enhances the cytotoxic activity of adenanthin on HCC, whereas the ectopic expression of Prx I and Prx II but not their mutants of adenanthin-bound cysteines can rescue adenanthin-induced cytotoxicity in Prxs-silenced HCC cells. Taken together, our results propose that adenanthin targets Prx I/II to kill HCC cells and its therapeutic significance warrants to be further explored in HCC patients. PMID:25188510

  16. The Dual-Targeted Plant Sulfiredoxin Retroreduces the Sulfinic Form of Atypical Mitochondrial Peroxiredoxin1[W

    PubMed Central

    Iglesias-Baena, Iván; Barranco-Medina, Sergio; Sevilla, Francisca; Lázaro, Juan-José

    2011-01-01

    Sulfiredoxin (Srx) couples the energy of ATP hydrolysis to the energetically unfavorable process of reducing the inactive sulfinic form of 2-cysteine peroxiredoxins (Prxs) to regenerate its active form. In plants, Srx as well as typical 2-cysteine Prx have been considered as enzymes with exclusive chloroplast localization. This work explores the subcellular localization of Srx in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana). Immunocytochemistry, analysis of protein extracts from isolated intact organelles, and cell-free posttranslational import assays demonstrated that plant Srx also localizes to the mitochondrion in addition to plastids. The dual localization was in line with the prediction of a signal peptide for dual targeting. Activity tests and microcalorimetric data proved the interaction between Srx and its mitochondrial targets Prx IIF and thioredoxin. Srx catalyzed the retroreduction of the inactive sulfinic form of atypical Prx IIF using thioredoxin as reducing agent. Arabidopsis Srx also reduced overoxidized human Prx V. These results suggest that plant Srx could play a crucial role in the regulation of Prx IIF activity by controlling the regeneration of its overoxidized form in mitochondria, which are sites of efficient reactive oxygen species production in plants. PMID:21139087

  17. Characterization of a bacterioferritin comigratory protein family 1-Cys peroxiredoxin from Candidatus Liberibacter asiaticus.

    PubMed

    Singh, Anamika; Kumar, Narender; Tomar, Prabhat P S; Bhose, Sumit; Ghosh, Dilip Kumar; Roy, Partha; Sharma, Ashwani K

    2016-12-16

    To defend against the lethality of the reactive oxygen species (ROS), nature has armed microorganisms with a range of antioxidant proteins. These include peroxiredoxin (Prx) super family proteins which are ubiquitous cysteine-based non-heme peroxidases. The phytopathogenic bacterium Candidatus Liberibacter asiaticus (CLA), an etiological agent of citrus plants diseases, posses many genes for defense against oxidative stress. The bacterioferritin comigratory protein (BCP), a member of Prxs, is part of an oxidative stress defense system of CLA. The key residue of these enzymes is peroxidatic Cys (termed CPSH) which is contained within an absolutely conserved PXXX (T/S) XXC motif. In the present study, a 1-Cys Prx enzyme (CLa-BCP), having CPSH/sulfenic acid cysteine (C-46) but lacking the resolving cysteine (CRSH), was characterized from CLA. The peroxidase activity was demonstrated using a non-physiological electron donor DTT against varied substrates. The protein was shown to have the defensive role against peroxide-mediated cell killing and an antioxidant activity. In vitro DNA-binding studies showed that this protein can protect supercoiled DNA from oxidative damage. To the best of our knowledge, this is the first report on a 1-Cys BCPs to have an intracellular reactive oxygen species scavenging activity.

  18. Peroxiredoxin 1-mediated activation of TLR4/NF-κB pathway contributes to neuroinflammatory injury in intracerebral hemorrhage.

    PubMed

    Liu, Dong-Ling; Zhao, Li-Xue; Zhang, Shuang; Du, Jun-Rong

    2016-12-01

    The proinflammatory properties of extracellular peroxiredoxins (Prxs) via induction of Toll-like receptor 4 (TLR4) activation have been gradually revealed under diverse stress conditions, including cerebral ischemia but not hemorrhage. Prx1 is proposed to be a major hemorrhagic stress-inducible isoform of Prxs during acute and subacute phases of intracerebral hemorrhage (ICH). However, the potential of Prx1 in the neuroinflammatory injury after ICH remains unclear. This study investigated the proinflammatory effect and underlying mechanism of extracellular Prx1 in cultured murine macrophages and a collagenase-induced mouse ICH model. The current results show that incubation of exogenous Prx1 (0-50nM) with murine RAW264.7 macrophages resulted in increased expression of TLR4, nuclear translocation of nuclear factor κB (NF-κB) p65 and production of proinflammatory mediators (NO, TNF-a and IL-6) in a concentration-dependent manner. In addition, ICH induced murine neurological deficits, cerebral edema and neuropathological alterations, such as neuron injury, astrocyte and microglia/macrophage activation, and neutrophil and T lymphocyte invasion up to 72h after ICH. Moreover, ICH stimulated Prx1 expression and extracellular release, TLR4/NF-κB signaling activation, reflected by increases in TLR4 expression, extracellular signal-regulated kinase (ERK) 1/2 and NF-κB activation, and production of cytokines (TNF-α, IL-6 and IL-17). Taken together, these findings suggest that extracellular Prx1-mediated TLR4/NF-κB pathway activation probably contributes to neuroinflammatory injury after ICH, and thus blocking Prx1-TLR4 signaling might provide a novel anti-neuroinflammatory strategy with extended therapeutic window for hemorrhagic stroke.

  19. Peroxiredoxins and NADPH-Dependent Thioredoxin Systems in the Model Legume Lotus japonicus1[W][OA

    PubMed Central

    Tovar-Méndez, Alejandro; Matamoros, Manuel A.; Bustos-Sanmamed, Pilar; Dietz, Karl-Josef; Cejudo, Francisco Javier; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

    2011-01-01

    Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules. PMID:21562331

  20. Human peroxiredoxin PrxI is an orthologue of yeast Tsa1, capable of suppressing genome instability in Saccharomyces cerevisiae.

    PubMed

    Iraqui, Ismail; Faye, Gérard; Ragu, Sandrine; Masurel-Heneman, Amélie; Kolodner, Richard D; Huang, Meng-Er

    2008-02-15

    The peroxiredoxins (Prx) are conserved antioxidant proteins that use cysteine as the primary site of oxidation during the reduction of peroxides. Many organisms have more than one isoform of Prx. Deletion of TSA1, one of five Prxs in yeast Saccharomyces cerevisiae, results in accumulation of a broad spectrum of mutations including gross chromosomal rearrangements. Deletion of TSA1 is synthetically lethal with mutations in RAD6 and several key genes involved in DNA double-strand break repair. Here, we have examined the function of human PrxI and PrxII, which share a high degree of sequence identity with Tsa1, by expressing them in S. cerevisiae cells under the control of the native TSA1 promoter. We found that expression of PrxI, but not PrxII, was capable of complementing a tsa1Delta mutant for a variety of defects including genome instability, the synthetic lethality observed in rad6 Delta tsa1Delta and rad51 Delta tsa1Delta double mutants, and mutagen sensitivity. Moreover, expression of either Tsa1 or PrxI prevented Bax-induced cell death. These data indicate that PrxI is an orthologue of Tsa1. PrxI and Tsa1 seem to act on the same substrates in vivo and share similar mechanisms of function. The observation that PrxI is involved in suppressing genome instability and protecting against cell death potentially provides a better understanding of the consequences of PrxI dysfunction in human cells. The S. cerevisiae system described here could provide a sensitive tool to uncover the mechanisms that underlie the function of human Prxs.

  1. The Effects of Hexavalent Chromium on Thioredoxin Reductase and Peroxiredoxins in Human Bronchial Epithelial Cells

    PubMed Central

    Myers, Judith M.; Myers, Charles R.

    2009-01-01

    Inhalational exposure to hexavalent chromium [Cr(VI)] compounds (e.g. chromates) is of concern in many Cr-related industries and their surrounding environments. The bronchial epithelium is directly exposed to inhaled Cr(VI). Cr(VI) species gain easy access inside cells where they are reduced to reactive Cr species which may also contribute to the generation of reactive oxygen species (ROS). The thioredoxin (Trx) system promotes cell survival and has a major role in maintaining intracellular thiol redox balance. Previous studies with normal human bronchial epithelial cells (BEAS-2B) demonstrated that chromates cause dose- and time-dependent oxidation of Trx1 and Trx2. The Trxs keep many intracellular proteins reduced including the peroxiredoxins (Prx). Prx1 (cytosolic) and Prx3 (mitochondrial) were oxidized by Cr(VI) treatments that oxidized all, or nearly all, of the respective Trxs. Prx oxidation is therefore likely the result of a lack of reducing equivalents from Trx. Trx reductases (TrxR) maintain the Trxs largely in the reduced state. Cr(VI) caused pronounced inhibition of TrxR, but the levels of TrxR protein remained unchanged. The inhibition of TrxR was not reversed by removal of residual Cr(VI) or by NADPH, the endogenous electron donor for TrxR. In contrast, the oxidation of Trx1, Trx2, and Prx3 were reversible by disulfide reductants. Prolonged inhibition of TrxR in Cr(VI)-treated cells might contribute to the sustained oxidation of Trxs and Prxs. Reduced Trx binds to an N-terminal domain of apoptosis signaling kinase (ASK1), keeping ASK1 inactive. Cr(VI) treatments that significantly oxidized Trx1 resulted in pronounced dissociation of Trx1 from ASK1. Overall, the effects of Cr(VI) on the redox state and function of the Trxs, Prxs, and TrxR in the bronchial epithelium could have important implications for redox-sensitive cell signaling and tolerance to oxidant insults. PMID:19703554

  2. A Zn(II)2Cys6 DNA binding protein regulates the sirodesmin PL biosynthetic gene cluster in Leptosphaeria maculans

    PubMed Central

    Fox, Ellen M.; Gardiner, Donald M.; Keller, Nancy P.; Howlett, Barbara J.

    2008-01-01

    A gene, sirZ, encoding a Zn(II)2Cys6 DNA binding protein is present in a cluster of genes responsible for the biosynthesis of the epipolythiodioxopiperazine (ETP) toxin, sirodesmin PL in the ascomycete plant pathogen, Leptosphaeria maculans. RNA-mediated silencing of sirZ gives rise to transformants that produce only residual amounts of sirodesmin PL and display a decrease in the transcription of several sirodesmin PL biosynthetic genes. This indicates that SirZ is a major regulator of this gene cluster. Proteins similar to SirZ are encoded in the gliotoxin biosynthetic gene cluster of Aspergillus fumigatus (gliZ) and in an ETP-like cluster in Penicillium lilacinoechinulatum (PlgliZ). Despite its high level of sequence similarity to gliZ, PlgliZ is unable to complement the gliotoxin-deficiency of a mutant of gliZ in A. fumigatus. Putative binding sites for these regulatory proteins in the promoters of genes in these clusters were predicted using bioinformatic analysis. These sites are similar to those commonly bound by other proteins with Zn(II)2Cys6 DNA binding domains. PMID:18023597

  3. Ovariectomy upregulated the expression of Peroxiredoxin 1 & 5 in osteoblasts of mice

    PubMed Central

    Du, Juan; Feng, Wei; Sun, Jing; Kang, Cuijie; Amizuka, Norio; Li, Minqi

    2016-01-01

    Peroxiredoxin (PRX), a family of peroxidases, is associated with various biological processes such as the detoxification of oxidants and cell apoptosis. Besides, the anti-apoptosis effect of estrogen results partially from its anti-oxidant function. The purpose of this study was to investigate the expression of PRXs in ovariectomy (OVX) mice and the related anti-oxidative mechanism of estrogen. Eight-week-old mice were subjected to ovariectomy. MC3T3-E1 cells were pretreatment with 17b-estradiol and N-acetyl cysteine followed by oxidative injury induced with H2O2. Western blot and real time-PCR were applied to clarify the expressions of PRX1 and caspase-3, with both wild-type and PRX1 knockout MC3T3-E1 cells generated by CRISPR/Cas9 technology. The results showed PRX1 and PRX5 were upregulated in osteoblasts in the proximal tibial metaphysis of ovariectomy mice. Interestingly, PRX1 and PRX5 showed different distribution patterns, with PRX1 mainly accumulated in cell nuclei and PRX5 in the cytoplasm. Gene expression analysis showed significantly reduced expressions of PRX1 and caspase-3 in the pretreatment groups when compared with cells treated with H2O2 alone. Also, a decrease of caspase-3 expressions was observed in PRX1 knockout MC3T3-E1 cells with or without H2O2 in comparison to wild-type cells. These findings suggested that PRX may play important roles in estrogen-deficient osteoporosis. (200 words). PMID:27786251

  4. Molecular and functional properties of three different peroxiredoxin isotypes in Chinese cabbage.

    PubMed

    Kim, Sun Young; Jung, Young Jun; Shin, Mi Rim; Park, Jung Hoon; Nawkar, Ganesh M; Maibam, Punyakishore; Lee, Eun Seon; Kim, Kang-San; Paeng, Seol Ki; Kim, Woe Yeon; Lee, Kyun Oh; Yun, Dae-Jin; Kang, Chang Ho; Lee, Sang Yeol

    2012-01-01

    Peroxiredoxins (Prxs), which are classified into three isotypes in plants, play important roles in protection systems as peroxidases or molecular chaperones. The three Prx isotypes of Chinese cabbage, namely C1C-Prx, C2C-Prx, and C-PrxII, have recently been identified and characterized. The present study compares their molecular properties and biochemical functions to gain insights into their concerted roles in plants. The three Prx isotype genes were differentially expressed in tissue- and developmental stage-specific manners. The transcript level of the C1C-Prx gene was abundant at the seed stage, but rapidly decreased after imbibitions. In contrast, the C2C-Prx transcript was not detected in the seeds, but its expression level increased at germination and was maintained thereafter. The C-PrxII transcript level was mild at the seed stage, rapidly increased for 10 days after imbibitions, and gradually disappeared thereafter. In the localization analysis using GFP-fusion proteins, the three isotypes showed different cellular distributions. C1C-Prx was localized in the cytosol and nucleus, whereas C2C-Prx and C-Prx were found mainly in the chloroplast and cytosol, respectively. In vitro thiol-dependent antioxidant assays revealed that the relative peroxidase activities of the isotypes were CPrxII > C2C-Prx > C1C-Prx. C1C-Prx and C2C-Prx, but not C-PrxII, prevented aggregation of malate dehydrogenase as a molecular chaperone. Taken together, these results suggest that the three isotypes of Prx play specific roles in the cells in timely and spatially different manners, but they also cooperate with each other to protect the plant.

  5. Kinetic analysis of structural influences on the susceptibility of peroxiredoxins 2 and 3 to hyperoxidation

    PubMed Central

    Poynton, Rebecca A.; Peskin, Alexander V.; Haynes, Alexina C.; Lowther, W. Todd; Hampton, Mark B.; Winterbourn, Christine C.

    2016-01-01

    Mammalian 2-cysteine peroxiredoxins (Prxs) are susceptible to hyperoxidation by excess H2O2. The cytoplasmic family member Prx2 hyperoxidizes more readily than mitochondrial Prx3 due to slower dimerization of the sulfenic acid (SpOH) intermediate. Four variant amino acids near the C-terminus have been shown to contribute to this difference. We have performed kinetic analysis of the relationship between hyperoxidation and disulfide formation, using whole-protein MS and comparing wild-type (WT) Prx2 and Prx3 with tail-swap mutants in which the four amino acids were reversed. These changes make Prx3 more sensitive and Prx2 less sensitive to hyperoxidation and accounted for ~70% of the difference between the two proteins. The tail swap mutant of Prx3 was also more susceptible when expressed in the mitochondria of HeLa cells. The hyperoxidized product at lower excesses of H2O2 was a semi-hyperoxidized dimer with one active site disulfide and the other a sulfinic acid. For Prx2, increasing the H2O2 concentration resulted in complete hyperoxidation. In contrast, only approximately half the Prx3 active sites underwent hyperoxidation and, even with high H2O2, the predominant product was the hyperoxidized dimer. Size exclusion chromatography (SEC) showed that the oligomeric forms of all redox states of Prx3 dissociated more readily into dimeric units than their Prx2 counterparts. Notably the species with one disulfide and one hyperoxidized active site was decameric for Prx2 and dimeric for Prx3. Reduction and re-oxidation of the hyperoxidized dimer of Prx3 produced hyperoxidized monomers, implying dissociation and rearrangement of the subunits of the functional homodimer. PMID:26614766

  6. A peroxiredoxin cDNA from Taiwanofungus camphorata: role of Cys31 in dimerization.

    PubMed

    Huang, Chih-Yu; Chen, Yu-Ting; Wen, Lisa; Sheu, Dey-Chyi; Lin, Chi-Tsai

    2014-01-01

    Peroxiredoxins (Prxs) play important roles in antioxidant defense and redox signaling pathways. A Prx isozyme cDNA (TcPrx2, 745 bp, EF552425) was cloned from Taiwanofungus camphorata and its recombinant protein was overexpressed. The purified protein was shown to exist predominantly as a dimer by sodium dodecyl sulfate-polyacrylamide gel electrolysis in the absence of a reducing agent. The protein in its dimeric form showed no detectable Prx activity. However, the protein showed increased Prx activity with increasing dithiothreitol concentration which correlates with dissociation of the dimer into monomer. The TcPrx2 contains two Cys residues. The Cys(60) located in the conserved active site is the putative active peroxidatic Cys. The role of Cys(31) was investigated by site-directed mutagenesis. The C31S mutant (C(31) → S(31)) exists predominantly as a monomer with noticeable Prx activity. The Prx activity of the mutant was higher than that of the corresponding wild-type protein by nearly twofold at 12 μg/mL. The substrate preference of the mutant was H2O2 > cumene peroxide > t-butyl peroxide. The Michaelis constant (K M) value for H2O2 of the mutant was 0.11 mM. The mutant enzyme was active under a broad pH range from 6 to 10. The results suggest a role of Cys(31) in dimerization of the TcPrx2, a role which, at least in part, may be involved in determining the activity of Prx. The C(31) residue does not function as a resolving Cys and therefore the TcPrx2 must follow the reaction mechanism of 1-Cys Prx. This TcPrx2 represents a new isoform of Prx family.

  7. The sulfiredoxin-peroxiredoxin (Srx-Prx) axis in cell signal transduction and cancer development.

    PubMed

    Mishra, Murli; Jiang, Hong; Wu, Lisha; Chawsheen, Hedy A; Wei, Qiou

    2015-10-01

    Redox signaling is a critical component of cell signaling pathways that are involved in the regulation of cell growth, metabolism, hormone signaling, immune regulation and variety of other physiological functions. Peroxiredoxin (Prx) is a family of thiol-based peroxidase that acts as a regulator of redox signaling. Members of Prx family can act as antioxidants and chaperones. Sulfiredoxin (Srx) is an antioxidant protein that exclusively reduces over-oxidized typical 2-Cys Prx. Srx has different affinities for individual Prx and it also catalyzes the deglutathionylation of variety of substrates. Individual component of the Srx-Prx system plays critical role in carcinogenesis by modulating cell signaling pathways involved in cell proliferation, migration and metastasis. Expression levels of individual component of the Srx-Prx axis have been correlated with patient survival outcome in multiple cancer types. This review will summarize the molecular basis of differences in the affinity of Srx for individual Prx and the role of individual component of the Srx-Prx system in tumor progression and metastasis. This enhanced understanding of molecular aspects of Srx-Prx interaction and its role in cell signal transduction will help define the Srx-Prx system as a future therapeutic target in human cancer.

  8. Peroxiredoxin 5 modulates immune response in Drosophila

    PubMed Central

    Radyuk, Svetlana N.; Michalak, Katarzyna; Klichko, Vladimir I.; Benes, Judith; Orr, William C.

    2010-01-01

    Background Peroxiredoxins are redox-sensing enzymes with multiple cellular functions. Previously, we reported on the potent antioxidant function of Drosophila peroxiredoxin 5 (dPrx5). Studies with mammalian and human cells suggest that peroxiredoxins can modulate immune-related signaling. Methods Survivorship studies and bacteriological analysis were used to determine resistance of flies to fungal and bacterial infections. RT-PCR and immunoblot analyses determined expression of dPrx5 and immunity factors in response to bacterial challenge. Double mutants for dprx5 gene and genes comprising the Imd/Relish and dTak1/Basket branches of the immune signaling pathways were used in epistatic analysis. Results The dprx5 mutant flies were more resistant to bacterial infection than controls, while flies overexpressing dPrx5 were more susceptible. The enhanced resistance to bacteria was accompanied by rapid induction of the Imd-dependent antimicrobial peptides, phosphorylation of the JNK kinase Basket and altered transcriptional profiling of the transient response genes, puckered, ets21C and relish, while the opposite effects were observed in flies over-expressing dPrx5. Epistatic analysis of double mutants, using attacin D and Puckered as read outs of activation of the Imd and JNK pathways, implicated dPrx5 function in the control of the dTak1-JNK arm of immune signaling. Conclusions Differential effects on fly survivorship suggested a trade-off between the antioxidant and immune functions of dPrx5. Molecular and epistatic analyses identified dPrx5 as a negative regulator in the dTak1-JNK arm of immune signaling. General significance Our findings suggest that peroxiredoxins play an important modulatory role in the Drosophila immune response. PMID:20600624

  9. Metal-induced self-assembly of peroxiredoxin as a tool for sorting ultrasmall gold nanoparticles into one-dimensional clusters

    NASA Astrophysics Data System (ADS)

    Ardini, Matteo; Giansanti, Francesco; di Leandro, Luana; Pitari, Giuseppina; Cimini, Annamaria; Ottaviano, Luca; Donarelli, Maurizio; Santucci, Sandro; Angelucci, Francesco; Ippoliti, Rodolfo

    2014-06-01

    Nanomanipulation of matter to create responsive, ordered materials still remains extremely challenging. Supramolecular chemistry has inspired new strategies by which such nanomaterials can be synthesized step by step by exploiting the self-recognition properties of molecules. In this work, the ring-shaped architecture of the 2-Cys peroxiredoxin I protein from Schistosoma mansoni, engineered to have metal ion-binding sites, is used as a template to build up 1D nanoscopic structures through metal-induced self-assembly. Chromatographic and microscopic analyses demonstrate the ability of the protein rings to stack directionally upon interaction with divalent metal ions and form well-defined nanotubes by exploiting the intrinsic recognition properties of the ring surfaces. Taking advantage of such behavior, the rings are then used to capture colloidal Ni2+-functionalized ultrasmall gold nanoparticles and arrange them into 1D arrays through stacking into peapod-like complexes. Finally, as the formation of such nano-peapods strictly depends on nanoparticle dimensions, the peroxiredoxin template is used as a colloidal cut-off device to sort by size the encapsulated nanoparticles. These results open up possibilities in developing Prx-based methods to synthesize new advanced functional materials.Nanomanipulation of matter to create responsive, ordered materials still remains extremely challenging. Supramolecular chemistry has inspired new strategies by which such nanomaterials can be synthesized step by step by exploiting the self-recognition properties of molecules. In this work, the ring-shaped architecture of the 2-Cys peroxiredoxin I protein from Schistosoma mansoni, engineered to have metal ion-binding sites, is used as a template to build up 1D nanoscopic structures through metal-induced self-assembly. Chromatographic and microscopic analyses demonstrate the ability of the protein rings to stack directionally upon interaction with divalent metal ions and form well

  10. Thioredoxin 1 protects astrocytes from oxidative stress by maintaining peroxiredoxin activity

    PubMed Central

    WANG, MENGFEI; ZHU, KUNTING; ZHANG, LUYU; LI, LINGYU; ZHAO, JING

    2016-01-01

    Previous studies have demonstrated that thioredoxin 1 (Trx1) exerts neuroprotective effects against cerebral ischemia/reperfusion injury caused by oxidative stress. While Trx1 is known to maintain the anti-oxidant activity of 2-Cys peroxiredoxins (Prdxs), the underlying mechanisms of its protective effects have remained to be elucidated, which was the aim of the present study. For this, an in vitro ischemic model of hypoxemia lasting for 4 h, followed by 24 h of reperfusion was used. Primary astrocytes from neonatal rats were pre-treated with small interfering RNA targeting Trx1 prior to oxygen glucose deprivation/reperfusion (OGD/R). MTS and lactate dehydrogenase assays were performed to evaluate cell viability. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were employed to assess the mRNA and protein expression levels of Prdx1-4 and Prdx-SO3. Furthermore, a dual luciferase reporter assay was used to assess the interaction between activator protein-1 (AP-1) and Trx1. The present study demonstrated that OGD/R decreased the cell viability and increased cellular damage, which was more marked following Trx1 knockdown. The expression of Prdx1-4 and Prdx-SO3 protein was higher in the cells subjected to OGD/R. Knockdown of Trx1 markedly decreased the levels of Prdx1-4 but increased Prdx-SO3 mRNA and protein levels. The results of the present study also suggested that AP-1 directly activated the expression of Trx1. The present study demonstrated that Trx1 exerts its neuroprotective effects by preventing oxidative stress in astrocytes via maintaining Prdx expression. PMID:26846911

  11. The role of peroxiredoxins in cancer

    PubMed Central

    Nicolussi, Arianna; D'Inzeo, Sonia; Capalbo, Carlo; Giannini, Giuseppe; Coppa, Anna

    2017-01-01

    Peroxiredoxins (PRDXs) are a ubiquitously expressed family of small (22–27 kDa) non-seleno peroxidases that catalyze the peroxide reduction of H2O2, organic hydroperoxides and peroxynitrite. They are highly involved in the control of various physiological functions, including cell growth, differentiation, apoptosis, embryonic development, lipid metabolism, the immune response, as well as cellular homeostasis. Although the protective role of PRDXs in cardiovascular and neurological diseases is well established, their role in cancer remains controversial. Increasing evidence suggests the involvement of PRDXs in carcinogenesis and in the development of drug resistance. Numerous types of cancer cells, in fact, are characterized by an increase in reactive oxygen species (ROS) production, and often exhibit an altered redox environment compared with normal cells. The present review focuses on the complex association between oxidant balance and cancer, and it provides a brief account of the involvement of PRDXs in tumorigenesis and in the development of chemoresistance. PMID:28357082

  12. Kinetic Approaches to Measuring Peroxiredoxin Reactivity

    PubMed Central

    Winterbourn, Christine C.; Peskin, Alexander V.

    2016-01-01

    Peroxiredoxins are ubiquitous thiol proteins that catalyse the breakdown of peroxides and regulate redox activity in the cell. Kinetic analysis of their reactions is required in order to identify substrate preferences, to understand how molecular structure affects activity and to establish their physiological functions. Various approaches can be taken, including the measurement of rates of individual steps in the reaction pathway by stopped flow or competitive kinetics, classical enzymatic analysis and measurement of peroxidase activity. Each methodology has its strengths and they can often give complementary information. However, it is important to understand the experimental conditions of the assay so as to interpret correctly what parameter is being measured. This brief review discusses different kinetic approaches and the information that can be obtained from them. PMID:26813658

  13. Characterization of a cDNA of peroxiredoxin II responding to hydrogen peroxide and phagocytosis in Amoeba proteus.

    PubMed

    Park, Miey; Shin, Hae J; Lee, Soo Y; Ahn, Tae I

    2005-01-01

    Phagocytic cells have defense systems against reactive oxygen species generated as the first non-specific defense mechanism against invading pathogens or microorganisms. We cloned a cDNA encoding a 21.69-kDa protein in Amoeba proteus homologous to 2-Cys peroxiredoxin (Prx-Ap). In the disk inhibition assay using H2O2 as an oxidizing agent, Escherichia coli overproducing Prx-Ap showed better viability than did E. coli transformed with pBluescript II SK for control. Monoclonal antibodies (mAb) produced against Prx-Ap reacted with a 22.5-kDa protein and several minor proteins. In Western blot analysis, levels of the 22.5-kDa protein in amoebae treated with 2-mM H2O2 for 1 h increased about 2-fold over those in control cells. Immunofluorescence scattered throughout the cytoplasm also increased after H2O2 treatment. In Northern blot analysis using the cDNA as a probe, the level of transcripts also changed with H2O2 treatment. When amoebae were fed with Tetrahymena, the intensity of immunofluorescence increased from 15 min and persisted until 2 h after phagocytosis. These results suggest that the 22.5-kDa protein of A. proteus is a Prx protein and that it has an antioxidant property responding to phagocytosis.

  14. Structural changes upon peroxynitrite-mediated nitration of peroxiredoxin 2; nitrated Prx2 resembles its disulfide-oxidized form.

    PubMed

    Randall, Lía; Manta, Bruno; Nelson, Kimberly J; Santos, Javier; Poole, Leslie B; Denicola, Ana

    2016-01-15

    Peroxiredoxins are cys-based peroxidases that function in peroxide detoxification and H2O2-induced signaling. Human Prx2 is a typical 2-Cys Prx arranged as pentamers of head-to-tail homodimers. During the catalytic mechanism, the active-site cysteine (CP) cycles between reduced, sulfenic and disulfide state involving conformational as well as oligomeric changes. Several post-translational modifications were shown to affect Prx activity, in particular CP overoxidation which leads to inactivation. We have recently reported that nitration of Prx2, a post-translational modification on non-catalytic tyrosines, unexpectedly increases its peroxidase activity and resistance to overoxidation. To elucidate the cross-talk between this post-translational modification and the enzyme catalysis, we investigated the structural changes of Prx2 after nitration. Analytical ultracentrifugation, UV absorption, circular dichroism, steady-state and time-resolved fluorescence were used to connect catalytically relevant redox changes with tyrosine nitration. Our results show that the reduced nitrated Prx2 structurally resembles the disulfide-oxidized native form of the enzyme favoring a locally unfolded conformation that facilitates disulfide formation. These results provide structural basis for the kinetic analysis previously reported, the observed increase in activity and the resistance to overoxidation of the peroxynitrite-treated enzyme.

  15. The Dof domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain.

    PubMed

    Umemura, Yoshimi; Ishiduka, Tomoko; Yamamoto, Rie; Esaka, Muneharu

    2004-03-01

    The Dof (DNA-binding with one finger) proteins are plant transcription factors that have a highly conserved DNA-binding domain, called the Dof domain. The Dof domain, which is composed of 52 amino acid residues, is similar to the Cys2/Cys2 zinc finger DNA-binding domain of GATA1 and steroid hormone receptors, but has a longer putative loop than that in the case of these zinc finger domains. The DNA-binding function of ascorbate oxidase gene binding protein (AOBP), a Dof protein, was investigated by gel retardation analysis. When Cys was replaced by His, the Dof domain could not function as a Cys3/His- or a Cys2/His2-type zinc finger. The characteristic longer loop was essential for DNA-binding activity. Furthermore, heavy metals such as Co(II), Ni(II), Cd(II), Cu(II), Hg(II), Fe(II), and Fe(III) inhibited the DNA-binding activity of the Dof domain. Manganese ion as well as zinc ion was coordinated by the Dof domain in vitro. On the other hand, the analysis using inductively coupled argon plasma mass spectrometry (ICP-MS) showed that the Dof domain contained zinc ion but not manganese ion. Thus, the Dof domain was proved to function as a Cys2/Cys2 zinc finger domain.

  16. Circadian rhythm of hyperoxidized peroxiredoxin II is determined by hemoglobin autoxidation and the 20S proteasome in red blood cells.

    PubMed

    Cho, Chun-Seok; Yoon, Hyun Ju; Kim, Jeong Yeon; Woo, Hyun Ae; Rhee, Sue Goo

    2014-08-19

    The catalytic cysteine of the typical 2-Cys Prx subfamily of peroxiredoxins is occasionally hyperoxidized to cysteine sulfinic acid during the peroxidase catalytic cycle. Sulfinic Prx (Prx-SO2H) is reduced back to the active form of the enzyme by sulfiredoxin. The abundance of Prx-SO2H was recently shown to oscillate with a period of ∼24 h in human red blood cells (RBCs). We have now investigated the molecular mechanism and physiological relevance of such oscillation in mouse RBCs. Poisoning of RBCs with CO abolished Prx-SO2H formation, implicating H2O2 produced from hemoglobin autoxidation in Prx hyperoxidation. RBCs express the closely related PrxI and PrxII isoforms, and analysis of RBCs deficient in either isoform identified PrxII as the hyperoxidized Prx in these cells. Unexpectedly, RBCs from sulfiredoxin-deficient mice also exhibited circadian oscillation of Prx-SO2H. Analysis of the effects of protease inhibitors together with the observation that the purified 20S proteasome degraded PrxII-SO2H selectively over nonhyperoxidized PrxII suggested that the 20S proteasome is responsible for the decay phase of PrxII-SO2H oscillation. About 1% of total PrxII undergoes daily oscillation, resulting in a gradual loss of PrxII during the life span of RBCs. PrxII-SO2H was detected in cytosolic and ghost membrane fractions of RBCs, and the amount of membrane-bound PrxII-SO2H oscillated in a phase opposite to that of total PrxII-SO2H. Our results suggest that membrane association of PrxII-SO2H is a tightly controlled process and might play a role in the tuning of RBC function to environmental changes.

  17. The mitochondrial type II peroxiredoxin F is essential for redox homeostasis and root growth of Arabidopsis thaliana under stress.

    PubMed

    Finkemeier, Iris; Goodman, Megan; Lamkemeyer, Petra; Kandlbinder, Andrea; Sweetlove, Lee J; Dietz, Karl-Josef

    2005-04-01

    Peroxiredoxins (Prx) have recently moved into the focus of plant and animal research in the context of development, adaptation, and disease, as they function both in antioxidant defense by reducing a broad range of toxic peroxides and in redox signaling relating to the adjustment of cell redox and antioxidant metabolism. At-PrxII F is one of six type II Prx identified in the genome of Arabidopsis thaliana and the only Prx that is targeted to the plant mitochondrion. Therefore, it might be assumed to have functions similar to the human 2-Cys Prx (PRDX3) and type II Prx (PRDX5) and yeast 1-Cys Prx that likewise have mitochondrial localizations. This paper presents a characterization of PrxII F at the level of subcellular distribution, activity, and reductive regeneration by mitochondrial thioredoxin and glutaredoxin. By employing tDNA insertion mutants of A. thaliana lacking expression of AtprxII F (KO-AtPrxII F), it is shown that under optimal environmental conditions the absence of PrxII F is almost fully compensated for, possibly by increases in activity of mitochondrial ascorbate peroxidase and glutathione-dependent peroxidase. However, a stronger inhibition of root growth in KO-AtPrxII F seedlings as compared with wild type is observed under stress conditions induced by CdCl2 as well as after administration of salicylhydroxamic acid, an inhibitor of cyanide-insensitive respiration. Simultaneously, major changes in the abundance of both nuclear and mitochondria-encoded transcripts were observed. These results assign a principal role to PrxII F in antioxidant defense and possibly redox signaling in plants cells.

  18. Molecular characterization and functional analysis of a peroxiredoxin 1 cDNA from golden pompano (Trachinotus ovatus).

    PubMed

    Wang, Long; Guo, Huayang; Zhang, Nan; Ma, Zhenhua; Jiang, Shigui; Zhang, Dianchang

    2015-08-01

    Peroxiredoxin 1 (Prx 1) is an important antioxidant protein that can protect organisms against the toxicity of reactive oxygen species. In this study, a full-length Prx 1 cDNA sequence (ToPrx 1) was identified from golden pompano (Trachinotus ovatus). The ToPrx 1 cDNA was 1049 base pairs (bp) long and contained a 5'-untranslated region (UTR) of 127 nucleotides, a 3'-UTR of 328 nucleotides, and a 594 bp open reading frame (ORF) encoding a 197 amino acid polypeptide. The ToPrx 1 protein showed strong homology (79-91%) with Prx 1 proteins from other species and contained the conserved Prx domain and the signature of the peroxidase catalytic center. Phylogenetic analysis revealed that ToPrx 1 was in the fish Prx 1 subgroup, which suggests that ToPrx 1 could belong to the 2-Cys Prx subgroup. ToPrx 1 mRNA was ubiquitously detected in all tested tissues, and its expression was comparatively high in the fin, spleen, kidney, intestine, eye, gill, and blood. The expression levels of ToPrx 1 mRNA were significantly up-regulated in liver, spleen, kidney, and intestine of golden pompano injected with Photobacterium damselae. The recombinant ToPrx 1 protein (rToPrx 1) was expressed and purified through affinity chromatography and refolded successfully using ion-exchange chromatography. The antioxidant activity assay of rToPrx 1 showed that it could reduce insulin in the presence of dithiothreitol, which suggests that the antioxidant function of rToPrx 1 is thiol dependent. This study provides useful information to help further understand the functional mechanism of Prx 1 in marine fish immunity.

  19. The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences.

    PubMed

    Sautel, Céline F; Ortet, Philippe; Saksouk, Nehmé; Kieffer, Sylvie; Garin, Jérôme; Bastien, Olivier; Hakimi, Mohamed-Ali

    2009-01-01

    The ability of living cells to alter their gene expression patterns in response to environmental changes is essential for viability. Oxidative stress represents a common threat for all aerobic life. In normally growing cells, in which hydrogen peroxide generation is transient or pulsed, the antioxidant systems efficiently control its concentration. Intracellular parasites must also protect themselves against the oxidative burst imposed by the host. In this work, we have investigated the role of KMTox, a new histone lysine methyltransferase, in the obligate intracellular parasite Toxoplasma gondii. KMTox is a nuclear protein that holds a High Mobility Group domain, which is thought to recognize bent DNA. The enzyme methylates both histones H4 and H2A in vitro with a great preference for the substrate in reduced conditions. Importantly, KMTox interacts specifically with the typical 2-cys peroxiredoxin-1 and the binding is to some extent enhanced upon oxidation. It appears that the cellular functions that are primarily regulated by the KMTox are antioxidant defences and maintenance of cellular homeostasis. KMTox may regulate gene expression in T. gondii by providing the rapid re-arrangement of chromatin domains and by interacting with the redox-sensor TgPrx1 contribute to establish the antioxidant 'firewall' in T. gondii.

  20. Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2

    PubMed Central

    De Haes, Wouter; Frooninckx, Lotte; Van Assche, Roel; Smolders, Arne; Depuydt, Geert; Billen, Johan; Braeckman, Bart P.; Schoofs, Liliane; Temmerman, Liesbet

    2014-01-01

    The antiglycemic drug metformin, widely prescribed as first-line treatment of type II diabetes mellitus, has lifespan-extending properties. Precisely how this is achieved remains unclear. Via a quantitative proteomics approach using the model organism Caenorhabditis elegans, we gained molecular understanding of the physiological changes elicited by metformin exposure, including changes in branched-chain amino acid catabolism and cuticle maintenance. We show that metformin extends lifespan through the process of mitohormesis and propose a signaling cascade in which metformin-induced production of reactive oxygen species increases overall life expectancy. We further address an important issue in aging research, wherein so far, the key molecular link that translates the reactive oxygen species signal into a prolongevity cue remained elusive. We show that this beneficial signal of the mitohormetic pathway is propagated by the peroxiredoxin PRDX-2. Because of its evolutionary conservation, peroxiredoxin signaling might underlie a general principle of prolongevity signaling. PMID:24889636

  1. ZCF32, a fungus specific Zn(II)2 Cys6 transcription factor, is a repressor of the biofilm development in the human pathogen Candida albicans

    PubMed Central

    Kakade, Pallavi; Sadhale, Parag; Sanyal, Kaustuv; Nagaraja, Valakunja

    2016-01-01

    As a human fungal pathogen, Candida albicans can cause a wide variety of disease conditions ranging from superficial to systemic infections. Many of these infections are caused by an inherent ability of the pathogen to form biofilms on medical devices resulting in high mortality. Biofilms formed by C. albicans are a complex consortium of yeast and hyphal cells embedded in an extracellular matrix and are regulated by a network of transcription factors. Here, we report the role of a novel Zn(II)2-Cys6 binuclear cluster transcription factor, ZCF32, in the regulation of biofilm formation. Global transcriptome analysis reveals that biofilm development is the most altered pathway in the zcf32 null mutant. To delineate the functional correlation between ZCF32 and biofilm development, we determined the set of genes directly regulated by Zcf32. Our data suggests that Zcf32 regulates biofilm formation by repressing the expression of adhesins, chitinases and a significant number of other GPI-anchored proteins. We establish that there is the lesser recruitment of Zcf32 on the promoters of biofilm genes in biofilm condition compared to the planktonic mode of growth. Taking together, we propose that the transcription factor ZCF32 negatively regulates biofilm development in C. albicans. PMID:27498700

  2. The Fusarium verticillioides FUM Gene Cluster Encodes a Zn(II)2Cys6 Protein That Affects FUM Gene Expression and Fumonisin Production▿

    PubMed Central

    Brown, Daren W.; Butchko, Robert A. E.; Busman, Mark; Proctor, Robert H.

    2007-01-01

    Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Δfum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Δfum21 mutant with a wild-type copy of the gene restored fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis. PMID:17483290

  3. Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma.

    PubMed

    Trzeciecka, Anna; Klossowski, Szymon; Bajor, Malgorzata; Zagozdzon, Radoslaw; Gaj, Pawel; Muchowicz, Angelika; Malinowska, Agata; Czerwoniec, Anna; Barankiewicz, Joanna; Domagala, Antoni; Chlebowska, Justyna; Prochorec-Sobieszek, Monika; Winiarska, Magdalena; Ostaszewski, Ryszard; Gwizdalska, Iwonna; Golab, Jakub; Nowis, Dominika; Firczuk, Malgorzata

    2016-01-12

    Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease.

  4. Peroxiredoxin 1 is involved in disassembly of flagella and cilia.

    PubMed

    Gong, Fanghua; Liu, Hongtao; Li, Jie; Xue, Lexun; Zhang, Mingzhi

    2014-02-14

    Cilia/flagella are evolutionarily conserved cellular organelles. In this study, we demonstrated that Dunaliella salina Peroxiredoxin 1 (DsPrdx1) localized to the flagella and basal bodies, and was involved in flagellar disassembly. The link between DsPrdx1 and flagella of Dunaliella salina (D. salina) encouraged us to explore the function of its human homologue, Homo sapiens Peroxiredoxin 1 (HsPrdx1) in development and physiology. Our results showed that HsPrdx1 was overexpressed, and cilia were lost in esophageal squamous cell carcinoma (ESCC) cells compared with the non-cancerous esophageal epithelial cells Het-1A. Furthermore, when HsPrdx1 was knocked down by short hairpin RNA (shRNA) lentivirus in ESCC cells, the phenotype of cilia lost can be reversed, and the expression levels of tumor suppressor genes LKB1 and p-AMPK were increased, and the activity of the oncogene Aurora A was inhibited compared with those in cells transfected with scrambe-shRNA lentivirus. These findings firstly showed that Prdx1 is involved in disassembly of flagella and cilia, and suggested that the abnormal expression of the cilia-related gene including Prdx1 may affect both ciliogenesis and cancernogenesis.

  5. Engineering functional artificial hybrid proteins between poplar peroxiredoxin II and glutaredoxin or thioredoxin

    SciTech Connect

    Rouhier, Nicolas . E-mail: nrouhier@scbiol.uhp-nancy.fr; Gama, Filipe; Wingsle, Gunnar; Gelhaye, Eric; Gans, Pierre; Jacquot, Jean-Pierre

    2006-03-24

    The existence of natural peroxiredoxin-glutaredoxin hybrid enzymes in several bacteria is in line with previous findings indicating that poplar peroxiredoxin II can use glutaredoxin as an electron donor. This peroxiredoxin remains however unique since it also uses thioredoxin with a quite good efficiency. Based on the existing fusions, we have created artificial enzymes containing a poplar peroxiredoxin module linked to glutaredoxin or thioredoxin modules. The recombinant fusion enzymes folded properly into non-covalently bound homodimers or homotetramers. Two of the three protein constructs exhibit peroxidase activity, a reaction where the two modules need to function together, but they also display enzymatic activities specific of each module. In addition, mass spectrometry analyses indicate that the Prx module can be both glutathiolated or overoxidized in vitro. This is discussed in the light of the Prx reactivity.

  6. Hexameric oligomerization of mitochondrial peroxiredoxin PrxIIF and formation of an ultrahigh affinity complex with its electron donor thioredoxin Trx-o

    PubMed Central

    Barranco-Medina, Sergio; Krell, Tino; Bernier-Villamor, Laura; Sevilla, Francisca; Lázaro, Juan-José; Dietz, Karl-Josef

    2008-01-01

    Mitochondria from plants, yeast, and animals each contain at least one peroxiredoxin (Prx) that is involved in peroxide detoxification and redox signalling. The supramolecular dynamics of atypical type II Prx targeted to the mitochondrion was addressed in pea. Microcalorimetric (ITC) titrations identified an extremely high-affinity binding between the mitochondrial PsPrxIIF and Trx-o with a KD of 126±14 pM. Binding was driven by a favourable enthalpy change (ΔH= –60.6 kcal mol−1) which was counterbalanced by unfavourable entropy changes (TΔS= –47.1 kcal mol−1). This is consistent with the occurrence of large conformational changes during binding which was abolished upon site-directed mutaganesis of the catalytic C59S and C84S. The redox-dependent interaction was confirmed by gel filtration of mitochondrial extracts and co-immunoprecipitation from extracts. The heterocomplex of PsPrxIIF and Trx-o reduced peroxide substrates more efficiently than free PsPrxIIF suggesting that Trx-o serves as an efficient and specific electron donor to PsPrxIIF in vivo. Other Trx-s tested by ITC analysis failed to interact with PsPrxIIF indicating a specific recognition of PsPrxIIF by Trx-o. PsPrxIIF exists primarily as a dimer or a hexamer depending on the redox state. In addition to the well-characterized oligomerization of classical 2-Cys Prx the results also show that atypical Prx undergo large structural reorganization with implications for protein–protein interaction and function. PMID:18632730

  7. Essential role of the flexible linker on the conformational equilibrium of bacterial peroxiredoxin reductase for effective regeneration of peroxiredoxin.

    PubMed

    Kamariah, Neelagandan; Eisenhaber, Birgit; Eisenhaber, Frank; Gruber, Gerhard

    2017-03-07

    Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. AhpF is a flexible multi-domain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. A flexible linker connecting the N-terminal domain (NTD) and C-terminal domain (CTD) of AhpF suggests that the enzyme adopts a large-scale domain motion that alternates between the closed and open states to shuttle electrons from the CTD via the NTD to AhpC. Here, we conducted comprehensive mutational, biochemical, and biophysical analyses to gain insights into the role of the flexible linker and the residues critical for the domain motions of Escherichia coli AhpF (EcAhpF) during electron transfer. Small-angle X-ray scattering studies of linker mutants revealed that a group of charged residues, 200EKR202, is crucial for the swiveling motion of the NTD. Moreover, NADH binding significantly affected EcAhpF flexibility and the movement of the NTD relative to the CTD. The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. We propose that a concerted movement involving the NTD, C-terminal NADH and FAD domains, and the flexible linker between them is essential for optimal intra-domain crosstalk and for efficient electron transfer to the redox partner AhpC required for peroxidation.

  8. The pcz1 Gene, which Encodes a Zn(II)2Cys6 Protein, Is Involved in the Control of Growth, Conidiation, and Conidial Germination in the Filamentous Fungus Penicillium roqueforti

    PubMed Central

    Medina, Exequiel; Vaca, Inmaculada; García-Rico, Ramón O.; Villagrán, Sebastián; Levicán, Gloria; Chávez, Renato

    2015-01-01

    Proteins containing Zn(II)2Cys6 domains are exclusively found in fungi and yeasts. Genes encoding this class of proteins are broadly distributed in fungi, but few of them have been functionally characterized. In this work, we have characterized a gene from the filamentous fungus Penicillium roqueforti that encodes a Zn(II)2Cys6 protein, whose function to date remains unknown. We have named this gene pcz1. We showed that the expression of pcz1 is negatively regulated in a P. roqueforti strain containing a dominant active Gαi protein, suggesting that pcz1 encodes a downstream effector that is negatively controlled by Gαi. More interestingly, the silencing of pcz1 in P. roqueforti using RNAi-silencing technology resulted in decreased apical growth, the promotion of conidial germination (even in the absence of a carbon source), and the strong repression of conidiation, concomitant with the downregulation of the genes of the central conidiation pathway brlA, abaA and wetA. A model for the participation of pcz1 in these physiological processes in P. roqueforti is proposed. PMID:25811807

  9. Linking Peroxiredoxin and Vacuolar-ATPase Functions in Calorie Restriction-Mediated Life Span Extension.

    PubMed

    Molin, Mikael; Demir, Ayse Banu

    2014-01-01

    Calorie restriction (CR) is an intervention extending the life spans of many organisms. The mechanisms underlying CR-dependent retardation of aging are still poorly understood. Despite mechanisms involving conserved nutrient signaling pathways proposed, few target processes that can account for CR-mediated longevity have so far been identified. Recently, both peroxiredoxins and vacuolar-ATPases were reported to control CR-mediated retardation of aging downstream of conserved nutrient signaling pathways. In this review, we focus on peroxiredoxin-mediated stress-defence and vacuolar-ATPase regulated acidification and pinpoint common denominators between the two mechanisms proposed for how CR extends life span. Both the activities of peroxiredoxins and vacuolar-ATPases are stimulated upon CR through reduced activities in conserved nutrient signaling pathways and both seem to stimulate cellular resistance to peroxide-stress. However, whereas vacuolar-ATPases have recently been suggested to control both Ras-cAMP-PKA- and TORC1-mediated nutrient signaling, neither the physiological benefits of a proposed role for peroxiredoxins in H2O2-signaling nor downstream targets regulated are known. Both peroxiredoxins and vacuolar-ATPases do, however, impinge on mitochondrial iron-metabolism and further characterization of their impact on iron homeostasis and peroxide-resistance might therefore increase our understanding of the beneficial effects of CR on aging and age-related diseases.

  10. Inhibitory role of peroxiredoxin II (Prx II) on cellular senescence.

    PubMed

    Han, Ying-Hao; Kim, Hyun-Sun; Kim, Jin-Man; Kim, Sang-Keun; Yu, Dae-Yeul; Moon, Eun-Yi

    2005-08-29

    Reactive oxygen species (ROS) were generated in all oxygen-utilizing organisms. Peroxiredoxin II (Prx II) as one of antioxidant enzymes may play a protective role against the oxidative damage caused by ROS. In order to define the role of Prx II in organismal aging, we evaluated cellular senescence in Prx II(-/-) mouse embryonic fibroblast (MEF). As compared to wild type MEF, cellular senescence was accelerated in Prx II(-/-) MEF. Senescence-associated (SA)-beta-galactosidase (Gal)-positive cell formation was about 30% higher in Prx II(-/-) MEF. N-Acetyl-l-cysteine (NAC) treatment attenuated SA-beta-Gal-positive cell formation. Prx II(-/-) MEF exhibited the higher G2/M (41%) and lower S (1.6%) phase cells as compared to 24% and 7.3% [corrected] in wild type MEF, respectively. A high increase in the p16 and a slight increase in the p21 and p53 levels were detected in PrxII(-/-) MEF cells. The cellular senescence of Prx II(-/-) MEF was correlated with the organismal aging of Prx II(-/-) mouse skin. While extracellular signal-regulated kinase (ERK) and p38 activation was detected in Prx II(-/-) MEF, ERK and c-Jun N-terminal kinase (JNK) activation was detected in Prx II(-/-) skin. These results suggest that Prx II may function as an enzymatic antioxidant to prevent cellular senescence and skin aging.

  11. Chloramines and hypochlorous acid oxidize erythrocyte peroxiredoxin 2.

    PubMed

    Stacey, Melissa M; Peskin, Alexander V; Vissers, Margreet C; Winterbourn, Christine C

    2009-11-15

    Peroxiredoxin 2 (Prx2) is an abundant thiol protein that is readily oxidized in erythrocytes exposed to hydrogen peroxide. We investigated its reactivity in human erythrocytes with hypochlorous acid (HOCl) and chloramines, relevant oxidants in inflammation. Prx2 was oxidized to a disulfide-linked dimer by HOCl, glycine chloramine (GlyCl), and monochloramine (NH(2)Cl) in a dose-dependent manner. In the absence of added glucose, Prx2 and GSH showed similar sensitivities. Second-order rate constants for the reactions of Prx2 with NH(2)Cl and GlyCl were 1.5 x 10(4) and 8 M(-1) s(-1), respectively. The NH(2)Cl value is approximately 10 times higher than that for GSH, whereas Prx2 is approximately 30 times less sensitive than GSH to GlyCl. Thus, the relative sensitivity of Prx2 to GlyCl is greater in the erythrocyte. Oxidation of erythrocyte Prx2 and GSH was less in the presence of glucose, probably because of recycling. High doses of NH(2)Cl resulted in incomplete regeneration of reduced Prx2, suggesting impairment of the recycling mechanism. Our results show that, although HOCl and chloramines are less selective than H(2)O(2), they nevertheless oxidize Prx2. Exposure to these inflammatory oxidants will result in Prx2 oxidation and could compromise the erythrocyte's ability to resist damaging oxidative insult.

  12. Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane.

    PubMed

    Bayer, Simone B; Low, Felicia M; Hampton, Mark B; Winterbourn, Christine C

    2016-12-01

    Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H2O2, but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H2O2 or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.

  13. Prognostic significance of peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 in cholangiocarcinoma.

    PubMed

    Yonglitthipagon, Ponlapat; Pairojkul, Chawalit; Chamgramol, Yaovalux; Loukas, Alex; Mulvenna, Jason; Bethony, Jeffrey; Bhudhisawasdi, Vajarabhongsa; Sripa, Banchob

    2012-10-01

    We performed a comparative proteomic analysis of protein expression profiles in 4 cholangiocarcinoma cell lines: K100, M156, M213, and M139. The H69 biliary cell line was used as a control. Peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 were selected for further validation by immunohistochemistry using a cholangiocarcinoma tissue microarray (n = 301) to assess their prognostic value in this cancer. Both peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 were overexpressed in cholangiocarcinoma tissues compared with normal liver tissues. Of the 301 cholangiocarcinoma cases, overexpression of peroxiredoxin 1 in 103 (34.3%) was associated with an age-related effect in young patients (P = .011) and the absence of cholangiocarcinoma in lymphatic vessels and perineural tissues (P = .004 and P = .037, respectively). Expression of radixin-moesin-binding phosphoprotein 50 correlated with histopathologic type, with 180 (59.8%) of moderately or poorly differentiated tumors (P = .039) being higher, and was associated with the presence of cholangiocarcinoma in lymphatic and vascular vessels (P < .001 and P < .001, respectively). The high expression of radixin-moesin-binding phosphoprotein 50 and the low expression of peroxiredoxin 1 correlated with reduced survival by univariate analysis (P = .017 and P = .048, respectively). Moreover, the impact of peroxiredoxin 1 and radixin-moesin-binding phosphoprotein 50 expression on patient survival was an independent predictor in multivariate analyses (P = .004 and P = .025, respectively). Therefore, altered expression of peroxiredoxin 1 and radixin-moesin-binding phosphoprotein 50 may be used as prognostic markers in cholangiocarcinoma.

  14. Redox balance mechanisms in Schistosoma mansoni rely on peroxiredoxins and albumin and implicate peroxiredoxins as novel drug targets.

    PubMed

    Sayed, Ahmed A; Cook, Shawna K; Williams, David L

    2006-06-23

    Schistosoma mansoni, a causative agent of schistosomiasis, resides in the hepatic portal circulation of their human host up to 30 years without being eliminated by the host immune attack. Production of an antioxidant "firewall," which would neutralize the oxidative assault generated by host immune defenses, is one proposed survival mechanism of the parasite. Schistosomes lack catalase, the main H2O2-neutralizing enzyme of many organisms, and their glutathione peroxidases are in the phospholipid class with poor reactivity toward H2O2. Evidence implicates peroxiredoxins (Prx) as providing the main enzymatic activity to reduce H2O2 in the parasite. Quantitative monitoring of Prx mRNAs during parasite life cycle indicated that Prx proteins are differentially expressed, with highest expression occurring in adult stages (oxidative resistant stages). Incubation of schistosomula with Prx1 double-stranded RNA knocked down total Prx enzymatic activity and resulted in lowered survival of cultured parasites compared with controls demonstrating that Prx are essential parasite proteins. These results represent the first report of lethal gene silencing in Schistosoma. Investigation of downstream effects of Prx silencing revealed an abrupt increase of lipid peroxides and the generation of several oxidized proteins. Using mass spectrometry, parasite albumin and actin were identified as the main oxidized proteins. Gene expression analysis showed that schistosome albumin was induced by oxidative stress. This study highlights Prx proteins as essential parasite proteins and potential new targets for anti-schistosome drug development and albumin as a novel, sacrificial oxidant scavenging protein in parasite redox regulation.

  15. The multiple conformational charge states of zinc(II) coordination by 2His-2Cys oligopeptide investigated by ion mobility-mass spectrometry, density functional theory and theoretical collision cross sections.

    PubMed

    Wagoner, Stephanie M; Deeconda, Manogna; Cumpian, Kayleah L; Ortiz, Rafael; Chinthala, Swetha; Angel, Laurence A

    2016-12-01

    Whether traveling wave ion mobility-mass spectrometry (IM-MS), B3LYP/LanL2DZ density functional theory, and ion size scaled Lennard-Jones (LJ) collision cross sections (CCS) from the B3LYP optimized structures could be used to determine the type of Zn(II) coordination by the oligopeptide acetyl-His1 -Cys2 -Gly3 -Pro4 -Tyr5 -His6 -Cys7 (amb5 ) was investigated. The IM-MS analyses of a pH titration of molar equivalents of Zn(II):amb5 showed that both negatively and positively charged complexes formed and coordination of Zn(II) increased as the His and Cys deprotonated near their pKa values. The B3LYP method was used to generate a series of alternative coordination structures to compare with the experimental results. The method predicted that the single negatively charged complex coordinated Zn(II) in a distorted tetrahedral geometry via the 2His-2Cys substituent groups, whereas, the double negatively charged and positively charged complexes coordinated Zn(II) via His, carbonyl oxygens and the C-terminus. The CCS of the B3LYP complexes were calculated using the LJ method and compared with those measured by IM-MS for the various charge state complexes. The LJ method provided CCS that agreed with five of the alternative distorted tetrahedral and trigonal bipyramidal coordinations for the doubly charged complexes, but provided CCS that were 15 to 31 Å(2) larger than those measured by IM-MS for the singly charged complexes. Collision-induced dissociation of the Zn(II) complexes and a further pH titration study of amb5B , which included amidation of the C-terminus, suggested that the 2His-2Cys coordination was more significant than coordinations that included the C-terminus. Copyright © 2016 John Wiley & Sons, Ltd.

  16. The role of Peroxiredoxin 4 in inflammatory response and aging

    PubMed Central

    Klichko, Vladimir I.; Orr, William C.; Radyuk, Svetlana N.

    2015-01-01

    In prior studies, we determined that moderate overexpression of the Drosophila endoplasmic reticulum (ER)-localized peroxiredoxin (Prx), dPrx4, reduced oxidative damage and conferred beneficial effects on lifespan, while high level expression increased the incidence of tissue-specific apoptosis and dramatically shortened longevity. The detrimental pro-apoptotic and life-shortening effects were attributed to aberrant localization of dPrx4 and the apparent ER stress elicited by dPrx4 overexpression. In addition, activation of both the NF-κB- and JAK/STAT- mediated stress responses was detected, although it wasn’t clear whether these served as functional alarm signals. Here we extend these findings to show that activation of the NF-κB -dependent immunity-related/inflammatory genes, associated with lifespan shortening effects, is dependent on the activity of a Drosophila NF-κB ortholog, Relish. In the absence of Relish, the pro-inflammatory effects typically elicited by dPrx4 overexpression were not detected. The absence of Relish not only prevented hyperactivation of the immunity-related genes but also significantly rescued the severe shortening of lifespan normally observed in dPrx4 over-expressors. Overactivation of the immune/inflammatory responses was also lessened by JAK/STAT signaling. In addition we found that cellular immune/pro-inflammatory responses provoked by the oxidant paraquat but not bacteria are mediated via dPrx4 activity in the ER, as up-regulation of the immune-related genes was eliminated in flies underexpressing dPrx4 whereas immune responses triggered by bacteria were unaffected. Finally, efforts to reveal critical tissues where dPrx4 modulates longevity showed that broad targeting of dPrx4 to neuronal tissue had strong beneficial effects, while targeting expression to the fat body had deleterious effects. PMID:26689888

  17. Abolition of Peroxiredoxin-5 Mitochondrial Targeting during Canid Evolution

    PubMed Central

    Van der Eecken, Valérie; Clippe, André; Dekoninck, Sophie; Goemaere, Julie; Walbrecq, Geoffroy; Van Veldhoven, Paul P.; Knoops, Bernard

    2013-01-01

    In human, the subcellular targeting of peroxiredoxin-5 (PRDX5), a thioredoxin peroxidase, is dependent on the use of multiple alternative transcription start sites and two alternative in-frame translation initiation sites, which determine whether or not the region encoding a mitochondrial targeting sequence (MTS) is translated. In the present study, the abolition of PRDX5 mitochondrial targeting in dog is highlighted and the molecular mechanism underlying the loss of mitochondrial PRDX5 during evolution is examined. Here, we show that the absence of mitochondrial PRDX5 is generalized among the extant canids and that the first events leading to PRDX5 MTS abolition in canids involve a mutation in the more 5′ translation initiation codon as well as the appearance of a STOP codon. Furthermore, we found that PRDX5 MTS functionality is maintained in giant panda and northern elephant seal, which are phylogenetically closely related to canids. Also, the functional consequences of the restoration of mitochondrial PRDX5 in dog Madin-Darby canine kidney (MDCK) cells were investigated. The restoration of PRDX5 mitochondrial targeting in MDCK cells, instead of protecting, provokes deleterious effects following peroxide exposure independently of its peroxidase activity, indicating that mitochondrial PRDX5 gains cytotoxic properties under acute oxidative stress in MDCK cells. Altogether our results show that, although mitochondrial PRDX5 cytoprotective function against oxidative stress has been clearly demonstrated in human and rodents, PRDX5 targeting to mitochondria has been evolutionary lost in canids. Moreover, restoration of mitochondrial PRDX5 in dog MDCK cells, instead of conferring protection against peroxide exposure, makes them more vulnerable. PMID:24023783

  18. Peroxiredoxins: hidden players in the antioxidant defence of human spermatozoa.

    PubMed

    O'Flaherty, Cristian

    2014-01-01

    Spermatozoon is a cell with a precious message to deliver: the paternal DNA. Its motility machinery must be working perfectly and it should be able to acquire fertilizing ability in order to accomplish this mission. Infertility touches 1 in 6 couples worldwide and in half of the cases the causes can be traced to men. A variety of conditions such as infections of the male genital tract, varicocele, drugs, environmental factors, diseases, smoking, etc., are associated with male infertility and a common feature among them is the oxidative stress in semen that occurs when reactive oxygen species (ROS) are produced at high levels and/or when the antioxidant systems are decreased in the seminal plasma and/or spermatozoa. ROS-dependent damage targets proteins, lipids, and DNA, thus compromising sperm function and survival. Elevated ROS in spermatozoa are associated with DNA damage and decreased motility. Paradoxically, ROS, at very low levels, regulate sperm activation for fertilization. Therefore, the regulation of redox signaling in the male reproductive tract is essential for fertility. Peroxiredoxins (PRDXs) play a central role in redox signaling being both antioxidant enzymes and modulators of ROS action and are essential for pathological and physiological events. Recent studies from our lab emphasize the importance of PRDXs in the protection of spermatozoa as infertile men have significant low levels of PRDXs in semen and with little enzymatic activity available for ROS scavenging. The relationships between sperm DNA damage, motility and lipid peroxidation and high levels of thiol-oxidized PRDXs suggest the enhanced susceptibility of spermatozoa to oxidative stress and further support the importance of PRDXs in human sperm physiology. This review aims to characterize PRDXs, hidden players of the sperm antioxidant system and highlight the central role of PRDXs isoforms in the protection against oxidative stress to assure a proper function and DNA integrity of human

  19. Oxidation of archaeal peroxiredoxin involves a hypervalent sulfur intermediate

    PubMed Central

    Nakamura, Tsutomu; Yamamoto, Takahiko; Abe, Manabu; Matsumura, Hiroyoshi; Hagihara, Yoshihisa; Goto, Tadashi; Yamaguchi, Takafumi; Inoue, Tsuyoshi

    2008-01-01

    The oxidation of thiol groups in proteins is a common event in biochemical processes involving disulfide bond formation and in response to an increased level of reactive oxygen species. It has been widely accepted that the oxidation of a cysteine side chain is initiated by the formation of cysteine sulfenic acid (Cys-SOH). Here, we demonstrate a mechanism of thiol oxidation through a hypervalent sulfur intermediate by presenting crystallographic evidence from an archaeal peroxiredoxin (Prx), the thioredoxin peroxidase from Aeropyrum pernix K1 (ApTPx). The reaction of Prx, which is the reduction of a peroxide, depends on the redox active cysteine side chains. Oxidation by hydrogen peroxide converted the active site peroxidatic Cys-50 of ApTPx to a cysteine sulfenic acid derivative, followed by further oxidation to cysteine sulfinic and sulfonic acids. The crystal structure of the cysteine sulfenic acid derivative was refined to 1.77 Å resolution with Rcryst and Rfree values of 18.8% and 22.0%, respectively. The refined structure, together with quantum chemical calculations, revealed that the sulfenic acid derivative is a type of sulfurane, a hypervalent sulfur compound, and that the Sγ atom is covalently linked to the Nδ1 atom of the neighboring His-42. The reaction mechanism is revealed by the hydrogen bond network around the peroxidatic cysteine and the motion of the flexible loop covering the active site and by quantum chemical calculations. This study provides evidence that a hypervalent sulfur compound occupies an important position in biochemical processes. PMID:18436649

  20. Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes.

    PubMed

    Collins, John A; Wood, Scott T; Nelson, Kimberly J; Rowe, Meredith A; Carlson, Cathy S; Chubinskaya, Susan; Poole, Leslie B; Furdui, Cristina M; Loeser, Richard F

    2016-03-25

    Oxidative stress-mediated post-translational modifications of redox-sensitive proteins are postulated as a key mechanism underlying age-related cellular dysfunction and disease progression. Peroxiredoxins (PRX) are critical intracellular antioxidants that also regulate redox signaling events. Age-related osteoarthritis is a common form of arthritis that has been associated with mitochondrial dysfunction and oxidative stress. The objective of this study was to determine the effect of aging and oxidative stress on chondrocyte intracellular signaling, with a specific focus on oxidation of cytosolic PRX2 and mitochondrial PRX3. Menadione was used as a model to induce cellular oxidative stress. Compared with chondrocytes isolated from young adult humans, chondrocytes from older adults exhibited higher levels of PRX1-3 hyperoxidation basally and under conditions of oxidative stress. Peroxiredoxin hyperoxidation was associated with inhibition of pro-survival Akt signaling and stimulation of pro-death p38 signaling. These changes were prevented in cultured human chondrocytes by adenoviral expression of catalase targeted to the mitochondria (MCAT) and in cartilage explants from MCAT transgenic mice. Peroxiredoxin hyperoxidation was observedin situin human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism.

  1. Peroxiredoxins and tropomyosins as plasma biomarkers for lung cancer and asbestos exposure.

    PubMed

    Rostila, Annina; Puustinen, Anne; Toljamo, Tuula; Vuopala, Katri; Lindström, Irmeli; Nyman, Tuula A; Oksa, Panu; Vehmas, Tapio; Anttila, Sisko L

    2012-08-01

    The prognosis of lung cancer is poor due to late diagnosis, the lack of established screening programs, and the paucity of early biomarkers for high-risk populations. Plasma proteome analysis was used to identify novel biomarkers for diagnosing lung cancer, and to unravel the mechanisms of underlying pathogenesis. Plasma proteins obtained from asbestos-exposed lung cancer cases detected by CT screening, asbestos-exposed subjects, clinical lung cancer patients, and healthy tobacco smokers, 5-6 cases in each group, were separated by two-dimensional gel electrophoresis, and identified with tandem mass spectrometry (LC-MS/MS). Nine proteins were selected for immunological confirmation in a test or validation set of plasma samples from an additional 49 clinical lung cancer cases, 66 asbestos-exposed patients, and 107 healthy tobacco smokers. Twenty-eight unique proteins were differentially expressed between the four study groups (p<0.05). Peroxiredoxin 1 (PRX1) was detected as a novel plasma marker for lung cancer (p=0.001). We also confirmed the previously found association of serum amyloid A with lung cancer (p<0.001). High plasma levels of tropomyosin 4 (TPM4: p<0.001) and peroxiredoxins 1 and 2 (PRX2: p<0.001) correlated with asbestos exposure or a diagnosis of asbestosis. PRX1 and PRX2 exhibited an inverse correlation with tobacco smoking (p<0.001). Plasma peroxiredoxins 1 and 2, and tropomyosin 4 were shown to associate with asbestos-exposure, and peroxiredoxin 1 with lung cancer. High plasma levels of peroxiredoxin 1 may result from genetic damage caused by reactive oxygen species. This study has identified several biomarkers worthy of further investigation in lung cancer and asbestos-related diseases.

  2. [Xenopus laevis peroxiredoxins: Gene expression during development and characterization of the enzymes].

    PubMed

    Sharapov, M G; Novoselov, V I; Ravin, V K

    2016-01-01

    Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1-6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10-63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1-6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1-6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development.

  3. A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions

    PubMed Central

    Shimizu, Kiminori; Paul, Sanjoy; Ohba, Ayumi; Gonoi, Tohru; Watanabe, Akira; Gomi, Katsuya

    2017-01-01

    Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B). PMID:28052140

  4. Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in β-thalassemic erythropoiesis

    PubMed Central

    De Franceschi, Lucia; Bertoldi, Mariarita; De Falco, Luigia; Santos Franco, Sara; Ronzoni, Luisa; Turrini, Franco; Colancecco, Alessandra; Camaschella, Clara; Cappellini, Maria Domenica; Iolascon, Achille

    2011-01-01

    Background β-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to β-thalassemic anemia is only partially understood. Design and Methods We studied erythroid precursors from normal and β-thalassemic peripheral CD34+ cells in two-phase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of δ-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced peroxiredoxin-2 to H2O2 and generated a recombinant peroxiredoxin-2 for kinetic measurements in the presence of H2O2 or hemin. Results In β-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of peroxiredoxin-2. In agreement with these observations in β-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, δ-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant δ-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in β-thalassemic cells. We then addressed the question of the protective role of peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing peroxiredoxin-2 in human erythroleukemia K562 cells. We found that peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of peroxiredoxin-2 in β-thalassemia. Conclusions In β-thalassemic erythroid cells the reduction of

  5. The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents.

    PubMed

    Peskin, Alexander V; Low, Felicia M; Paton, Louise N; Maghzal, Ghassan J; Hampton, Mark B; Winterbourn, Christine C

    2007-04-20

    Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H(2)O(2). Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 x 10(7) M(-1) s(-1) for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.

  6. Functional analysis and expression characteristics of chloroplastic Prx IIE.

    PubMed

    Gama, Filipe; Bréhélin, Claire; Gelhaye, Eric; Meyer, Yves; Jacquot, Jean-Pierre; Rey, Pascal; Rouhier, Nicolas

    2008-07-01

    Peroxiredoxins (Prxs) are ubiquitous thiol-dependent peroxidases capable of eliminating a variety of peroxides through reactive catalytic cysteines, which are regenerated by reducing systems. Based on amino acid sequences and their mode of catalysis, five groups of thiol peroxidases have been distinguished in plants, and type II Prx is one of them with representatives in many sub-cellular compartments. The mature form of poplar chloroplastic Prx IIE was expressed as a recombinant protein in Escherichia coli. The protein is able to reduce H2O2 and tert-butyl hydroperoxide and is regenerated by both glutaredoxin (Grx) and thioredoxin (Trx) systems. Nevertheless, compared with Trxs, Grxs, and more especially chloroplastic Grx S12, are far more efficient reductants towards Prx IIE. The expression of Prx IIE at both the mRNA and protein levels as a function of organ type and abiotic stress conditions was investigated. Western blot analysis revealed that Prx IIE gene is constitutively expressed in Arabidopsis thaliana, mostly in young and mature leaves and in flowers. Under photo-oxidative treatment and water deficit, almost no change was observed in the abundance of Prx IIE in A. thaliana, while the level of Prx Q (one of the two other chloroplastic Prxs with 2-Cys Prx) increased in response to both stresses, indicating that plastidic members of the Prx family exhibit specific patterns of expression under stress.

  7. Targeting catalase but not peroxiredoxins enhances arsenic trioxide-induced apoptosis in K562 cells.

    PubMed

    Song, Li-Li; Tu, Yao-Yao; Xia, Li; Wang, Wei-Wei; Wei, Wei; Ma, Chun-Min; Wen, Dong-Hua; Lei, Hu; Xu, Han-Zhang; Wu, Ying-Li

    2014-01-01

    Despite considerable efficacy of arsenic trioxide (As2O3) in acute promyelocytic leukemia (APL) treatment, other non-APL leukemias, such as chronic myeloid leukemia (CML), are less sensitive to As2O3 treatment. However, the underlying mechanism is not well understood. Here we show that relative As2O3-resistant K562 cells have significantly lower ROS levels than As2O3-sensitive NB4 cells. We compared the expression of several antioxidant enzymes in these two cell lines and found that peroxiredoxin 1/2/6 and catalase are expressed at high levels in K562 cells. We further investigated the possible role of peroxirdoxin 1/2/6 and catalase in determining the cellular sensitivity to As2O3. Interestingly, knockdown of peroxiredoxin 1/2/6 did not increase the susceptibility of K562 cells to As2O3. On the contrary, knockdown of catalase markedly enhanced As2O3-induced apoptosis. In addition, we provide evidence that overexpression of BCR/ABL cannot increase the expression of PRDX 1/2/6 and catalase. The current study reveals that the functional role of antioxidant enzymes is cellular context and treatment agents dependent; targeting catalase may represent a novel strategy to improve the efficacy of As2O3 in CML treatment.

  8. Prokaryotic ancestry and gene fusion of a dual localized peroxiredoxin in malaria parasites

    PubMed Central

    Djuika, Carine F.; Huerta-Cepas, Jaime; Przyborski, Jude M.; Deil, Sophia; Sanchez, Cecilia P.; Doerks, Tobias; Bork, Peer; Lanzer, Michael; Deponte, Marcel

    2015-01-01

    Horizontal gene transfer has emerged as a crucial driving force for the evolution of eukaryotes. This also includes Plasmodium falciparum and related economically and clinically relevant apicomplexan parasites, whose rather small genomes have been shaped not only by natural selection in different host populations but also by horizontal gene transfer following endosymbiosis. However, there is rather little reliable data on horizontal gene transfer between animal hosts or bacteria and apicomplexan parasites. Here we show that apicomplexan homologues of peroxiredoxin 5 (Prx5) have a prokaryotic ancestry and therefore represent a special subclass of Prx5 isoforms in eukaryotes. Using two different immunobiochemical approaches, we found that the P. falciparum Prx5 homologue is dually localized to the parasite plastid and cytosol. This dual localization is reflected by a modular Plasmodium-specific gene architecture consisting of two exons. Despite the plastid localization, our phylogenetic analyses contradict an acquisition by secondary endosymbiosis and support a gene fusion event following a horizontal prokaryote-to-eukaryote gene transfer in early apicomplexans. The results provide unexpected insights into the evolution of apicomplexan parasites as well as the molecular evolution of peroxiredoxins, an important family of ubiquitous, usually highly concentrated thiol-dependent hydroperoxidases that exert functions as detoxifying enzymes, redox sensors and chaperones. PMID:28357258

  9. Lack of protective efficacy in buffaloes vaccinated with Fasciola gigantica leucine aminopeptidase and peroxiredoxin recombinant proteins.

    PubMed

    Raina, O K; Nagar, Gaurav; Varghese, Anju; Prajitha, G; Alex, Asha; Maharana, B R; Joshi, P

    2011-06-01

    Gene coding for leucine aminopeptidase (LAP), a metalloprotease, was identified in the tropical liver fluke, Fasciola gigantica; that on sequence analysis showed a close homology (98.6%) with leucine aminopeptidase of the temperate liver fluke, Fasciola hepatica. The recombinant leucine aminopeptidase protein was expressed in Escherichia coli. F. gigantica peroxiredoxin, a hydrogen peroxide scavenger and an immunomodulating protein, was also cloned and expressed in E. coli. A vaccination trial in buffaloes was conducted with these two recombinant proteins, with 150 and 300 μg of leucine aminopeptidase and a cocktail of 150 μg each of recombinant leucine aminopeptidase and peroxiredoxin in three groups, respectively. Both Th1- and Th2-associated humoral immune responses were elicited to immunization with these antigens. A challenge study with 400 metacercariae did not show a significant protection in terms of reduction in the worm burden (8.4%) or anti-fecundity/embryonation effect in the immunized groups, as to the non-immunized control animals. Our observations in this buffalo vaccination trial are contrary to the earlier promise shown by leucine aminopeptidase of F. hepatica as a leading candidate vaccine molecule. Identification of leucine aminopeptidase gene and evaluation of the protein for its protective efficacy in buffaloes is the first scientific report on this protein in F. gigantica.

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

    PubMed

    Matté, Alessandro; Pantaleo, Antonella; Ferru, Emanuela; Turrini, Franco; Bertoldi, Mariarita; Lupo, Francesca; Siciliano, Angela; Ho Zoon, Chae; De Franceschi, Lucia

    2014-11-01

    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.

  11. Quinone- and nitroreductase reactions of Thermotoga maritima peroxiredoxin-nitroreductase hybrid enzyme.

    PubMed

    Anusevičius, Žilvinas; Misevičienė, Lina; Šarlauskas, Jonas; Rouhier, Nicolas; Jacquot, Jean-Pierre; Čėnas, Narimantas

    2012-12-01

    Thermotoga maritima peroxiredoxin-nitroreductase hybrid enzyme (Prx-NR) consists of a FMN-containing nitroreductase (NR) domain fused to a peroxiredoxin (Prx) domain. These domains seem to function independently as no electron transfer occurs between them. The reduction of quinones and nitroaromatics by NR proceeded in a two-electron manner, and follows a 'ping-pong' scheme with sometimes pronounced inhibition by quinone substrate. The comparison of steady- and presteady-state kinetic data shows that in most cases, the oxidative half-reaction may be rate-limiting in the catalytic cycle of NR. The enzyme was inhibited by dicumarol, a classical inhibitor of oxygen-insensitive nitroreductases. The reduction of quinones and nitroaromatic compounds by Prx-NR was characterized by the linear dependence of their reactivity (logk(cat)/K(m)) on their single-electron reduction potentials E(7)(1), while the reactivity of quinones markedly exceeded the one with nitroaromatics. It shows that NR lacks the specificity for the particular structure of these oxidants, except their single-electron accepting potency and the rate of electron self-exchange. It points to the possibility of a single-electron transfer step in a net two-electron reduction of quinones and nitroaromatics by T. maritima Prx-NR, and to a significant diversity of the structures of flavoenzymes which may perform the two-electron reduction of quinones and nitroaromatics.

  12. Prdx1-encoded peroxiredoxin is important for vascular development in zebrafish.

    PubMed

    Huang, Po-Chun; Chiu, Chien-Chih; Chang, Hsueh-Wei; Wang, Yi-Shan; Syue, Hai-Hong; Song, Yi-Chun; Weng, Zhi-Hong; Tai, Ming-Hong; Wu, Chang-Yi

    2017-02-23

    Genetic signaling and redox homeostasis are required for proper growth of blood vessels. Here, we report a novel function of peroxiredoxin1 (Prdx1) in vascular development in zebrafish. Knockdown of prdx1 impairs the growth of intersegmental vessel (ISV) and caudal vein plexus (CVP), and reduces the expression of vascular markers, thus suggesting a role for prdx1 in vasculature and indicating that the antioxidant function of prdx1 is important. We found that H2 O2 -treated embryos also have CVP defects and observed synergistic effects when prdx1 knockdown was combined with H2 O2 treatment. Moreover, N-acetyl-cysteine (NAC) treatment rescues the vascular defects in prdx1 morphants. These results suggest that oxidative stress disturbs vascularization. Furthermore, we show that the regulation of prdx1 is mediated by Notch and BMP signals. This article is protected by copyright. All rights reserved.

  13. Dynamic Regulation of Ero1α and Peroxiredoxin 4 Localization in the Secretory Pathway*

    PubMed Central

    Kakihana, Taichi; Araki, Kazutaka; Vavassori, Stefano; Iemura, Shun-ichiro; Cortini, Margherita; Fagioli, Claudio; Natsume, Tohru; Sitia, Roberto; Nagata, Kazuhiro

    2013-01-01

    In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis. PMID:23979138

  14. Dynamic regulation of Ero1α and peroxiredoxin 4 localization in the secretory pathway.

    PubMed

    Kakihana, Taichi; Araki, Kazutaka; Vavassori, Stefano; Iemura, Shun-ichiro; Cortini, Margherita; Fagioli, Claudio; Natsume, Tohru; Sitia, Roberto; Nagata, Kazuhiro

    2013-10-11

    In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.

  15. Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

    PubMed Central

    Manevich, Yefim; Shuvaeva, Tea; Dodia, Chandra; Kazi, Altaf; Feinstein, Sheldon I.; Fisher, Aron B.

    2010-01-01

    Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A2 (PLA2) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA2 activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO2(3) antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes. PMID:19236840

  16. RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

    SciTech Connect

    Kim, Ji-Hee; Lee, Jeong-Mi; Lee, Hae Na; Kim, Eun-Kyung; Ha, Bin; Ahn, Sung-Min; Jang, Ho Hee; Lee, Sang Yeol

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer hPrx1 has RNA-binding properties. Black-Right-Pointing-Pointer hPrx1 exhibits helix-destabilizing activity. Black-Right-Pointing-Pointer Cold stress increases hPrx1 level in the nuclear fraction. Black-Right-Pointing-Pointer hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwound nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem-loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.

  17. Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention.

    PubMed

    Yang, Kai; Li, De-Feng; Wang, Xi'e; Liang, Jinzhao; Sitia, Roberto; Wang, Chih-Chen; Wang, Xi

    2016-10-04

    ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds the oxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes the essential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis.

  18. Redox-dependent Regulation of Gluconeogenesis by a Novel Mechanism Mediated by a Peroxidatic Cysteine of Peroxiredoxin

    PubMed Central

    Irokawa, Hayato; Tachibana, Tsuyoshi; Watanabe, Toshihiko; Matsuyama, Yuka; Motohashi, Hozumi; Ogasawara, Ayako; Iwai, Kenta; Naganuma, Akira; Kuge, Shusuke

    2016-01-01

    Peroxiredoxin is an abundant peroxidase, but its non-peroxidase function is also important. In this study, we discovered that Tsa1, a major peroxiredoxin of budding yeast cells, is required for the efficient flux of gluconeogenesis. We found that the suppression of pyruvate kinase (Pyk1) via the interaction with Tsa1 contributes in part to gluconeogenic enhancement. The physical interactions between Pyk1 and Tsa1 were augmented during the shift from glycolysis to gluconeogenesis. Intriguingly, a peroxidatic cysteine in the catalytic center of Tsa1 played an important role in the physical Tsa1-Pyk1 interactions. These interactions are enhanced by exogenous H2O2 and by endogenous reactive oxygen species, which is increased during gluconeogenesis. Only the peroxidatic cysteine, but no other catalytic cysteine of Tsa1, is required for efficient growth during the metabolic shift to obtain maximum yeast growth (biomass). This Tsa1 function is separable from the peroxidase function as an antioxidant. This is the first report to demonstrate that peroxiredoxin has a novel nonperoxidase function as a redox-dependent target modulator and that pyruvate kinase is modulated via an alternative mechanism. PMID:27634403

  19. Expression of peroxiredoxin I in plasma cells of oral inflammatory diseases.

    PubMed

    Demasi, Ana P D; Ceratti, Daniela; Furuse, Cristiane; Cury, Patrícia; Junqueira, José L C; Araújo, Vera C

    2007-08-01

    The participation of reactive oxygen species (ROS) in the immune response, both as pathogen killers and as mediators of signaling pathways, is well established. However, little is known about the enzymes responsible for ROS elimination in immune cells. Peroxiredoxin I (PrdxI) is a multifunctional enzyme that exhibits thioredoxin-dependent peroxidase activity. It has been described as a major hydrogen peroxide (H(2)O(2))-inducible protein in mouse peritoneal macrophages. In order to characterize its participation in the antioxidant defense of inflammatory/immune cells in greater detail, we evaluated its expression at sites of the oral cavity affected by inflammatory disorders induced by different agents (infectious, chemical, mechanical or tumor). In this study we demonstrated, by immunohistochemistry, that PrdxI is expressed in plasma cells, but not in B lymphocytes, regardless of the inflammation-inducing agent. We suggest that PrdxI induction could be considered a crucial part of the cellular adaptive response to the B-cell differentiation process to cope with the additional H(2)O(2) associated with massive disulfide bond formation during immunoglobulin folding in the endoplasmic reticulum of plasma cells. PrdxI could diminish the tissue damage that accompanies inflammation.

  20. Peroxiredoxin-3 Is Involved in Bactericidal Activity through the Regulation of Mitochondrial Reactive Oxygen Species

    PubMed Central

    Lee, Sena; Wi, Sae Mi; Min, Yoon

    2016-01-01

    Peroxiredoxin-3 (Prdx3) is a mitochondrial protein of the thioredoxin family of antioxidant peroxidases and is the principal peroxidase responsible for metabolizing mitochondrial hydrogen peroxide. Recent reports have shown that mitochondrial reactive oxygen species (mROS) contribute to macrophage-mediated bactericidal activity in response to Toll-like receptors. Herein, we investigated the functional effect of Prdx3 in bactericidal activity. The mitochondrial localization of Prdx3 in HEK293T cells was confirmed by cell fractionation and confocal microscopy analyses. To investigate the functional role of Prdx3 in bactericidal activity, Prdx3-knockdown (Prdx3KD) THP-1 cells were generated. The mROS levels in Prdx3KD THP-1 cells were significantly higher than those in control THP-1 cells. Moreover, the mROS levels were markedly increased in response to lipopolysaccharide. Notably, the Salmonella enterica serovar Typhimurium infection assay revealed that the Prdx3KD THP-1 cells were significantly resistant to S. Typhimurium infection, as compared with control THP-1 cells. Taken together, these results indicate that Prdx3 is functionally important in bactericidal activity through the regulation of mROS. PMID:28035213

  1. T lymphocytes and dendritic cells are activated by the deletion of peroxiredoxin II (Prx II) gene.

    PubMed

    Moon, Eun-Yi; Noh, Young-Wook; Han, Ying-Hao; Kim, Sun-Uk; Kim, Jin-Man; Yu, Dae-Yeul; Lim, Jong-Seok

    2006-02-15

    Peroxiredoxin II (Prx II) is a member of antioxidant enzyme family and it plays a protective role against oxidative damage. Constitutive production of endogenous reactive oxygen species was detected in spleen and bone marrow cells lacking Prx II. Here, we investigated the role of Prx II in immune responses. The total number of splenocytes (especially, the population of S-phase cells and CD3(+) T cells) was significantly higher in Prx II(-/-) mice than in wild type. Number of peripheral blood mononuclear cells (PBMCs) in Prx II(-/-) mice was also higher than wild type. Differentiation of Prx II(-/-) mouse bone marrow cells into CD11c-positive dendritic cells was greater than that of wild type. Transplantation of Prx II(-/-) bone marrow cells into wild type mice increased PBMCs in blood and bone marrow-derived dendritic cells. Prx II deletion enhances concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction (MLR) activity of bone marrow-derived CD11c-positive dendritic cells to stimulate recipient splenocytes. Collectively, these data suggest that Prx II inhibits the immune cell responsiveness, which may be regulated by scavenging the low amount of reactive oxygen species (ROS).

  2. Secretory expression and characterization of a novel peroxiredoxin for zearalenone detoxification in Saccharomyces cerevisiae.

    PubMed

    Tang, Yuqian; Xiao, Junmei; Chen, Yi; Yu, Yigang; Xiao, Xinglong; Yu, Yuanshan; Wu, Hui

    2013-01-15

    Zearalenone (ZEN) is a Fusarium mycotoxin, which is considered to be an oestrogenic endocrine disruptor found to cause severe morphological and functional disorders of reproductive organs in livestock. Increasing attention has been paid to the development of an effective strategy for ZEN decontamination. ZEN is oxidized into smaller estrogenic metabolites by a novel peroxiredoxin (Prx) isolated from Acinetobacter sp. SM04. The Prx coding gene was cloned in a secretory vector pYES2-alpha (pYα) with an alpha (α) signal peptide gene inserted into the multiple cloning site of pYES2. The recombinant Prx was secreted from Saccharomyces cerevisiae INVSc1 after inducing with 2% (w/v) galactose for 72 h, and was found to be nearly 20 kDa through 12% SDS-PAGE. The expressed amount of recombinant Prx was 0.24 mg/mL in the extracellular supernatant. Recombinant Prx showed a gradient increase at the beginning of ZEN degradation. The final ZEN degradation amount was 0.43 μg by one unit recombinant Prx after 12 h. Furthermore, the temperature, H(2)O(2) concentration, and pH for highest peroxidase activity of recombinant Prx were 80°C, 20 mM and 9.0, respectively. When compared with other peroxidases, the thermal stability and alkali resistance of recombinant Prx were much better. The results suggest that recombinant Prx is successfully expressed in S. cerevisiae.

  3. Quantitative HDL Proteomics Identifies Peroxiredoxin-6 as a Biomarker of Human Abdominal Aortic Aneurysm

    PubMed Central

    Burillo, Elena; Jorge, Inmaculada; Martínez-López, Diego; Camafeita, Emilio; Blanco-Colio, Luis Miguel; Trevisan-Herraz, Marco; Ezkurdia, Iakes; Egido, Jesús; Michel, Jean-Baptiste; Meilhac, Olivier; Vázquez, Jesús; Martin-Ventura, Jose Luis

    2016-01-01

    High-density lipoproteins (HDLs) are complex protein and lipid assemblies whose composition is known to change in diverse pathological situations. Analysis of the HDL proteome can thus provide insight into the main mechanisms underlying abdominal aortic aneurysm (AAA) and potentially detect novel systemic biomarkers. We performed a multiplexed quantitative proteomics analysis of HDLs isolated from plasma of AAA patients (N = 14) and control study participants (N = 7). Validation was performed by western-blot (HDL), immunohistochemistry (tissue), and ELISA (plasma). HDL from AAA patients showed elevated expression of peroxiredoxin-6 (PRDX6), HLA class I histocompatibility antigen (HLA-I), retinol-binding protein 4, and paraoxonase/arylesterase 1 (PON1), whereas α-2 macroglobulin and C4b-binding protein were decreased. The main pathways associated with HDL alterations in AAA were oxidative stress and immune-inflammatory responses. In AAA tissue, PRDX6 colocalized with neutrophils, vascular smooth muscle cells, and lipid oxidation. Moreover, plasma PRDX6 was higher in AAA (N = 47) than in controls (N = 27), reflecting increased systemic oxidative stress. Finally, a positive correlation was recorded between PRDX6 and AAA diameter. The analysis of the HDL proteome demonstrates that redox imbalance is a major mechanism in AAA, identifying the antioxidant PRDX6 as a novel systemic biomarker of AAA. PMID:27934969

  4. Identification of H7 as a novel peroxiredoxin I inhibitor to induce differentiation of leukemia cells

    PubMed Central

    Qin, Dongjun; Chen, Yingyi; Liu, Chuanxu; Xia, Li; Wang, Tongdan; Lei, Hu; Yu, Yun; Huang, Min; Tong, Yin; Xu, Hanzhang; Gao, Fenghou

    2016-01-01

    Identifying novel targets to enhance leukemia-cell differentiation is an urgent requirment. We have recently proposed that inhibiting the antioxidant enzyme peroxiredoxin I (Prdx I) may induce leukemia-cell differentiation. However, this concept remains to be confirmed. In this work, we identified H7 as a novel Prdx I inhibitor through virtual screening, in vitro activity assay, and surface plasmon resonance assay. Cellular thermal shift assay showed that H7 directly bound to Prdx I but not to Prdxs II–V in cells. H7 treatment also increased reactive oxygen species (ROS) level and cell differentiation in leukemia cells, as reflected by the upregulation of the cell surface differentiation marker CD11b/CD14 and the morphological maturation of cells. The differentiation-induction effect of H7 was further observed in some non-acute promyelocytic leukemia (APL) and primary leukemia cells apart from APL NB4 cells. Moreover, the ROS scavenger N-acetyl cysteine significantly reversed the H7-induced cell differentiation. We demonstrated as well that H7-induced cell differentiation was associated with the activation of the ROS-Erk1/2-C/EBPβ axis. Finally, we showed H7 treatment induced cell differentiation in an APL mouse model. All of these data confirmed that Prdx I was novel target for inducing leukemia-cell differentiation and that H7 was a novel lead compound for optimizing Prdx I inhibition. PMID:26716647

  5. Cigarette smoke extract inhibits expression of peroxiredoxin V and increases airway epithelial permeability.

    PubMed

    Serikov, Vladimir B; Leutenegger, Christian; Krutilina, Raisa; Kropotov, Andrei; Pleskach, Nadezhda; Suh, Jung H; Tomilin, Nikolay V

    2006-01-01

    Inhaled cigarette smoke induces oxidative stress in the epithelium of airways. Peroxiredoxin V (PRXV) is a potent antioxidant protein, highly expressed in cells of the airway epithelium. The goal of our study was to determine whether cigarette smoke extract (CSE) influenced expression of this protein in airway epithelia in vivo and in vitro. In Sprague-Dawley rats, we determined effects of CSE on airway epithelial permeability, mRNA levels and expression of PRXV protein. Exposure of isolated tracheal segment in vitro to 20% CSE for 4 h resulted in development of increased permeability to albumin, significantly reduced mRNA levels for PRXV, and reduced amounts of PRXV protein in the epithelium. In cultures of the airway epithelial cell lines (Calu-3, JME), primary airway cell culture (cow), and alveolar epithelial cells A549, CSE also significantly decreased transepithelial electrical resistance and expression of PRXV protein, and induced glutathione and protein oxidation. To demonstrate functional importance of PRXV, we exposed clones of HeLa cells with siRNA-downregulated PRXV to hydrogen peroxide, which resulted in increased rate of cell death and protein oxidation. CSE directly downregulates expression of functionally important antioxidant enzyme PRXV in the epithelial cells of airways, which represents one pathophysiological mechanism of cigarette smoke toxicity.

  6. Peroxiredoxin 2 functions as a noncatalytic scavenger of low-level hydrogen peroxide in the erythrocyte.

    PubMed

    Low, Felicia M; Hampton, Mark B; Peskin, Alexander V; Winterbourn, Christine C

    2007-03-15

    Peroxiredoxin 2 (Prx2), a thiol-dependent peroxidase, is the third most abundant protein in the erythrocyte, and its absence in knock-out mice gives rise to hemolytic anemia. We have found that in human erythrocytes, Prx2 was extremely sensitive to oxidation by H(2)O(2), as dimerization was observed after exposure of 5 x 10(6) cells/mL to 0.5 muM H(2)O(2). In contrast to Prx2 in Jurkat T lymphocytes, Prx2 was resistant to overoxidation (oxidation of the cysteine thiol to a sulfinic/sulfonic acid) in erythrocytes. Reduction of dimerized Prx2 in the erythrocyte occurred very slowly, with reversal occurring gradually over a 20-minute period. Very low thioredoxin reductase activity was detected in hemolysates. We postulate that this limits the rate of Prx2 regeneration, and this inefficiency in recycling prevents the overoxidation of Prx2. We also found that Prx2 was oxidized by endogenously generated H(2)O(2), which was mainly derived from hemoglobin autoxidation. Our results demonstrate that in the erythrocyte Prx2 is extremely efficient at scavenging H(2)O(2) noncatalytically. Although it does not act as a classical antioxidant enzyme, its high concentration and substrate sensitivity enable it to handle low H(2)O(2) concentrations efficiently. These unique redox properties may account for its nonredundant role in erythrocyte defense against oxidative stress.

  7. Nitration Transforms a Sensitive Peroxiredoxin 2 into a More Active and Robust Peroxidase*

    PubMed Central

    Randall, Lía M.; Manta, Bruno; Hugo, Martín; Gil, Magdalena; Batthyàny, Carlos; Trujillo, Madia; Poole, Leslie B.; Denicola, Ana

    2014-01-01

    Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H2O2-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H2O2 accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling. PMID:24719319

  8. Thiol peroxiredoxin, a novel allergen from Bombyx mori, modulates functions of macrophages and dendritic cells

    PubMed Central

    Wang, Hui; Hu, Wei; Liang, Zhiling; zeng, Lu; Li, Jianjie; Yan, Hao; Yang, Pingchang; Liu, Zhigang; Wang, Lianglu

    2016-01-01

    Bombyx mori (B.mori, also known as silkworm) plays a role in the pathogenesis of allergic diseases. However, its allergens are to be characterized. The aim of this paper is to identify new silkworm allergens. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry were employed to separate and identify potential allergens from silkworm pupa. Six potential allergens were identified in this study. Thiol peroxiredoxin (TP), one of the 6 allergens, reacted to serum IgE from patients sensitized to silkworm. By sensitizing with TP allergic asthma like symptoms were induced in mice, including elevation of the levels of serum IgE, IL-4 from bronchoalveolar lavage fluid and culture supernatant of spleen cells. In vitro experiments showed that TP significantly induced RAW264.7 cells (a macrophage cell line) apoptosis via modulating the BCL2 and Caspase9 pathways. The levels of CD80, CD40, CD83 and TNF-α in DC2.4 cells (a dendritic cell line) were increased in the culture after exposure to TP. In summary, TP is an allergic component of silkworm. It induces allergic asthma, and modulates the functions of macrophages and dendritic cells. PMID:28078005

  9. Hydrogen Peroxide Modifies Human Sperm Peroxiredoxins in a Dose-Dependent Manner1

    PubMed Central

    O'Flaherty, Cristian; Rico de Souza, Angela

    2010-01-01

    Low levels of reactive oxygen species (ROS) modulate signaling pathways required for human sperm activation, but high levels impair sperm function, leading to infertility. Peroxiredoxins (PRDXs) are enzymes with a dual role as ROS scavengers and modulators of ROS-dependent signaling. The present study aimed to characterize PRDXs in human spermatozoa and possible modifications resulting from hydrogen peroxide (H2O2). We found PRDX1, PRDX4, PRDX5, and PRDX6 in both seminal plasma and spermatozoa. Using immunocytochemistry, we demonstrated that these PRDXs are differentially localized in the head, acrosome, mitochondrial sheath, and flagellum. These observations were confirmed by immunoblotting using cytosolic, Triton-soluble and -insoluble, and head and flagella sperm fractions. PRDXs are dose-dependently modified by H2O2, as seen by the formation of disulfide bridges and high-molecular-mass complexes. This first study, to our knowledge, on PRDXs in human spermatozoa indicates that PRDX1, PRDX4, PRDX5, and PRDX6 are modified when spermatozoa are challenged with H2O2. This suggests that PRDXs may protect these cells at high levels of H2O2 but could also control H2O2 levels within different cell compartments so that normal sperm activation can occur. PMID:20864641

  10. Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling.

    PubMed

    Sobotta, Mirko C; Liou, Willy; Stöcker, Sarah; Talwar, Deepti; Oehler, Michael; Ruppert, Thomas; Scharf, Annette N D; Dick, Tobias P

    2015-01-01

    Hydrogen peroxide (H(2)O(2)) acts as a signaling messenger by oxidatively modifying distinct cysteinyl thiols in distinct target proteins. However, it remains unclear how redox-regulated proteins, which often have low intrinsic reactivity towards H(2)O(2) (k(app) ∼1-10 M(-1) s(-1)), can be specifically and efficiently oxidized by H(2)O(2). Moreover, cellular thiol peroxidases, which are highly abundant and efficient H(2)O(2) scavengers, should effectively eliminate virtually all of the H(2)O(2) produced in the cell. Here, we show that the thiol peroxidase peroxiredoxin-2 (Prx2), one of the most H(2)O(2)-reactive proteins in the cell (k(app) ∼10(7)-10(8) M(-1) s(-1)), acts as a H(2)O(2) signal receptor and transmitter in transcription factor redox regulation. Prx2 forms a redox relay with the transcription factor STAT3 in which oxidative equivalents flow from Prx2 to STAT3. The redox relay generates disulfide-linked STAT3 oligomers with attenuated transcriptional activity. Cytokine-induced STAT3 signaling is accompanied by Prx2 and STAT3 oxidation and is modulated by Prx2 expression levels.

  11. Revisiting sulfur H-bonds in proteins: The example of peroxiredoxin AhpE

    PubMed Central

    van Bergen, Laura A. H.; Alonso, Mercedes; Palló, Anna; Nilsson, Lennart; De Proft, Frank; Messens, Joris

    2016-01-01

    In many established methods, identification of hydrogen bonds (H-bonds) is primarily based on pairwise comparison of distances between atoms. These methods often give rise to systematic errors when sulfur is involved. A more accurate method is the non-covalent interaction index, which determines the strength of the H-bonds based on the associated electron density and its gradient. We applied the NCI index on the active site of a single-cysteine peroxiredoxin. We found a different sulfur hydrogen-bonding network to that typically found by established methods, and we propose a more accurate equation for determining sulfur H-bonds based on geometrical criteria. This new algorithm will be implemented in the next release of the widely-used CHARMM program (version 41b), and will be particularly useful for analyzing water molecule-mediated H-bonds involving different atom types. Furthermore, based on the identification of the weakest sulfur-water H-bond, the location of hydrogen peroxide for the nucleophilic attack by the cysteine sulfur can be predicted. In general, current methods to determine H-bonds will need to be reevaluated, thereby leading to better understanding of the catalytic mechanisms in which sulfur chemistry is involved. PMID:27468924

  12. Quantitative HDL Proteomics Identifies Peroxiredoxin-6 as a Biomarker of Human Abdominal Aortic Aneurysm.

    PubMed

    Burillo, Elena; Jorge, Inmaculada; Martínez-López, Diego; Camafeita, Emilio; Blanco-Colio, Luis Miguel; Trevisan-Herraz, Marco; Ezkurdia, Iakes; Egido, Jesús; Michel, Jean-Baptiste; Meilhac, Olivier; Vázquez, Jesús; Martin-Ventura, Jose Luis

    2016-12-09

    High-density lipoproteins (HDLs) are complex protein and lipid assemblies whose composition is known to change in diverse pathological situations. Analysis of the HDL proteome can thus provide insight into the main mechanisms underlying abdominal aortic aneurysm (AAA) and potentially detect novel systemic biomarkers. We performed a multiplexed quantitative proteomics analysis of HDLs isolated from plasma of AAA patients (N = 14) and control study participants (N = 7). Validation was performed by western-blot (HDL), immunohistochemistry (tissue), and ELISA (plasma). HDL from AAA patients showed elevated expression of peroxiredoxin-6 (PRDX6), HLA class I histocompatibility antigen (HLA-I), retinol-binding protein 4, and paraoxonase/arylesterase 1 (PON1), whereas α-2 macroglobulin and C4b-binding protein were decreased. The main pathways associated with HDL alterations in AAA were oxidative stress and immune-inflammatory responses. In AAA tissue, PRDX6 colocalized with neutrophils, vascular smooth muscle cells, and lipid oxidation. Moreover, plasma PRDX6 was higher in AAA (N = 47) than in controls (N = 27), reflecting increased systemic oxidative stress. Finally, a positive correlation was recorded between PRDX6 and AAA diameter. The analysis of the HDL proteome demonstrates that redox imbalance is a major mechanism in AAA, identifying the antioxidant PRDX6 as a novel systemic biomarker of AAA.

  13. Peroxiredoxin isoforms are associated with cardiovascular risk factors in type 2 diabetes mellitus.

    PubMed

    El Eter, E; Al-Masri, A A

    2015-05-01

    The production of oxygen free radicals in type 2 diabetes mellitus contributes to the development of complications, especially the cardiovascular-related ones. Peroxiredoxins (PRDXs) are antioxidant enzymes that combat oxidative stress. The aim of this study was to investigate the associations between the levels of PRDX isoforms (1, 2, 4, and 6) and cardiovascular risk factors in type 2 diabetes mellitus. Fifty-three patients with type 2 diabetes mellitus (28F/25M) and 25 healthy control subjects (7F/18M) were enrolled. We measured the plasma levels of each PRDX isoform and analyzed their correlations with cardiovascular risk factors. The plasma PRDX1, -2, -4, and -6 levels were higher in the diabetic patients than in the healthy control subjects. PRDX2 and -6 levels were negatively correlated with diastolic blood pressure, fasting blood sugar, and hemoglobin A1c. In contrast, PRDX1 levels were positively correlated with low-density lipoprotein and C-reactive protein levels. PRDX4 levels were negatively correlated with triglycerides. In conclusion, PRDX1, -2, -4, and -6 showed differential correlations with a variety of traditional cardiovascular risk factors. These results should encourage further research into the crosstalk between PRDX isoforms and cardiovascular risk factors.

  14. Nitration transforms a sensitive peroxiredoxin 2 into a more active and robust peroxidase.

    PubMed

    Randall, Lía M; Manta, Bruno; Hugo, Martín; Gil, Magdalena; Batthyàny, Carlos; Trujillo, Madia; Poole, Leslie B; Denicola, Ana

    2014-05-30

    Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H2O2-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H2O2 accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.

  15. Diagnostic performance of peroxiredoxin 1 to determine time-of-onset of acute cerebral infarction

    PubMed Central

    Richard, Sébastien; Lapierre, Vanessa; Girerd, Nicolas; Bonnerot, Mathieu; Burkhard, Pierre R.; Lagerstedt, Linnéa; Bracard, Serge; Debouverie, Marc; Turck, Natacha; Sanchez, Jean-Charles

    2016-01-01

    Accurately determining time-of-onset of cerebral infarction is important to clearly identify patients who could benefit from reperfusion therapies. We assessed the kinetics of peroxiredoxin 1 (PRDX1), a protein involved in oxidative stress during the acute phase of ischemia, and its ability to determine stroke onset in a population of patients with known onset of less than 24 hours and in a control group. Median PRDX1 levels were significantly higher in stroke patients compared to controls. PRDX1 levels were also higher from blood samples withdrawn before vs. after 3 hours following stroke onset, and before vs. after 6 hours. ROC analysis with area under the curve (AUC), sensitivity (Se) and specificity (Sp) determined from the Youden index was performed to assess the ability of PRDX1 levels to determine onset. Diagnostic performances of PRDX1 levels were defined by an AUC of 69%, Se of 53% and Sp of 86% for identifying cerebral infarction occurring <3 hours, and an AUC of 68%, Se of 49% and Sp of 88% for cerebral infarction occurring <6 hours. These first results suggest that PRDX1 levels could be the basis of a new method using biomarkers for determining cerebral infarction onset. PMID:27924073

  16. Inhibition of lung tumor growth and augmentation of radiosensitivity by decreasing peroxiredoxin I expression

    SciTech Connect

    Chen, M.-F.; Keng, Peter C.; Shau Hungyi; Wu, C.-T.; Hu, Y.-C.; Liao, S.-K.; Chen, W.-C. . E-mail: miaofen@adm.cgmh.org.tw

    2006-02-01

    Purpose: In this study, we examined the role of peroxiredoxin I (Prx I) in lung cancer cell growth in vitro and in vivo and its influence on these tumor cells' sensitivity to radiotherapy. Methods and materials: We established stable transfectants of A549 (p53+) and H1299 (p53-) lung carcinoma cell lines with Prx I antisense to downregulate their Prx I protein. We then examined their in vitro biologic changes and used nude mice xenografts of these cell lines to compare tumor invasion, spontaneous metastatic capacity, and sensitivity to radiotherapy. Results: The Prx I antisense transfectants of both cell lines showed a significant reduction in Prx I protein production. Prx I antisense transfectants grew more slowly than did the wild type. As xenografts in mice, A549 Prx I antisense transfectants showed a threefold delay in the generation of palpable tumors. The incidence of spontaneous metastasis of Prx I antisense transfectants was significantly less than that of the wild-type cells. Furthermore, irradiation of Prx I antisense transfectants caused more than twice the growth delay compared with the wild type. Conclusion: The results of these studies suggest that inactivation of Prx I may be a promising approach to improve the treatment outcome of patients with lung cancer.

  17. Peroxiredoxin isoforms are associated with cardiovascular risk factors in type 2 diabetes mellitus

    PubMed Central

    El Eter, E.; Al-Masri, A.A.

    2015-01-01

    The production of oxygen free radicals in type 2 diabetes mellitus contributes to the development of complications, especially the cardiovascular-related ones. Peroxiredoxins (PRDXs) are antioxidant enzymes that combat oxidative stress. The aim of this study was to investigate the associations between the levels of PRDX isoforms (1, 2, 4, and 6) and cardiovascular risk factors in type 2 diabetes mellitus. Fifty-three patients with type 2 diabetes mellitus (28F/25M) and 25 healthy control subjects (7F/18M) were enrolled. We measured the plasma levels of each PRDX isoform and analyzed their correlations with cardiovascular risk factors. The plasma PRDX1, -2, -4, and -6 levels were higher in the diabetic patients than in the healthy control subjects. PRDX2 and -6 levels were negatively correlated with diastolic blood pressure, fasting blood sugar, and hemoglobin A1c. In contrast, PRDX1 levels were positively correlated with low-density lipoprotein and C-reactive protein levels. PRDX4 levels were negatively correlated with triglycerides. In conclusion, PRDX1, -2, -4, and -6 showed differential correlations with a variety of traditional cardiovascular risk factors. These results should encourage further research into the crosstalk between PRDX isoforms and cardiovascular risk factors. PMID:25742636

  18. Overproduction, purification, crystallization and preliminary X-ray analysis of the peroxiredoxin domain of a larger natural hybrid protein from Thermotoga maritima

    SciTech Connect

    Barbey, Carole; Rouhier, Nicolas; Haouz, Ahmed; Navaza, Alda; Jacquot, Jean-Pierre

    2008-01-01

    Crystals of the peroxiredoxin domain of a larger natural hybrid protein from T. maritima were obtained which diffracted to 2.9 Å resolution on a synchrotron source. Thermotoga maritima contains a natural hybrid protein constituted of two moieties: a peroxiredoxin domain at the N-terminus and a nitroreductase domain at the C-terminus. The peroxiredoxin (Prx) domain has been overproduced and purified from Escherichia coli cells. The recombinant Prx domain, which is homologous to bacterial Prx BCP and plant Prx Q, folds properly into a stable protein that possesses biological activity. The recombinant protein was crystallized and synchrotron data were collected to 2.9 Å resolution. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 176.67, c = 141.20 Å.

  19. Expression of Cytosolic Peroxiredoxins in Plasmodium berghei Ookinetes Is Regulated by Environmental Factors in the Mosquito Bloodmeal

    PubMed Central

    Tasch, James J.; Zalewski, Angelika; Kooistra, Rachel; Schroeter, Eric H.; Sharma, Sapna; Kawazu, Shin-Ichiro; Kanzok, Stefan M.

    2013-01-01

    The Plasmodium ookinete develops over several hours in the bloodmeal of its mosquito vector where it is exposed to exogenous stresses, including cytotoxic reactive oxygen species (ROS). How the parasite adapts to these challenging conditions is not well understood. We have systematically investigated the expression of three cytosolic antioxidant proteins, thioredoxin-1 (Trx-1), peroxiredoxin-1 (TPx-1), and 1-Cys peroxiredoxin (1-Cys Prx), in developing ookinetes of the rodent parasite Plasmodium berghei under various growth conditions. Transcriptional profiling showed that tpx-1 and 1-cys prx but not trx-1 are more strongly upregulated in ookinetes developing in the mosquito bloodmeal when compared to ookinetes growing under culture conditions. Confocal immunofluorescence imaging revealed comparable expression patterns on the corresponding proteins. 1-Cys Prx in particular exhibited strong expression in mosquito-derived ookinetes but was not detectable in cultured ookinetes. Furthermore, ookinetes growing in culture upregulated tpx-1 and 1-cys prx when challenged with exogenous ROS in a dose-dependent fashion. This suggests that environmental factors in the mosquito bloodmeal induce upregulation of cytosolic antioxidant proteins in Plasmodium ookinetes. We found that in a parasite line lacking TPx-1 (TPx-1KO), expression of 1-Cys Prx occurred significantly earlier in mosquito-derived TPx-1KO ookinetes when compared to wild type (WT) ookinetes. The protein was also readily detectable in cultured TPx-1KO ookinetes, indicating that 1-Cys Prx at least in part compensates for the loss of TPx-1 in vivo. We hypothesize that this dynamic expression of the cytosolic peroxiredoxins reflects the capacity of the developing Plasmodium ookinete to rapidly adapt to the changing conditions in the mosquito bloodmeal. This would significantly increase its chances of survival, maturation and subsequent escape. Our results also emphasize that environmental conditions must be taken

  20. Integration between anticipatory blocking and redox signaling by the peroxiredoxin/thioredoxin/thioredoxin-reductase system.

    PubMed

    Selvaggio, Gianluca; Coelho, Pedro M B M; Salvador, Armindo

    2014-10-01

    Cells are occasionally exposed to high H2O2 concentrations, often preceding exposure to other electrophylic compounds. Both H2O2 and these compounds can irreversibly modify protein thiols, with deleterious consequences. Induction of enzymatic defenses against those agents is too slow to avoid significant damage. Cells may solve this conundrum by reversibly "blocking" the thiols once H2O2 concentrations begin to increase. We term this mechanism "anticipatory blocking" because it acts in anticipation of irreversible damage upon detection of early signs of stress. Here we examine the design requirements for the Peroxiredoxin/Thioredoxin/Thioredoxin-Reductase/Protein-Dithiol System (PTTRDS) to effectively integrate H2O2 signaling and anticipatory blocking of protein dithiols as disulfides, and we compared them to the designs found in cells. To that effect, we developed a minimal model of the PTTRDS, and we defined a set of quantitative performance criteria that embody the requirements for (a) efficient scavenging capacity, (b) low NADPH consumption, (c) effective signal propagation, and (d) effective anticipatory blocking. We then sought the design principles (relationships among rate constants and species concentrations) that warrant fulfillment of all these criteria. Experimental data indicates that the design of the PTTRDS in human erythrocytes fulfills these principles and thus accomplishes effective integration between anticipatory blocking, antioxidant protection and redox signaling. A more general analysis suggests that the same principles hold in a wide variety of cell types and organisms. We acknowledge grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) financed by FEDER through the "Programa Operacional Factores de Competitividade, COMPETE" and by national funds through "FCT, Fundação para a Ciência e a Tecnologia".

  1. Mitochondrial Peroxiredoxin-3 protects against hyperglycemia induced myocardial damage in Diabetic cardiomyopathy.

    PubMed

    Arkat, Silpa; Umbarkar, Prachi; Singh, Sarojini; Sitasawad, Sandhya L

    2016-08-01

    Mitochondrial oxidative stress has emerged as a key contributor towards the development of diabetic cardiomyopathy. Peroxiredoxin-3 (Prx-3), a mitochondrial antioxidant, scavenges H2O2 and offers protection against ROS related pathologies. We observed a decrease in the expression of Prx-3 in the hearts of streptozotocin (STZ) induced diabetic rats, and also high glucose treated H9c2 cardiac cells, which may augment oxidative stress mediated damage. Hence we hypothesized that overexpression of Prx-3 could prevent the cardiac damage associated with diabetes. In this study we used quercetin (QUE) to achieve Prx-3 induction in vivo, while a Prx-3 overexpressing H9c2 cell line was employed for carrying out in vitro studies. Diabetes was induced in Wistar rats by a single intraperitoneal injection of STZ. Quercetin (50mg/kg body weight) was delivered orally to hyperglycemic and age matched control rats for 2 months. Quercetin treatment induced the myocardial expression of Prx-3 but not Prx-5 both in control and STZ rats. Prx-3 induction by quercetin prevented diabetes induced oxidative stress as confirmed by decrease in expression of markers such as 4-HNE and mitochondrial uncoupling protein, UCP-3. It was also successful in reducing cardiac cell apoptosis, hypertrophy and fibrosis leading to amelioration of cardiac contractility defects. Overexpression of Prx-3 in cultured H9c2 cardiac cells could significantly diminish high glucose inflicted mitochondrial oxidative damage and apoptosis, thus strengthening our hypothesis. These results suggest that diabetes induced cardiomyopathy can be prevented by elevating Prx-3 levels thereby providing extensive protection to the diabetic heart.

  2. Peroxiredoxin II Is Essential for Maintaining Stemness by Redox Regulation in Liver Cancer Cells.

    PubMed

    Kwon, Taeho; Bak, Yesol; Park, Young-Ho; Jang, Gyu-Beom; Nam, Jeong-Seok; Yoo, Jeong Eun; Park, Young Nyun; Bak, In Seon; Kim, Jin-Man; Yoon, Do-Young; Yu, Dae-Yeul

    2016-05-01

    Redox regulation in cancer stem cells (CSCs) is viewed as a good target for cancer therapy because redox status plays an important role in cancer stem-cell maintenance. Here, we investigated the role of Peroxiredoxin II (Prx II), an antioxidant enzyme, in association with maintenance of liver CSCs. Our study demonstrates that Prx II overexpressed in liver cancer cells has high potential for self-renewal activity. Prx II expression significantly corelated with expression of epithelial-cell adhesion molecules (EpCAM) and cytokerain 19 in liver cancer tissues of hepatocellular carcinoma (HCC) patients. Downregulation of Prx II in Huh7 cells with treatment of siRNA reduced expression of EpCAM and CD133 as well as Sox2 in accordance with increased ROS and apoptosis, which were reversed in Huh7-hPrx II cells. Huh7-hPrx II cells exhibited strong sphere-formation activity compared with mock cells. Vascular endothelial growth factor (VEGF) exposure enhanced sphere formation, cell-surface expression of EpCAM and CD133, and pSTAT3 along with activation of VEGF receptor 2 in Huh7-hPrx II cells. The result also emerged in Huh7-H-ras(G12V) and SK-HEP-1-H-ras(G12V) cells with high-level expression of Prx II. Prx II was involved in regulation of VEGF driving cancer stem cells through VEGFR-2/STAT3 signaling to upregulate Bmi1 and Sox2. In addition, knockdown of Prx II in Huh7-H-ras(G12V) cells showed significant reduction in cell migration in vitro and in tumorigenic potential in vivo. Taken together, all the results demonstrated that Prx II plays a key role in the CSC self-renewal of HCC cells through redox regulation. Stem Cells 2016;34:1188-1197.

  3. NADH oxidase and alkyl hydroperoxide reductase subunit C (peroxiredoxin) from Amphibacillus xylanus form an oligomeric assembly.

    PubMed

    Arai, Toshiaki; Kimata, Shinya; Mochizuki, Daichi; Hara, Keita; Zako, Tamotsu; Odaka, Masafumi; Yohda, Masafumi; Arisaka, Fumio; Kanamaru, Shuji; Matsumoto, Takashi; Yajima, Shunsuke; Sato, Junichi; Kawasaki, Shinji; Niimura, Youichi

    2015-01-01

    The NADH oxidase-peroxiredoxin (Prx) system of Amphibacillus xylanus reduces hydroperoxides with the highest turnover rate among the known hydroperoxide-scavenging enzymes. The high electron transfer rate suggests that there exists close interaction between NADH oxidase and Prx. Variant enzyme experiments indicated that the electrons from β-NADH passed through the secondary disulfide, Cys128-Cys131, of NADH oxidase to finally reduce Prx. We previously reported that ionic strength is essential for a system to reduce hydroperoxides. In this study, we analyzed the effects of ammonium sulfate (AS) on the interaction between NADH oxidase and Prx by surface plasmon resonance analysis. The interaction between NADH oxidase and Prx was observed in the presence of AS. Dynamic light scattering assays were conducted while altering the concentration of AS and the ratio of NADH oxidase to Prx in the solutions. The results revealed that the two proteins formed a large oligomeric assembly, the size of which depended on the ionic strength of AS. The molecular mass of the assembly converged at approximately 300 kDa above 240 mM AS. The observed reduction rate of hydrogen peroxide also converged at the same concentration of AS, indicating that a complex formation is required for activation of the enzyme system. That the complex generation is dependent on ionic strength was confirmed by ultracentrifugal analysis, which resulted in a signal peak derived from a complex of NADH oxidase and Prx (300 mM AS, NADH oxidase: Prx = 1:10). The complex formation under this condition was also confirmed structurally by small-angle X-ray scattering.

  4. Peroxiredoxin I is important for cancer-cell survival in Ras-induced hepatic tumorigenesis.

    PubMed

    Han, Bing; Shin, Hye-Jun; Bak, In Seon; Bak, Yesol; Jeong, Ye-Lin; Kwon, Taeho; Park, Young-Ho; Sun, Hu-Nan; Kim, Cheol-Hee; Yu, Dae-Yeul

    2016-10-18

    Peroxiredoxin I (Prx I), an antioxidant enzyme, has multiple functions in human cancer. However, the role of Prx I in hepatic tumorigenesis has not been characterized. Here we investigated the relevance and underlying mechanism of Prx I in hepatic tumorigenesis. Prx I increased in tumors of hepatocellular carcinoma (HCC) patients that aligned with overexpression of oncogenic H-ras. Prx I also increased in H-rasG12V transfected HCC cells and liver tumors of H-rasG12V transgenic (Tg) mice, indicating that Prx I may be involved in Ras-induced hepatic tumorigenesis. When Prx I was knocked down or deleted in HCC-H-rasG12V cells or H-rasG12V Tg mice, cell colony or tumor formation was significantly reduced that was associated with downregulation of pERK pathway as well as increased intracellular reactive oxygen species (ROS) induced DNA damage and cell death. Overexpressing Prx I markedly increased Ras downstream pERK/FoxM1/Nrf2 signaling pathway and inhibited oxidative damage in HCC cells and H-rasG12V Tg mice. In this study, we found Nrf2 was transcriptionally activated by FoxM1, and Prx I was activated by the H-rasG12V/pERK/FoxM1/Nrf2 pathway and suppressed ROS-induced hepatic cancer-cell death along with formation of a positive feedback loop with Ras/ERK/FoxM1/Nrf2 to promote hepatic tumorigenesis.

  5. Peroxiredoxin 3 is a redox-dependent target of thiostrepton in malignant mesothelioma cells.

    PubMed

    Newick, Kheng; Cunniff, Brian; Preston, Kelsey; Held, Paul; Arbiser, Jack; Pass, Harvey; Mossman, Brooke; Shukla, Arti; Heintz, Nicholas

    2012-01-01

    Thiostrepton (TS) is a thiazole antibiotic that inhibits expression of FOXM1, an oncogenic transcription factor required for cell cycle progression and resistance to oncogene-induced oxidative stress. The mechanism of action of TS is unclear and strategies that enhance TS activity will improve its therapeutic potential. Analysis of human tumor specimens showed FOXM1 is broadly expressed in malignant mesothelioma (MM), an intractable tumor associated with asbestos exposure. The mechanism of action of TS was investigated in a cell culture model of human MM. As for other tumor cell types, TS inhibited expression of FOXM1 in MM cells in a dose-dependent manner. Suppression of FOXM1 expression and coincidental activation of ERK1/2 by TS were abrogated by pre-incubation of cells with the antioxidant N-acetyl-L-cysteine (NAC), indicating its mechanism of action in MM cells is redox-dependent. Examination of the mitochondrial thioredoxin reductase 2 (TR2)-thioredoxin 2 (TRX2)-peroxiredoxin 3 (PRX3) antioxidant network revealed that TS modifies the electrophoretic mobility of PRX3. Incubation of recombinant human PRX3 with TS in vitro also resulted in PRX3 with altered electrophoretic mobility. The cellular and recombinant species of modified PRX3 were resistant to dithiothreitol and SDS and suppressed by NAC, indicating that TS covalently adducts cysteine residues in PRX3. Reduction of endogenous mitochondrial TRX2 levels by the cationic triphenylmethane gentian violet (GV) promoted modification of PRX3 by TS and significantly enhanced its cytotoxic activity. Our results indicate TS covalently adducts PRX3, thereby disabling a major mitochondrial antioxidant network that counters chronic mitochondrial oxidative stress. Redox-active compounds like GV that modify the TR2/TRX2 network may significantly enhance the efficacy of TS, thereby providing a combinatorial approach for exploiting redox-dependent perturbations in mitochondrial function as a therapeutic approach in

  6. An unexplored role for Peroxiredoxin in exercise-induced redox signalling?

    PubMed Central

    Wadley, Alex J.; Aldred, Sarah; Coles, Steven J.

    2015-01-01

    Peroxiredoxin (PRDX) is a ubiquitous oxidoreductase protein with a conserved ionised thiol that permits catalysis of hydrogen peroxide (H2O2) up to a million times faster than any thiol-containing signalling protein. The increased production of H2O2 within active tissues during exercise is thought to oxidise conserved cysteine thiols, which may in turn facilitate a wide variety of physiological adaptations. The precise mechanisms linking H2O2 with the oxidation of signalling thiol proteins (phosphates, kinases and transcription factors) are unclear due to these proteins' low reactivity with H2O2 relative to abundant thiol peroxidases such as PRDX. Recent work has shown that following exposure to H2O2 in vitro, the sulfenic acid of the PRDX cysteine can form mixed disulphides with transcription factors associated with cell survival. This implicates PRDX as an ‘active’ redox relay in transmitting the oxidising equivalent of H2O2 to downstream proteins. Furthermore, under oxidative stress, PRDX can form stable oxidised dimers that can be secreted into the extracellular space, potentially acting as an extracellular ‘stress’ signal. There is extensive literature assessing non-specific markers of oxidative stress in response to exercise, however the PRDX catalytic cycle may offer a more robust approach for measuring changes in redox balance following exercise. This review discusses studies assessing PRDX-mediated cellular signalling and integrates the recent advances in redox biology with investigations that have examined the role of PRDX during exercise in humans and animals. Future studies should explore the role of PRDX as a key regulator of peroxide mediated-signal transduction during exercise in humans. PMID:26748042

  7. The biological importance of glutathione peroxidase and peroxiredoxin backup systems in bivalves during peroxide exposure.

    PubMed

    Trevisan, Rafael; Mello, Danielle Ferraz; Uliano-Silva, Marcela; Delapedra, Gabriel; Arl, Miriam; Dafre, Alcir Luiz

    2014-10-01

    Organic peroxide elimination in eukaryotes essentially depends on glutathione peroxidase (GPx) and peroxiredoxin (Prx) enzymes, which are supported by their respective electron donors, glutathione (GSH) and thioredoxin (Trx). This system depends on the ancillary enzymes glutathione reductase (GR) and thioredoxin reductase (TrxR) to maintain GSH and Trx in their reduced state. This study discusses the biological importance of GR and TrxR in supporting GPx and Prx during cumene hydroperoxide (CHP) exposure in brown mussel Perna perna. ZnCl2 or 1-chloro-2,4-dinitrobenze (CDNB) was used to decrease GR and TrxR activities in gills, as already reported with mammals and bivalves. ZnCl2 exposure lowered GR activity (28%), impaired the in vivo CHP decomposition and decreased the survival rates under CHP exposure. CDNB decreased GR (54%) and TrxR (73%) activities and induced glutathione depletion (99%), promoting diminished peroxide elimination and survival rates at a greater extent than ZnCl2. CDNB also increased the susceptibility of hemocytes to CHP toxicity. Despite being toxic and causing mortality at longer exposures, short (2 h) exposure to CHP promoted an up regulation of GSH (50 and 100 μM CHP) and protein-thiol (100 μM CHP) levels, which was blocked by ZnCl2 or CDNB pre-exposure. Results highlight the biological importance of GSH, GR and TrxR in supporting GPx and Prx activities, contributing to organic peroxides elimination and mussel survival under oxidative challenges. To our knowledge, this is the first work that demonstrates, albeit indirectly, the biological importance of GPx/GR/GSH and Prx/TrxR/Trx systems on in vivo organic peroxide elimination in bivalves.

  8. Effect of peroxiredoxin II on the quality and mitochondrial activity of pre-implantation bovine embryos.

    PubMed

    Fakruzzaman, Md; Ghanem, Nasser; Bang, Jae-Il; Ha, A-Na; Lee, Kyeong-Lim; Sohn, Sea-Hwan; Wang, Zhongde; Lee, Dong-Seok; Kong, Il-Keun

    2015-08-01

    Endogenous peroxiredoxin II (PRDX II) protein plays a vital role in early embryonic development. This study assessed the beneficial effects of exogenous PRDX II on bovine embryo development at the cellular and molecular levels. To this end, in vitro maturation (IVM) medium was supplemented with various concentrations of PRDX II (0, 6.25, 12.5, 25, 50, and 100μg/mL). Of these, 12.5μg/mL PRDX II was the most effective and significantly promoted embryonic development. Therefore, this concentration of PRDX II was used in subsequent experiments. The percentage of embryos that developed into Day 8 blastocysts and the total number of cells per blastocyst (38.2% and 150.6±5.1) was higher in the PRDX II-treated group than in the control (26.4% and 128.9±3.9, respectively). Moreover, the percent of TUNEL positive cells was higher (P<0.05) in the control than in the PRDX II-treated. Furthermore, PRDX II added to the IVM media increased mitochondria content in blastocysts and decreased the intracellular ROS levels in oocytes and blastocysts compared with the control (P<0.05). The expression of genes associated with blastocyst quality (CDX2 and IFNτ), antioxidant activity (SOD2), and mitochondrial activity (TFAM) was higher, whereas the expression of a gene involved in the apoptotic pathway (c-FOS) was lower, in the PRDX II-treated than in the control group. In conclusion, supplementation of IVM medium with PRDX II promotes development to the blastocyst stage and improves blastocyst quality through reducing ROS, enhancing embryonic mitochondrial activity, and modulating development- related target genes expression.

  9. Mycothiol/mycoredoxin 1-dependent reduction of the peroxiredoxin AhpE from Mycobacterium tuberculosis.

    PubMed

    Hugo, Martín; Van Laer, Koen; Reyes, Aníbal M; Vertommen, Didier; Messens, Joris; Radi, Rafael; Trujillo, Madia

    2014-02-21

    Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H2O2-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.

  10. Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination

    PubMed Central

    Song, In-Kang; Lee, Jae-Jin; Cho, Jin-Hwan; Jeong, Jihye; Shin, Dong-Hae; Lee, Kong-Joo

    2016-01-01

    Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1. PMID:27703196

  11. Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination

    NASA Astrophysics Data System (ADS)

    Song, In-Kang; Lee, Jae-Jin; Cho, Jin-Hwan; Jeong, Jihye; Shin, Dong-Hae; Lee, Kong-Joo

    2016-10-01

    Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1.

  12. Dual role of the active-center cysteine in human peroxiredoxin 1: Peroxidase activity and heme binding.

    PubMed

    Watanabe, Yuta; Ishimori, Koichiro; Uchida, Takeshi

    2017-02-12

    HBP23, a 23-kDa heme-binding protein identified in rats, is a member of the peroxiredoxin (Prx) family, the primary peroxidases involved in hydrogen peroxide catabolism. Although HBP23 has a characteristic Cys-Pro heme-binding motif, the significance of heme binding to Prx family proteins remains to be elucidated. Here, we examined the effect of heme binding to human peroxiredoxin-1 (PRX1), which has 97% amino acid identity to HBP23. PRX1 was expressed in Escherichia coli and purified to homogeneity. Spectroscopic titration demonstrated that PRX1 binds heme with a 1:1 stoichiometry and a dissociation constant of 0.17 μM. UV-vis spectra of heme-PRX1 suggested that Cys52 is the axial ligand of ferric heme. PRX1 peroxidase activity was lost upon heme binding, reflecting the fact that Cys52 is not only the heme-binding site but also the active center of peroxidase activity. Interestingly, heme binding to PRX1 caused a decrease in the toxicity and degradation of heme, significantly suppressing H2O2-dependent heme peroxidase activity and degradation of PRX1-bound heme compared with that of free hemin. By virtue of its cytosolic abundance (∼20 μM), PRX1 thus functions as a scavenger of cytosolic hemin (<1 μM). Collectively, our results indicate that PRX1 has a dual role; Cys-dependent peroxidase activity and cytosolic heme scavenger.

  13. The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus.

    PubMed

    Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B; Bzymek, Krzysztof P; Williams, John C; Brakhage, Axel A; Kalkum, Markus

    2016-09-14

    Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target.

  14. The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus

    PubMed Central

    Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B.; Bzymek, Krzysztof P.; Williams, John C.; Brakhage, Axel A.; Kalkum, Markus

    2016-01-01

    Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3’s peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target. PMID:27624005

  15. Peroxiredoxin II promotes hepatic tumorigenesis through cooperation with Ras/Forkhead box M1 signaling pathway.

    PubMed

    Park, Y-H; Kim, S-U; Kwon, T-H; Kim, J-M; Song, I-S; Shin, H-J; Lee, B-K; Bang, D-H; Lee, S-J; Lee, D-S; Chang, K-T; Kim, B-Y; Yu, D-Y

    2016-07-07

    The current study was carried out to define the involvement of Peroxiredoxin (Prx) II in progression of hepatocellular carcinoma (HCC) and the underlying molecular mechanism(s). Expression and function of Prx II in HCC was determined using H-ras(G12V)-transformed HCC cells (H-ras(G12V)-HCC cells) and the tumor livers from H-ras(G12V)-transgenic (Tg) mice and HCC patients. Prx II was upregulated in H-ras(G12V)-HCC cells and H-ras(G12V)-Tg mouse tumor livers, the expression pattern of which highly similar to that of forkhead Box M1 (FoxM1). Moreover, either knockdown of FoxM1 or site-directed mutagenesis of FoxM1-binding site of Prx II promoter significantly reduced Prx II levels in H-ras(G12V)-HCC cells, indicating FoxM1 as a direct transcription factor of Prx II in HCC. Interestingly, the null mutation of Prx II markedly decreased the number and size of tumors in H-ras(G12V)-Tg livers. Consistent with this, knockdown of Prx II in H-ras(G12V)-HCC cells reduced the expression of cyclin D1, cell proliferation, anchorage-independent growth and tumor formation in athymic nude mice, whereas overexpression of Prx II increased or aggravated the tumor phenotypes. Importantly, the expression of Prx II was correlated with that of FoxM1 in HCC patients. The activation of extracellular signal-related kinase (ERK) pathway and the expression of FoxM1 and cyclin D1 were highly dependent on Prx II in H-ras(G12V)-HCC cells and H-ras(G12V)-Tg livers. Prx II is FoxM1-dependently-expressed antioxidant in HCC and function as an enhancer of Ras(G12V) oncogenic potential in hepatic tumorigenesis through activation of ERK/FoxM1/cyclin D1 cascade.

  16. A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway.

    PubMed

    Kim, So Yong; Kim, Tae Jin; Lee, Ki-Young

    2008-06-11

    We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.

  17. Peroxiredoxin I deficiency attenuates phagocytic capacity of macrophage in clearance of the red blood cells damaged by oxidative stress.

    PubMed

    Han, Ying-Hao; Kwon, Taeho; Kim, Sun-Uk; Ha, Hye-Lin; Lee, Tae-Hoon; Kim, Jin-Man; Jo, Eun-Kyeong; Kim, Bo Yeon; Yoon, Do Young; Yu, Dae-Yeul

    2012-10-01

    The role of peroxiredoxin (Prx) I as an erythrocyte antioxidant defense in red blood cells (RBCs) is controversial. Here we investigated the function of Prx I by using Prx I(-/-) and Prx I/II(-/-) mice. Prx I(-/-) mice exhibited a normal blood profile. However, Prx I/II(-/-) mice showed more significantly increased Heinz body formation as compared with Prx II(-/-) mice. The clearance rate of Heinz body-containing RBCs in Prx I(-/-) mice decreased significantly through the treatment of aniline hydrochloride (AH) compared with wild-type mice. Prx I deficiency decreased the phagocytic capacity of macrophage in clearing Heinz body-containing RBCs. Our data demonstrate that Prx I deficiency did not cause hemolytic anemia, but showed that further increased hemolytic anemia symptoms in Prx II(-/-) mice by attenuating phagocytic capacity of macrophage in oxidative stress damaged RBCs, suggesting a novel role of Prx I in phagocytosis of macrophage.

  18. The effect of peroxiredoxin 4 on fly physiology is a complex interplay of antioxidant and signaling functions

    PubMed Central

    Radyuk, Svetlana N.; Klichko, Vladimir I.; Michalak, Katarzyna; Orr, William C.

    2013-01-01

    Peroxiredoxin 4 (Prx4) has been implicated in a wide variety of biological processes, including development, progression of cancer, inflammation, and antioxidant function. The purpose of this study was to provide further insight into its multiple roles at the whole-animal level, using Drosophila. Reduced expression of dPrx4 (up to 90%) resulted in greater sensitivity to oxidative stress, an elevated H2O2 flux, and increases in lipid peroxidation, but no effect on longevity. Overexpression at low levels (<2-fold) gave reduced levels of oxidative damage and tended to show an increase in longevity. Flies expressing dPrx4 globally at high levels (>5-fold) had a dramatically reduced life span (by 20–80%) and increased apoptosis. Analysis of these overexpressors revealed an aberrant redistribution of the dPrx4 protein from the endoplasmic reticulum (ER) to cytosol and hemolymph. In addition to the known proapoptotic effects of the cytosolic form of dPrx4, dPrx4 overexpression triggered an NF-κB-mediated proinflammatory response, similar to that observed in cells under ER stress or when microbially challenged. Finally, we provide the first evidence that dPrx4, on secretion into the hemolymph, elicits a JAK/STAT-mediated response. The effects on fly survival and homeostasis appear to represent a combination of differential effects dictated in large part by dPrx4 subcellular and tissue-specific localization.—Radyuk, S. N., Klichko, V. I., Michalak, K., Orr, W. C. The effect of peroxiredoxin 4 on fly physiology is a complex interplay of antioxidant and signaling functions. PMID:23271054

  19. Peroxiredoxin-6 Negatively Regulates Bactericidal Activity and NF-κB Activity by Interrupting TRAF6-ECSIT Complex

    PubMed Central

    Min, Yoon; Wi, Sae M.; Shin, Dongwoo; Chun, Eunyoung; Lee, Ki-Young

    2017-01-01

    A TRAF6-ECSIT complex is crucial for the generation of mitochondrial reactive oxygen species (mROS) and nuclear factor-kappa B (NF-κB) activation induced by Toll-like receptor 4 (TLR4). Peroxiredoxin-6 (Prdx6) as a member of the peroxiredoxin family of antioxidant enzymes is involved in antioxidant protection and cell signaling. Here, we report on a regulatory role of Prdx6 in mROS production and NF-κB activation by TLR4. Prdx6 was translocated into the mitochondria by TLR4 stimulation and Prdx6-knockdown (Prdx6KD) THP-1 cells had increased level of mitochondrial reactive oxygen species levels and were resistant to Salmonella typhimurium infection. Biochemical studies revealed Prdx6 interaction with the C-terminal TRAF-C domain of TRAF6, which drove translocation into the mitochondria. Interestingly, Prdx6 competitively interacted with ECSIT to TRAF6 through its C-terminal TRAF-C domain, leading to the interruption of TRAF6-ECSIT interaction. The inhibitory effect was critically implicated in the activation of NF-κB induced by TLR4. Overexpression of Prdx6 led to the inhibition of NF-κB induced by TLR4, whereas Prdx6KD THP-1 cells displayed enhanced production of pro-inflammatory cytokines including interleukin-6 and -1β, and the up-regulation of NF-κB-dependent genes induced by TLR4 stimulation. Taken together, the data demonstrate that Prdx6 interrupts the formation of TRAF6-ECSIT complex induced by TLR4 stimulation, leading to suppression of bactericidal activity because of inhibited mROS production in mitochondria and the inhibition of NF-κB activation in the cytoplasm. PMID:28393051

  20. Mitochondrial peroxiredoxin-5 as potential modulator of mitochondria-ER crosstalk in MPP+-induced cell death.

    PubMed

    De Simoni, Stéphanie; Linard, Dominique; Hermans, Emmanuel; Knoops, Bernard; Goemaere, Julie

    2013-05-01

    Peroxiredoxin-5 (PRDX5) is an antioxidant enzyme which differs from the other peroxiredoxins with regards to its enzymatic mechanism, its high affinity for organic peroxides and peroxynitrite and its wide subcellular distribution. In particular, the mitochondrial isoform of PRDX5 confers a remarkable cytoprotection toward oxidative stress to mammalian cells. Mitochondrial dysfunction and disruption of Ca²⁺ homeostasis are implicated in neurodegeneration. Growing evidence supports that endoplasmic reticulum (ER) could operate in tandem with mitochondria to regulate intracellular Ca²⁺ fluxes in neurodegenerative processes. Here, we overexpressed mitochondrial PRDX5 in SH-SY5Y cells to dissect the role of this enzyme in 1-methyl-4-phenylpyridinium (MPP)⁺-induced cell death. Our data show that mitochondria-dependent apoptosis triggered by MPP⁺, assessed by the measurement of caspase-9 activation and mitochondrial DNA damage, is prevented by mitochondrial PRDX5 overexpression. Moreover, PRDX5 overexpression blocks the increase in intracellular Ca²⁺, Ca²⁺-dependent activation of calpains and Bax cleavage. Finally, using Ca²⁺ channel inhibitors (Nimodipine, Dantrolene and 2-APB), we show that Ca²⁺ release arises essentially from ER stores through 1,4,5-inositol-trisphosphate receptors (IP3 R). Altogether, our results suggest that the MPP⁺ mitochondrial pathway of apoptosis is regulated by mitochondrial PRDX5 in a process that could involve redox modulation of Ca²⁺ transporters via a crosstalk between mitochondria and ER.

  1. The identification of goat peroxiredoxin-5 and the evaluation and enhancement of its stability by nanoparticle formation

    NASA Astrophysics Data System (ADS)

    Feng, Xiaozhou; Liu, Juanjuan; Fan, Shuai; Liu, Fan; Li, Yadong; Jin, Yuanyuan; Bai, Liping; Yang, Zhaoyong

    2016-04-01

    An anticancer bioactive peptide (ACBP), goat peroxiredoxin-5 (gPRDX5), was identified from goat-spleen extract after immunizing the goat with gastric cancer-cell lysate. Its amino acid sequence was determined by employing 2D nano-LC-ESI-LTQ-Orbitrap MS/MS combined with Mascot database search in the goat subset of the Uniprot database. The recombinant gPRDX5 protein was acquired by heterogeneous expression in Escherichia coli. Subsequently, the anti-cancer bioactivity of the peptide was measured by several kinds of tumor cells. The results indicated that the gPRDX5 was a good anti-cancer candidate, especially for killing B16 cells. However, the peptide was found to be unstable without modification with pharmaceutical excipients, which would be a hurdle for future medicinal application. In order to overcome this problem and find an effective way to evaluate the gPRDX5, nanoparticle formation, which has been widely used in drug delivery because of its steadiness in application, less side-effects and enhancement of drug accumulation in target issues, was used here to address the issues. In this work, the gPRDX5 was dispersed into nanoparticles before delivered to B16 cells. By the nanotechnological method, the gPRDX5 was stabilized by a fast and accurate procedure, which suggests a promising way for screening the peptide for further possible medicinal applications.

  2. A genetic approach to study H2O2 scavenging in fission yeast--distinct roles of peroxiredoxin and catalase.

    PubMed

    Paulo, Esther; García-Santamarina, Sarela; Calvo, Isabel A; Carmona, Mercè; Boronat, Susanna; Domènech, Alba; Ayté, José; Hidalgo, Elena

    2014-04-01

    The main peroxiredoxin in Schizosaccharomyces pombe, Tpx1, is important to sustain aerobic growth, and cells lacking this protein are only able to grow on solid plates under anaerobic conditions. We have found that deletion of the gene coding for thioredoxin reductase, trr1, is a suppressor of the sensitivity to aerobic growth of Δtpx1 cells, so that cells lacking both proteins are able to grow on solid plates in the presence of oxygen. We have investigated this suppression effect, and determined that it depends on the presence of catalase, which is constitutively expressed in Δtrr1 cells in a transcription factor Pap1-dependent manner. A complete characterization of the repertoire of hydrogen peroxide scavenging activities in fission yeast suggests that Tpx1 is the only enzyme with sufficient sensitivity for peroxides and cellular abundance as to control the low levels produced during aerobic growth, catalase being the next barrier of detoxification when the steady-state levels of peroxides are increased in Δtpx1 cells. Gpx1, the only glutathione peroxidase encoded by the S. pombe genome, only has a minor secondary role when extracellular peroxides are added. Our study proposes non-overlapping roles for the different hydrogen peroxide scavenging activities of this eukaryotic organism.

  3. Tubular Peroxiredoxin 3 as a Predictor of Renal Recovery from Acute Tubular Necrosis in Patients with Chronic Kidney Disease.

    PubMed

    Wu, Chia-Lin; Su, Tzu-Cheng; Chang, Chia-Chu; Kor, Chew-Teng; Chang, Chung-Ho; Yang, Tao-Hsiang; Chiu, Ping-Fang; Tarng, Der-Cherng

    2017-02-27

    Peroxiredoxin 3 (PRX3) is a mitochondrial antioxidant that regulates apoptosis in various cancers. However, whether tubular PRX3 predicts recovery of renal function following acute kidney injury (AKI) remains unknown. This retrospective cohort study included 54 hospitalized patients who had AKI with biopsy-proven acute tubular necrosis (ATN). The study endpoint was renal function recovery within 6 months. Of the 54 enrolled patients, 25 (46.3%) had pre-existing chronic kidney disease (CKD) and 33 (61%) recovered renal function. Tubular PRX3 expression was higher in patients with ATN than in those without renal function recovery. The level of tubular but not glomerular PRX3 expression predicted renal function recovery from AKI (AUROC = 0.76). In multivariate Cox regression analysis, high PRX3 expression was independently associated with a higher probability of renal function recovery (adjusted hazard ratio = 8.99; 95% CI 1.13-71.52, P = 0.04). Furthermore, the discriminative ability of the clinical model for AKI recovery was improved by adding tubular PRX3. High tubular PRX3 expression was associated with a higher probability of renal function recovery from ATN. Therefore, tubular PRX3 in combination with conventional predictors can further improve recovery prediction and may help with risk stratification in AKI patients with pre-existing CKD.

  4. Laminar shear stress up-regulates peroxiredoxins (PRX) in endothelial cells: PRX 1 as a mechanosensitive antioxidant.

    PubMed

    Mowbray, Amy L; Kang, Dong-Hoon; Rhee, Sue Goo; Kang, Sang Won; Jo, Hanjoong

    2008-01-18

    Shear stress plays a significant role in endothelial cell biology and atherosclerosis development. Previous work by our group has shown that fluid flow stimulates important functional changes in cells through protein expression regulation. Peroxiredoxins (PRX) are a family of antioxidant enzymes but have yet to be investigated in response to shear stress. Studies have shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, whereas laminar shear stress (LS) blocks this response. We hypothesized that PRX are responsible for the anti-oxidative effect of LS. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to LS (15 dyn/cm(2)), OS (+/-5 dyn/cm(2), 1 Hz), or static conditions for 24 h. Using Western blot and immunofluorescence staining, all six isoforms of PRX were identified in BAEC. When compared with OS and static, exposure to chronic LS up-regulated PRX 1 levels intracellularly. LS also increased expression of PRX 5 relative to static controls, but not OS. PRX exhibited broad subcellular localization, with distribution in the cytoplasm, Golgi, mitochondria, and intermediate filaments. In addition, PRX 1 knock down, using specific small interference RNA, attenuated LS-dependent reactive oxygen species reduction in BAEC. However, PRX 5 depletion did not. Together, these results suggest that PRX 1 is a novel mechanosensitive antioxidant, playing an important role in shear-dependent regulation of endothelial biology and atherosclerosis.

  5. Measurement of peroxiredoxin-4 serum levels in rat tissue and its use as a potential marker for hepatic disease.

    PubMed

    Ito, Ritsu; Takahashi, Motoko; Ihara, Hideyuki; Tsukamoto, Hiroki; Fujii, Junichi; Ikeda, Yoshitaka

    2012-08-01

    Peroxiredoxin (Prx)-4, a secretable endoplasmic reticulum (ER)-resident isoform of the mammalian Prx family, functions as a thioredoxin-dependent peroxidase. It is acknowledged that Prx-4 plays a role in the detoxification of hydrogen peroxide, and potentially other peroxides, which may be generated during the oxidative folding of proteins and oxidative stress in the ER. The present study was undertaken in order to specifically quantify the tissue levels of Prx-4. To accomplish this, an enzyme-linked immunosorbent assay was developed using a specific polyclonal antibody produced by immunizing a rabbit with native recombinant rat Prx-4 protein. The assay was used to detect Prx-4 in the range of 0.1 and 10 ng/ml, and to investigate tissue distribution in rats. Using this immunoassay, we found that the serum levels of Prx-4 were substantially lower in asymptomatic Long-Evans Cinnamon rats, a rat model of Wilson's disease, compared to normal rats. In addition, the treatment of rat hepatoma cells with N-acetylcysteine led to a significant increase in the release of Prx-4 protein into the medium; thus, it appears likely that the secretion of Prx-4 is associated with the redox state within cells. These results suggest that serum Prx-4 has potential for use as a biomarker for hepatic oxidative stress.

  6. The identification of goat peroxiredoxin-5 and the evaluation and enhancement of its stability by nanoparticle formation

    PubMed Central

    Feng, Xiaozhou; Liu, Juanjuan; Fan, Shuai; Liu, Fan; Li, Yadong; Jin, Yuanyuan; Bai, Liping; Yang, Zhaoyong

    2016-01-01

    An anticancer bioactive peptide (ACBP), goat peroxiredoxin-5 (gPRDX5), was identified from goat-spleen extract after immunizing the goat with gastric cancer-cell lysate. Its amino acid sequence was determined by employing 2D nano-LC-ESI-LTQ-Orbitrap MS/MS combined with Mascot database search in the goat subset of the Uniprot database. The recombinant gPRDX5 protein was acquired by heterogeneous expression in Escherichia coli. Subsequently, the anti-cancer bioactivity of the peptide was measured by several kinds of tumor cells. The results indicated that the gPRDX5 was a good anti-cancer candidate, especially for killing B16 cells. However, the peptide was found to be unstable without modification with pharmaceutical excipients, which would be a hurdle for future medicinal application. In order to overcome this problem and find an effective way to evaluate the gPRDX5, nanoparticle formation, which has been widely used in drug delivery because of its steadiness in application, less side-effects and enhancement of drug accumulation in target issues, was used here to address the issues. In this work, the gPRDX5 was dispersed into nanoparticles before delivered to B16 cells. By the nanotechnological method, the gPRDX5 was stabilized by a fast and accurate procedure, which suggests a promising way for screening the peptide for further possible medicinal applications. PMID:27074889

  7. Renal peroxiredoxin 6 interacts with anion exchanger 1 and plays a novel role in pH homeostasis

    PubMed Central

    Johnstone, Duncan B.; Frankl, Fiona E. Karet

    2015-01-01

    Peroxiredoxin 6 (PRDX6) is one of six members of the PRDX family, which have peroxidase and antioxidant activity. PRDX6 is unique, containing only one conserved cysteine residue (C47) rather than the two found in other PRDXs. A yeast two-hybrid screen found PRDX6 to be a potential binding partner of the C-terminal tail of anion exchanger 1 (AE1), a Cl−/HCO3− exchanger basolaterally expressed in renal α-intercalated cells. PRDX6 immunostaining in human kidney was both cytoplasmic and peripheral and co-localized with AE1. Analysis of native protein showed it was largely monomeric, whereas expressed tagged protein was more dimeric. Two methionine oxidation sites were identified. In vitro and ex vivo pulldowns and immunoprecipitation assays confirmed interaction with AE1, but mutation of the conserved cysteine resulted in loss of interaction. Prdx6 knockout mice had a baseline acidosis with a major respiratory component and greater AE1 expression than wild type animals. After an oral acid challenge, PRDX6 expression increased in wild type mice, with preservation of AE1. However, AE1 expression was significantly decreased in knockout animals. Kidneys from acidified mice showed widespread proximal tubular vacuolation in wild type but not knockout animals. Knockdown of PRDX6 by siRNA in mammalian cells reduced both total and cell membrane AE1 levels. Thus, PRDX6-AE1 interaction contributes to maintenance of AE1 during cellular stress such as during metabolic acidosis. PMID:26398495

  8. Molecular characterization, immune response against white spot syndrome virus infection of peroxiredoxin 4 in Fenneropenaeus chinensis and its antioxidant activity.

    PubMed

    Zhang, Qingli; Huang, Jie; Li, Fuhua; Liu, Shuang; Liu, Qinghui; Wei, Jiankai; Liang, Gaofeng; Xiang, Jianhai

    2014-03-01

    Peroxiredoxins (Prx) are a family of antioxidant proteins and perform important functions in intracellular signal transduction. Here, we report a Prx gene from Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA of FcPrx gene contained an open reading frame of 735 bp encoding a polypeptide of 275 amino acids. The molecular mass of the deduced amino acid of FcPrx is 27445.43 Da with an estimated pI of 5.71. Sequence comparison showed that the FcPrx shares high identities with Prx IVs and it was named FcPrx4. A real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of FcPrx4 in different tissues and temporal expression in hemocytes and hepatopancreas of F. chinensis challenged by white spot syndrome virus (WSSV). Transcripts of FcPrx4 can be detected in all tissues examined. The expression of FcPrx4 showed significant up-regulation in shrimp hemocytes and hepatopancreas after artificial infection with WSSV. A fusion protein containing FcPrx4 was produced in vitro and was confirmed by Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) assay. And activity analysis indicated that the recombinant FcPrx4 proteins can reduce H2O2 in the presence of dithiothreitol.

  9. Metallothionein-I/II Knockout Mice Aggravate Mitochondrial Superoxide Production and Peroxiredoxin 3 Expression in Thyroid after Excessive Iodide Exposure

    PubMed Central

    Zhang, Na; Wang, Lingyan; Duan, Qi; Lin, Laixiang; Ahmed, Mohamed; Wang, Tingting; Yao, Xiaomei

    2015-01-01

    Purpose. We aim to figure out the effect of metallothioneins on iodide excess induced oxidative stress in the thyroid. Methods. Eight-week-old MT-I/II knockout (MT-I/II KO) mice and background-matched wild-type (WT) mice were used. Mitochondrial superoxide production and peroxiredoxin (Prx) 3 expression were measured. Results. In in vitro study, more significant increases in mitochondrial superoxide production and Prx 3 expression were detected in the MT-I/II KO groups. In in vivo study, significantly higher concentrations of urinary iodine level were detected in MT-I/II KO mice in 100 HI group. Compared to the NI group, there was no significant difference existing in serum thyroid hormones level in either groups (P > 0.05), while the mitochondrial superoxide production was significantly increased in 100 HI groups with significantly increased LDH activity and decreased relative cell viability. Compared to WT mice, more significant changes were detected in MT-I/II KO mice in 100 HI groups. No significant differences were detected between the NI group and 10 HI group in both the MT-I/II KO and WT mice groups (P > 0.05). Conclusions. Iodide excess in a thyroid without MT I/II protection may result in strong mitochondrial oxidative stress, which further leads to the damage of thyrocytes. PMID:26101557

  10. Fortilin potentiates the peroxidase activity of Peroxiredoxin-1 and protects against alcohol-induced liver damage in mice

    PubMed Central

    Chattopadhyay, Abhijnan; Pinkaew, Decha; Doan, Hung Q.; Jacob, Reed B.; Verma, Sunil K.; Friedman, Hana; Peterson, Alan C.; Kuyumcu-Martinez, Muge N.; McDougal, Owen M.; Fujise, Ken

    2016-01-01

    Fortilin, a pro-survival molecule, inhibits p53-induced apoptosis by binding to the sequence-specific DNA-binding domain of the tumor suppressor protein and preventing it from transcriptionally activating Bax. Intriguingly, fortilin protects cells against ROS-induced cell death, independent of p53. The signaling pathway through which fortilin protects cells against ROS-induced cell death, however, is unknown. Here we report that fortilin physically interacts with the antioxidant enzyme peroxiredoxin-1 (PRX1), protects it from proteasome-mediated degradation, and keeps it enzymatically active by blocking its deactivating phosphorylation by Mst1, a serine/threonine kinase. At the whole animal level, the liver-specific overexpression of fortilin reduced PRX1 phosphorylation in the liver, enhanced PRX1 activity, and protected the transgenic animals against alcohol-induced, ROS-mediated, liver damage. These data suggest the presence of a novel oxidative-stress-handling pathway where the anti-p53 molecule fortilin augments the peroxidase PRX1 by protecting it against degradation and inactivation of the enzyme. Fortilin-PRX1 interaction in the liver could be clinically exploited further to prevent acute alcohol-induced liver damage in humans. PMID:26726832

  11. Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma.

    PubMed

    Cunniff, Brian; Newick, Kheng; Nelson, Kimberly J; Wozniak, Alexandra N; Beuschel, Stacie; Leavitt, Bruce; Bhave, Anant; Butnor, Kelly; Koenig, Andreas; Chouchani, Edward T; James, Andrew M; Haynes, Alexina C; Lowther, W Todd; Murphy, Michael P; Shukla, Arti; Heintz, Nicholas H

    2015-01-01

    Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2--thioredoxin 2 (TRX2)--peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.

  12. Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma

    PubMed Central

    Wozniak, Alexandra N.; Beuschel, Stacie; Leavitt, Bruce; Bhave, Anant; Butnor, Kelly; Koenig, Andreas; Chouchani, Edward T.; James, Andrew M.; Haynes, Alexina C.; Lowther, W. Todd; Murphy, Michael P.; Shukla, Arti; Heintz, Nicholas H.

    2015-01-01

    Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2 - thioredoxin 2 (TRX2) - peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies. PMID:26011724

  13. Inhibitory effect of peroxiredoxin II (Prx II) on Ras-ERK-NFkappaB pathway in mouse embryonic fibroblast (MEF) senescence.

    PubMed

    Han, Ying-Hao; Kwon, Jeong-Hoon; Yu, Dae-Yeul; Moon, Eun-Yi

    2006-11-01

    Intracellular reactive oxygen species (ROS) were attenuated by the expression of peroxiredoxin II (Prx II). Cellular senescence as judged by senescence-associated (SA)-beta-galactosidase (Gal) positive cell formation was increased in Prx II-deficient mouse embryonic fibroblast (MEF). Ras expression was increased following passages. The level of Ras expression was higher in Prx II-/- MEF than wild type MEF. ERK activity was also augmented by the deletion of Prx II. SA-beta-Gal-positive cell formation was reduced by PD98059, ERK inhibitor. Activated nuclear transcription factor, nuclear factor-kappaB (NFkappaB) by the deletion of Prx II was inhibited by the treatment with PD98059. In contrast, no changes in SA-beta-Gal-positive cell formation were detected by NFkappaB inhibitor, N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Collectively, results suggest that Prx II deletion activate Ras-ERK-NFkappaB pathways and cellular senescence in Prx II-/- MEF cells was mediated by ERK activation but not by NFkappaB activation.

  14. Downregulation of peroxiredoxin-3 by hydrophobic bile acid induces mitochondrial dysfunction and cellular senescence in human trophoblasts

    PubMed Central

    Wu, Wei-Bin; Menon, Ramkumar; Xu, Yue-Ying; Zhao, Jiu-Ru; Wang, Yan-Lin; Liu, Yuan; Zhang, Hui-Juan

    2016-01-01

    Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disorder characterised by raised bile acids in foetal-maternal circulation, which threatens perinatal health. During the progression of ICP, the effect of oxidative stress is underscored. Peroxiredoxin-3 (PRDX3) is a mitochondrial antioxidant enzyme that is crucial to balance intracellular oxidative stress. However, the role of PRDX3 in placental trophoblast cells under ICP is not fully understood. We demonstrated that the level of PRDX3 was downregulated in ICP placentas as well as bile acids–treated trophoblast cells and villous explant in vitro. Toxic levels of bile acids and PRDX3 knockdown induced oxidative stress and mitochondrial dysfunction in trophoblast cells. Moreover, silencing of PRDX3 in trophoblast cell line HTR8/SVneo induced growth arrest and cellular senescence via activation of p38-mitogen-activated protein kinase (MAPK) and induction of p21WAF1/CIP and p16INK4A. Additionally, enhanced cellular senescence, determined by senescence-associated beta-galactosidase staining, was obviously attenuated by p38-MAPK inhibitor SB203580. Our data determined that exposure to bile acid decreased PRDX3 level in human trophoblasts. PRDX3 protected trophoblast cells against mitochondrial dysfunction and cellular senescence induced by oxidative stress. Our results suggest that decreased PRDX3 by excessive bile acids in trophoblasts plays a critical role in the pathogenesis and progression of ICP. PMID:27958341

  15. Peroxiredoxin 1 (Prx1) is a dual function enzyme by possessing Cys-independent catalase-like activity.

    PubMed

    Sun, Cen-Cen; Dong, Wei-Ren; Shao, Tong; Li, Jiang-Yuan; Zhao, Jing; Nie, Li; Xiang, Li-Xin; Zhu, Guan; Shao, Jian-Zhong

    2017-02-20

    Peroxiredoxin (Prx) was previously known as a Cys-dependent thioredoxin. However, we unexpected observed that Prx1 from the green spotted puffer fish Tetraodon nigroviridis (TnPrx1) was able to reduce H2O2 in a manner independent on the Cys peroxidation and reductants. This study aimed to validate the novel function for Prx1, delineate the biochemical features and explore its antioxidant role in cells. We have confirmed that Prx1 from the puffer fish and humans truly possesses a catalase-like activity that is independent of Cys residues and reductants, but dependent on iron. We have identified that the GVL motif was essential to the catalase-like (CAT) activity of Prx1, but not to the Cys-dependent thioredoxin peroxidase (POX) activity, and generated mutants lacking POX and/or CAT activities for individual functional validation. We discovered that the TnPrx1 POX and CAT activities possessed different kinetic features in reducing H2O2 The overexpression of wild-type TnPrx1 and mutants differentially regulated the intracellular levels of reactive oxygen species (ROS) and the phosphorylation of p38 in HEK-293T cells treated with H2O2 Prx1 is a dual function enzyme by acting as POX and CAT with varied affinities towards ROS. This study extends our knowledge on Prx1 and provides new opportunities to further study the biological roles of this family of antioxidants.

  16. Resemblance and Dissemblance of Arabidopsis Type II Peroxiredoxins: Similar Sequences for Divergent Gene Expression, Protein Localization, and Activity1

    PubMed Central

    Bréhélin, Claire; Meyer, Etienne H.; de Souris, Jean-Paul; Bonnard, Géraldine; Meyer, Yves

    2003-01-01

    The Arabidopsis type II peroxiredoxin (PRXII) family is composed of six different genes, five of which are expressed. On the basis of the nucleotide and protein sequences, we were able to define three subgroups among the PRXII family. The first subgroup is composed of AtPRXII-B, -C, and -D, which are highly similar and localized in the cytosol. AtPRXII-B is ubiquitously expressed. More striking is the specific expression of AtPRXII-C and AtPRXII-D localized in pollen. The second subgroup comprises the mitochondrial AtPRXII-F, the corresponding gene of which is expressed constitutively. We show that AtPRXII-E, belonging to the last subgroup, is expressed mostly in reproductive tissues and that its product is addressed to the plastid. By in vitro enzymatic experiments, we demonstrate that glutaredoxin is the electron donor of recombinant AtPRXII-B for peroxidase reaction, but the donors of AtPRXII-E and AtPRXII-F have still to be identified. PMID:12913160

  17. Tubular Peroxiredoxin 3 as a Predictor of Renal Recovery from Acute Tubular Necrosis in Patients with Chronic Kidney Disease

    PubMed Central

    Wu, Chia-Lin; Su, Tzu-Cheng; Chang, Chia-Chu; Kor, Chew-Teng; Chang, Chung-Ho; Yang, Tao-Hsiang; Chiu, Ping-Fang; Tarng, Der-Cherng

    2017-01-01

    Peroxiredoxin 3 (PRX3) is a mitochondrial antioxidant that regulates apoptosis in various cancers. However, whether tubular PRX3 predicts recovery of renal function following acute kidney injury (AKI) remains unknown. This retrospective cohort study included 54 hospitalized patients who had AKI with biopsy-proven acute tubular necrosis (ATN). The study endpoint was renal function recovery within 6 months. Of the 54 enrolled patients, 25 (46.3%) had pre-existing chronic kidney disease (CKD) and 33 (61%) recovered renal function. Tubular PRX3 expression was higher in patients with ATN than in those without renal function recovery. The level of tubular but not glomerular PRX3 expression predicted renal function recovery from AKI (AUROC = 0.76). In multivariate Cox regression analysis, high PRX3 expression was independently associated with a higher probability of renal function recovery (adjusted hazard ratio = 8.99; 95% CI 1.13–71.52, P = 0.04). Furthermore, the discriminative ability of the clinical model for AKI recovery was improved by adding tubular PRX3. High tubular PRX3 expression was associated with a higher probability of renal function recovery from ATN. Therefore, tubular PRX3 in combination with conventional predictors can further improve recovery prediction and may help with risk stratification in AKI patients with pre-existing CKD. PMID:28240739

  18. A New Family of Membrane Electron Transporters and Its Substrates, Including a New Cell Envelope Peroxiredoxin, Reveal a Broadened Reductive Capacity of the Oxidative Bacterial Cell Envelope

    PubMed Central

    Cho, Seung-Hyun; Parsonage, Derek; Thurston, Casey; Dutton, Rachel J.; Poole, Leslie B.; Collet, Jean-Francois; Beckwith, Jon

    2012-01-01

    ABSTRACT The Escherichia coli membrane protein DsbD functions as an electron hub that dispatches electrons received from the cytoplasmic thioredoxin system to periplasmic oxidoreductases involved in protein disulfide isomerization, cytochrome c biogenesis, and sulfenic acid reduction. Here, we describe a new class of DsbD proteins, named ScsB, whose members are found in proteobacteria and Chlamydia. ScsB has a domain organization similar to that of DsbD, but its amino-terminal domain differs significantly. In DsbD, this domain directly interacts with substrates to reduce them, which suggests that ScsB acts on a different array of substrates. Using Caulobacter crescentus as a model organism, we searched for the substrates of ScsB. We discovered that ScsB provides electrons to the first peroxide reduction pathway identified in the bacterial cell envelope. The reduction pathway comprises a thioredoxin-like protein, TlpA, and a peroxiredoxin, PprX. We show that PprX is a thiol-dependent peroxidase that efficiently reduces both hydrogen peroxide and organic peroxides. Moreover, we identified two additional proteins that depend on ScsB for reduction, a peroxiredoxin-like protein, PrxL, and a novel protein disulfide isomerase, ScsC. Altogether, our results reveal that the array of proteins involved in reductive pathways in the oxidative cell envelope is significantly broader than was previously thought. Moreover, the identification of a new periplasmic peroxiredoxin indicates that in some bacteria, it is important to directly scavenge peroxides in the cell envelope even before they reach the cytoplasm. PMID:22493033

  19. Development of Monoclonal Antibodies That Target 1-Cys Peroxiredoxin and Differentiate Plasmodium falciparum from P. vivax and P. knowlesi.

    PubMed

    Hakimi, Hassan; Nguyen, Thu-Thuy; Suganuma, Keisuke; Masuda-Suganuma, Hirono; Angeles, Jose Ma M; Inoue, Noboru; Kawazu, Shin-Ichiro

    2013-06-01

    Prompt and accurate diagnosis of malarial patients is a crucial factor in controlling the morbidity and mortality of the disease. Effective treatment decisions require a correct diagnosis among mixed-species malarial patients. Differential diagnosis is particularly important in cases of Plasmodium vivax, a species that shares endemicity with P. falciparum in most endemic areas. Moreover, it is difficult to identify P. knowlesi on the basis of morphology alone, and rapid diagnostic tests are still not available for this malaria species. Therefore, the development of diagnostic tests applicable to the field is urgently needed. 1-Cys peroxiredoxin (1-Cys-Prx) in P. falciparum is abundantly expressed in the mature asexual stages, making it a promising candidate as a diagnostic antigen. In this study, we produced five monoclonal antibodies (mAbs) against P. falciparum 1-Cys-Prx (Pf1-Cys-Prx) by immunizing BALB/c mice with recombinant Pf1-Cys-Prx and subsequent hybridoma production. Cross reactivity of established mAbs with the orthologous molecule of Pf1-Cys-Prx in P. vivax (Pv1-Cys-Prx) and P. knowlesi (Pk1-Cys-Prx) was examined. Western blot analyses showed that three mAbs reacted with Pv1-Cys-Prx and Pk1-Cys-Prx but two mAbs did not. These results indicate that the two mAbs were effective in differentiating P. falciparum from P. vivax and P. knowlesi and could be used in differential diagnosis as well as comparative molecular studies of human Plasmodium species.

  20. Role of reactive oxygene species, peroxiredoxins and thioredoxins in reaction of plants to hypergravity and oxidative stresses

    NASA Astrophysics Data System (ADS)

    Jadko, Sergiy

    Early increasing of reactive oxygen species (ROS) concentration, including H2O2, occur in plant cells under various impacts and these ROS can function as signaling molecules in starting of cell stress responses. Peroxiredoxins (Prx) and thioredoxins (Trx) are significant cell ROS/H2O2 sensors and transmitters. Prx besides its antioxidant activity, participate in creating of stress redox signals by destroying of H2O2 and reducing of Trx. Than these reduced Trx lead to activation of various redox sensitive proteins, transcription factors and MAP kinases. This study aimed to investigate early increasing of ROS and H2O2 contents and Prx and Trx activities in pea roots and arabidopsis tissue culture cells under hypergravity and oxidative stresses. Pea roots of 3-5 days old seedlings and 12 days old tissue culture of Arabidopsis thaliana from leaves were studied. Pea seedlings were grown on wet filter paper and the tissue culture was grown on MS medium in dark conditions under 24oC. Hypergravity stress was induced by centrifugation at 15 g. Chemiluminescence (ChL) intensity for ROS concentration, H2O2 content and Prx and Trx activities were determined. All experiments were repeated by 3-4 times. Early increasing of ChL intensity and H2O2 content in the pea roots and arabidopsis tissue culture cells took place under hypergravity and oxidative stresses and its were higher corresponding controls on average on 25, 21 and 17 percents to 30, 60 and 90 min. At the same time Prx and Trx activities increased on 7, 13 and 16 percents. Thus under hypergravity and oxidative stresses in both investigated plants take place early increasing of ROS and H2O2 contents which as second messengers can lead to ROS/H2O2-dependent increasing of Prx and Trx activities with creating of H2O2-Prx-Trx signaling pathway.

  1. Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7.

    PubMed

    Tae Lim, Young; Sup Song, Dong; Joon Won, Tae; Lee, Yun-Jung; Yoo, Jong-Sun; Eun Hyung, Kyeong; Won Yoon, Joo; Park, So-Young; Woo Hwang, Kwang

    2012-06-01

    Peroxiredoxin (PRX), a scavenger of H(2) O(2) and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti-oxidant roles, the involvement of PRX-1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX-1 having been uncovered only recently. In the present study, it was discovered that the PRX-1 deficient macrophage like cell line (RAW264.7) has anti-inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti-inflammatory cytokine, interleukin-10 (IL-10), in PRX-1 knock down RAW264.7 cells. Gene expression of pro-inflammatory cytokines IL-1β and tumor necrosis factor- α (TNF-α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX-1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL-10 was also increased in PRX-1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL-10 would result in decreased expression of IL-1β and TNF-α in PRX-1 knock-down cells. This was confirmed by blocking IL-10, which reestablished IL-1β and TNF-α secretion. We also observed that increased concentrations of IL-10 do not affect the NF-κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX-1 knockdown RAW264.7 cells. Up-regulation of IL-10 in PRX-1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down-regulation of PRX-1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.

  2. Overexpression of Peroxiredoxin 4 Affects Intestinal Function in a Dietary Mouse Model of Nonalcoholic Fatty Liver Disease

    PubMed Central

    Noguchi, Hirotsugu; Mazaki, Yuichi; Kurahashi, Toshihiro; Izumi, Hiroto; Wang, Ke-Yong; Guo, Xin; Uramoto, Hidetaka; Kohno, Kimitoshi; Taniguchi, Hatsumi; Tanaka, Yoshiya; Fujii, Junichi; Sasaguri, Yasuyuki; Tanimoto, Akihide; Nakayama, Toshiyuki

    2016-01-01

    Background Accumulating evidence has shown that methionine- and choline-deficient high fat (MCD+HF) diet induces the development of nonalcoholic fatty liver disease (NAFLD), in which elevated reactive oxygen species play a crucial role. We have reported that peroxiredoxin 4 (PRDX4), a unique secretory member of the PRDX antioxidant family, protects against NAFLD progression. However, the detailed mechanism and potential effects on the intestinal function still remain unclear. Methods & Results Two weeks after feeding mice a MCD+HF diet, the livers of human PRDX4 transgenic (Tg) mice exhibited significant suppression in the development of NAFLD compared with wild-type (WT) mice. The serum thiobarbituric acid reactive substances levels were significantly lower in Tg mice. In contrast, the Tg small intestine with PRDX4 overexpression showed more suppressed shortening of total length and villi height, and more accumulation of lipid in the jejunum, along with lower levels of dihydroethidium binding. The enterocytes exhibited fewer apoptotic but more proliferating cells, and inflammation was reduced in the mucosa. Furthermore, the small intestine of Tg mice had significantly higher expression of cholesterol absorption-regulatory factors, including liver X receptor-α, but lower expression of microsomal triglyceride-transfer protein. Conclusion Our present data provide the first evidence of the beneficial effects of PRDX4 on intestinal function in the reduction of the severity of NAFLD, by ameliorating oxidative stress-induced local and systemic injury. We can suggest that both liver and intestine are spared, to some degree, by the antioxidant properties of PRDX4. PMID:27035833

  3. In vivo oxidative stress alters thiol redox status of peroxiredoxin 1 and 6 and impairs rat sperm quality

    PubMed Central

    Liu, Yannan; O’Flaherty, Cristian

    2017-01-01

    Oxidative stress, the imbalance between the production of reactive oxygen species (ROS) and antioxidant activity is a major culprit of male infertility. Peroxiredoxins (PRDXs) are major antioxidant enzymes of mammalian spermatozoa and are thiol oxidized and inactivated by ROS in a dose-dependent manner. Their deficiency and/or inactivation have been associated with men infertility. The aim of this study was to elucidate the impact of oxidative stress, generated by the in vivo tert-butyl hydroperoxide (tert-BHP) treatment on rat epididymal spermatozoa during their maturation process. Adult Sprague-Dawley males were treated with μmoles tert-BHP/kg or saline (control) per day intraperitoneal for 15 days. Lipid peroxidation (2-thibarbituric acid reactive substances assay), total amount and thiol oxidation of PRDXs along with the total amount of superoxide dismutase (SOD), motility and DNA oxidation (8-hydroxy-deoxyguanosine) were determined in epididymal spermatozoa. Total amount of PRDXs and catalase and thiol oxidation of PRDXs were determined in caput and cauda epididymis. While animals were not affected by treatment, their epididymal spermatozoa have decreased motility, increased levels of DNA oxidation and lipid peroxidation along with increased PRDXs (and not SOD) amounts. Moreover, sperm PRDXs were highly thiol oxidized. There was a differential regulation in the expression of PRDX1 and PRDX6 in the epididymis that suggests a segment-specific role for PRDXs. In conclusion, PRDXs are increased in epididymal spermatozoa in an attempt to fight against the oxidative stress generated by tert-BHP in the epididymis. These findings highlight the role of PRDXs in the protection of sperm function and DNA integrity during epididymal maturation. PMID:26823067

  4. Peroxiredoxin 6 Fails to Limit Phospholipid Peroxidation in Lung from Cftr-Knockout Mice Subjected to Oxidative Challenge

    PubMed Central

    Trudel, Stéphanie; Kelly, Mairead; Fritsch, Janine; Nguyen-Khoa, Thao; Thérond, Patrice; Couturier, Martine; Dadlez, Michal; Debski, Janusz; Touqui, Lhousseine; Vallée, Benoit; Ollero, Mario; Edelman, Aleksander; Brouillard, Franck

    2009-01-01

    Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult. The results show that endogenous ROS and lipid peroxidation levels are higher in Cftr−/− lung when compared to wild-type (Cftr+/+) in basal conditions, despite a strong enzymatic antioxidant response involving superoxide dismutases, glutathione peroxidases and peroxiredoxin 6 (Prdx6). The latter has the unique capacity to directly reduce membrane phospholipid hydroperoxides (PL-OOH). A dramatic increase in PL-OOH levels in Cftr−/− lung consecutive to in vivo oxidative challenge by paraquat (PQ) unmasks a susceptibility to phospholipid peroxidation. PQ strongly decreases Prdx6 expression in Cftr−/− mice compared to Cftr+/+. Similar results were obtained after P. aeruginosa LPS challenge. Two-dimensional gel analysis of Prdx6 revealed one main molecular form in basal conditions and a PQ-induced form only detected in Cftr+/+ lung. Mass spectrometry experiments suggested that, as opposed to the main basal form, the one induced by PQ is devoid of overoxidized catalytic Cys47 and could correspond to a fully active form that is not induced in Cftr−/− lung. These results highlight a constitutive redox imbalance and a vulnerability to oxidative insult in Cftr−/− lung and present Prdx6 as a key component in CF antioxidant failure. This impaired PL-OOH detoxification mechanism may enhance oxidative damage and stress-related signaling, contributing to an exaggerated inflammatory response in CF lung. PMID:19562038

  5. Novel molecular insights into the function and the antioxidative stress response of a Peroxiredoxin Q protein from cyanobacteria.

    PubMed

    Tailor, Vijay; Ballal, Anand

    2017-01-31

    The Peroxiredoxin Q (PrxQ) proteins are thiol-based peroxidases that are important for maintaining redox homeostasis in several organisms. Activity of PrxQs is mediated by two cysteines, peroxidatic (Cp) and resolving (Cr), in association with a reducing partner. A PrxQ, Alr3183, from the cyanobacterium, Anabaena PCC 7120, was characterized in this study. Alr3183, which required thioredoxin A (TrxA) for peroxidase activity, was an intramolecular disulfide bond-containing monomeric protein. However, Alr3183 lacking Cp (Alr3183C46S) or Cr (Alr3183C51S) formed intermolecular disulfide linkages and was dimeric. Alr3183C46S was completely inactive, while Alr3183C51S required higher concentration of TrxA for peroxidase activity. Surface plasmon resonance analysis showed that unlike Alr3183 or Alr3183C46S, Alr3183C51S bound rather poorly to TrxA. Also, compared to the oxidized protein, the DTT-treated (reduced) Alr3183 displayed decreased interaction with TrxA. In vivo, Alr3183 was found to be induced in response to γ-radiation. On exposure to H2O2, Anabaena strain over-expressing Alr3183 showed reduced formation of ROS, intact photosynthetic pigments and consequently better survival than the wild-type, whereas overproduction of Alr3183C46S did not provide any protection. Significantly, this study (1) reveals the importance of Cr for interaction with thioredoxins and (2) demonstrates that over-expression of PrxQs can protect cyanobacteria from oxidative stresses.

  6. Protection of Xenopus laevis embryos against alcohol-induced delayed gut maturation and growth retardation by peroxiredoxin 5 and catalase.

    PubMed

    Peng, Ying; Yang, Pai-Hao; Ng, Samuel S M; Lum, Ching Tung; Kung, Hsiang-Fu; Lin, Marie C

    2004-07-16

    Accumulated evidence indicates that maternal alcohol consumption causes fetal enteric damage and growth retardation. In this study, we investigated the underlying molecular mechanisms in a Xenopus model of fetal alcohol exposure. We established a condition of transient alcohol exposure that produces tadpoles with delayed gut maturation and decreased body length. We then investigated the roles of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by microinjecting plasmids expressing catalase and peroxiredoxin 5 (PRDX5) into two-cell stage embryos. Finally, the effects of these enzymes on the expression of key gut developmental genes were determined by animal cap explant assay. We showed that exposure of Xenopus embryos to 0.5% alcohol from stage 13 to stage 22 produced tadpoles with delayed gut maturation, reduced growth, and down-regulation in several gut developmental genes, with VegT, Pax6 and Sox17 most vulnerable. We further demonstrated that microinjection of catalase attenuated alcohol-induced ROS production and restored the expression of VegT and Pax6, but protected the embryos from delayed gut development and retarded growth only partially. By contrast, microinjection of PRDX5 reduced both ROS and RNS production, and prevented the gut and growth defects, and restored VegT, Pax6 and Sox17 gene expression. A positive correlation was found between delayed gut maturation and reduced body length. These results indicate the crucial roles of both the ROS-Pax6 and RNS-Sox17 signaling axes in alcohol-induced fetal gut defects and growth retardation. In addition, they suggest strongly a cause-and-effect relationship between alcohol-induced delayed gut maturation and growth retardation.

  7. Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal

    PubMed Central

    Salzano, Sonia; Checconi, Paola; Hanschmann, Eva-Maria; Lillig, Christopher Horst; Bowler, Lucas D.; Chan, Philippe; Vaudry, David; Mengozzi, Manuela; Coppo, Lucia; Sacre, Sandra; Atkuri, Kondala R.; Sahaf, Bita; Herzenberg, Leonard A.; Herzenberg, Leonore A.; Mullen, Lisa; Ghezzi, Pietro

    2014-01-01

    The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the –SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases. PMID:25097261

  8. Detoxification of Mitochondrial Oxidants and Apoptotic Signaling Are Facilitated by Thioredoxin-2 and Peroxiredoxin-3 during Hyperoxic Injury

    PubMed Central

    Forred, Benjamin J.; Daugaard, Darwin R.; Titus, Brianna K.; Wood, Ryan R.; Floen, Miranda J.; Booze, Michelle L.

    2017-01-01

    Mitochondria play a fundamental role in the regulation of cell death during accumulation of oxidants. High concentrations of atmospheric oxygen (hyperoxia), used clinically to treat tissue hypoxia in premature newborns, is known to elicit oxidative stress and mitochondrial injury to pulmonary epithelial cells. A consequence of oxidative stress in mitochondria is the accumulation of peroxides which are detoxified by the dedicated mitochondrial thioredoxin system. This system is comprised of the oxidoreductase activities of peroxiredoxin-3 (Prx3), thioredoxin-2 (Trx2), and thioredoxin reductase-2 (TrxR2). The goal of this study was to understand the role of the mitochondrial thioredoxin system and mitochondrial injuries during hyperoxic exposure. Flow analysis of the redox-sensitive, mitochondrial-specific fluorophore, MitoSOX, indicated increased levels of mitochondrial oxidant formation in human adenocarcinoma cells cultured in 95% oxygen. Increased expression of Trx2 and TrxR2 in response to hyperoxia were not attributable to changes in mitochondrial mass, suggesting that hyperoxic upregulation of mitochondrial thioredoxins prevents accumulation of oxidized Prx3. Mitochondrial oxidoreductase activities were modulated through pharmacological inhibition of TrxR2 with auranofin and genetically through shRNA knockdown of Trx2 and Prx3. Diminished Trx2 and Prx3 expression was associated with accumulation of mitochondrial superoxide; however, only shRNA knockdown of Trx2 increased susceptibility to hyperoxic cell death and increased phosphorylation of apoptosis signal-regulating kinase-1 (ASK1). In conclusion, the mitochondrial thioredoxin system regulates hyperoxic-mediated death of pulmonary epithelial cells through detoxification of oxidants and regulation of redox-dependent apoptotic signaling. PMID:28045936

  9. Peroxiredoxin-glutaredoxin and catalase promote resistance of nontypeable Haemophilus influenzae 86-028NP to oxidants and survival within neutrophil extracellular traps.

    PubMed

    Juneau, Richard A; Pang, Bing; Armbruster, Chelsie E; Murrah, Kyle A; Perez, Antonia C; Swords, W Edward

    2015-01-01

    Nontypeable Haemophilus influenzae (NTHI) is a common commensal and opportunistic pathogen of the human airways. For example, NTHI is a leading cause of otitis media and is the most common cause of airway infections associated with chronic obstructive pulmonary disease (COPD). These infections are often chronic/recurrent in nature and involve bacterial persistence within biofilm communities that are highly resistant to host clearance. Our previous work has shown that NTHI within biofilms has increased expression of factors associated with oxidative stress responses. The goal of this study was to define the roles of catalase (encoded by hktE) and a bifunctional peroxiredoxin-glutaredoxin (encoded by pdgX) in resistance of NTHI to oxidants and persistence in vivo. Isogenic NTHI strain 86-028NP mutants lacking hktE and pdgX had increased susceptibility to peroxide. Moreover, these strains had persistence defects in the chinchilla infection model for otitis media, as well as in a murine model for COPD. Additional work showed that pdgX and hktE were important determinants of NTHI survival within neutrophil extracellular traps (NETs), which we have shown to be an integral part of NTHI biofilms in vivo. Based on these data, we conclude that catalase and peroxiredoxin-glutaredoxin are determinants of bacterial persistence during chronic/recurrent NTHI infections that promote bacterial survival within NETs.

  10. Life span extension and H(2)O(2) resistance elicited by caloric restriction require the peroxiredoxin Tsa1 in Saccharomyces cerevisiae.

    PubMed

    Molin, Mikael; Yang, Junsheng; Hanzén, Sarah; Toledano, Michel B; Labarre, Jean; Nyström, Thomas

    2011-09-02

    Caloric restriction (CR) extends the life span of organisms ranging from yeast to primates. Here, we show that the thiol-dependent peroxiredoxin Tsa1 and its partner sulfiredoxin, Srx1, are required for CR to extend the replicative life span of yeast cells. Tsa1 becomes hyperoxidized/inactive during aging, and CR mitigates such oxidation by elevating the levels of Srx1, which is required to reduce/reactivate hyperoxidized Tsa1. CR, by lowering cAMP-PKA activity, enhances Gcn2-dependent SRX1 translation, resulting in increased resistance to H(2)O(2) and life span extension. Moreover, an extra copy of the SRX1 gene is sufficient to extend the life span of cells grown in high glucose concentrations by 20% in a Tsa1-dependent and Sir2-independent manner. The data demonstrate that Tsa1 is required to ensure yeast longevity and that CR extends yeast life span, in part, by counteracting age-induced hyperoxidation of this peroxiredoxin.

  11. First report of a peroxiredoxin homologue in jellyfish: molecular cloning, expression and functional characterization of CcPrx4 from Cyanea capillata.

    PubMed

    Ruan, Zengliang; Liu, Guoyan; Wang, Beilei; Zhou, Yonghong; Lu, Jia; Wang, Qianqian; Zhao, Jie; Zhang, Liming

    2014-01-09

    We first identified and characterized a novel peroxiredoxin (Prx), designated as CcPrx4, from the cDNA library of the tentacle of the jellyfish Cyanea capillata. The full-length cDNA sequence of CcPrx4 consisted of 884 nucleotides with an open reading frame encoding a mature protein of 247 amino acids. It showed a significant homology to peroxiredoxin 4 (Prx4) with the highly conserved F-motif (93FTFVCPTEI101), hydrophobic region (217VCPAGW222), 140GGLG143 and 239YF240, indicating that it should be a new member of the Prx4 family. The deduced CcPrx4 protein had a calculated molecular mass of 27.2 kDa and an estimated isoelectric point of 6.3. Quantitative real-time PCR analysis showed that CcPrx4 mRNA could be detected in all the jellyfish tissues analyzed. CcPrx4 protein was cloned into the expression vector, pET-24a, and expressed in Escherichia coli Rosetta (DE3) pLysS. Recombinant CcPrx4 protein was purified by HisTrap High Performance chelating column chromatography and analyzed for its biological function. The results showed that the purified recombinant CcPrx4 protein manifested the ability to reduce hydrogen peroxide and protect supercoiled DNA from oxidative damage, suggesting that CcPrx4 protein may play an important role in protecting jellyfish from oxidative damage.

  12. Backbone chemical shift assignments for Xanthomonas campestris peroxiredoxin Q in the reduced and oxidized states: a dramatic change in backbone dynamics.

    PubMed

    Buchko, Garry W; Perkins, Arden; Parsonage, Derek; Poole, Leslie B; Karplus, P Andrew

    2016-04-01

    Peroxiredoxins (Prx) are ubiquitous enzymes that reduce peroxides as part of antioxidant defenses and redox signaling. While Prx catalytic activity and sensitivity to hyperoxidative inactivation depend on their dynamic properties, there are few examples where their dynamics has been characterized by NMR spectroscopy. Here, we provide a foundation for studies of the solution properties of peroxiredoxin Q from the plant pathogen Xanthomonas campestris (XcPrxQ) by assigning the observable (1)H(N), (15)N, (13)C(α), (13)C(β), and (13)C' chemical shifts for both the reduced (dithiol) and oxidized (disulfide) states. In the reduced state, most of the backbone amide resonances (149/152, 98 %) can be assigned in the XcPrxQ (1)H-(15)N HSQC spectrum. In contrast, a remarkable 51 % (77) of these amide resonances are not visible in the (1)H-(15)N HSQC spectrum of the disulfide state of the enzyme, indicating a substantial change in backbone dynamics associated with the formation of an intramolecular C48-C84 disulfide bond.

  13. A DNA-binding peroxiredoxin of Coxiella burnetii is involved in countering oxidative stress during exponential-phase growth.

    PubMed

    Hicks, Linda D; Raghavan, Rahul; Battisti, James M; Minnick, Michael F

    2010-04-01

    Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen's proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100 degrees C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain's ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P < 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen's early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that

  14. Cysteine Oxidation Targets Peroxiredoxins 1 and 2 for Exosomal Release through a Novel Mechanism of Redox-Dependent Secretion

    PubMed Central

    Mullen, Lisa; Hanschmann, Eva-Maria; Lillig, Christopher Horst; Herzenberg, Leonore A; Ghezzi, Pietro

    2015-01-01

    Nonclassical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of nonclassical secretion. We have shown recently that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms, as has been postulated for the inflammatory mediators interleukin-1β (IL-1β) and high mobility group box-1 (HMGB1). We show here that circulating Prdx1 and 2 are present exclusively as disulfide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α, and this release can be induced with an oxidant. By contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway, instead, both Prdx1 and 2 are released in exosomes from both human embryonic kidney (HEK) cells and monocytic cells. Serum Prdx1 and 2 also are associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signaling mechanisms in inflammation. PMID:25715249

  15. Novel links among peroxiredoxins, endothelial dysfunction, and severity of atherosclerosis in type 2 diabetic patients with peripheral atherosclerotic disease.

    PubMed

    El Eter, Eman; Al Masri, Abeer; Habib, Shahid; Al Zamil, Hana; Al Hersi, Ahmed; Al Hussein, Fawaz; Al Omran, Mohamed

    2014-03-01

    Peroxiredoxins, a group of antioxidant protein enzymes (PRDX1 to 6), are reported as antiatherogenic factors in animals; however, human studies are lacking. The present work aims to provide baseline data regarding the phenotype of PRDX1, 2, 4, and 6 in diabetic patients with peripheral atherosclerosis disease (PAD) and their relation to endothelial dysfunction (ED) and disease severity. Plasma levels of PRDX1, 2, 4, and 6 and markers of endothelial dysfunction (ICAM-1 and VCAM-1) were measured using ELISA in 55 type 2 diabetic patients having PAD and 25 healthy subjects. Ankle-brachial index (ABI), body mass index (BMI), triglycerides (TG), total cholesterol, HbA1c, and insulin resistance (HOMA IR) were measured. PRDX1, 2, 4, and 6 levels were significantly higher in patients compared to controls (PRDX1 21.9 ± 5.71 vs 16.8 ± 3.9 ng/ml, P < 0.001, PRDX2 36.5 ± 14.83 vs 20.4 ± 8.61 ng/ml, P < 0.001, PRDX4 3,840 ± 1,440 vs 2,696 ± 1,972 pg/ml, P < 0.005, PRDX6 311 ± 110 vs 287.9 ± 114 pg/ml, P < 0.05). PRDX1 and PRDX4 correlated negatively with ABI (r = -0.273, P < 0.05 and r = -0.28, P < 0.05, respectively), while PRDX1 and PRDX2 correlated positively with HOMA/IR and TG (r = 0.276, P < 0.01 and r = 0.295, P < 0.01, respectively). ICAM-1 was associated with PRDX2 and log PRDX6 (r = 0.345, P = 0.0037 and r = 0.344, P = 0.0038). Our results provide strong links among PRDXs, ED, and severity of PAD in diabetic patients which warrants further evaluation to clarify whether high circulating levels of PRDXs are a consequence of chronic atherosclerotic disease or a predisposing factor for later cardiovascular events.

  16. Nicotinamide nucleotide transhydrogenase (Nnt) links the substrate requirement in brain mitochondria for hydrogen peroxide removal to the thioredoxin/peroxiredoxin (Trx/Prx) system.

    PubMed

    Lopert, Pamela; Patel, Manisha

    2014-05-30

    Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H2O2) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H2O2 catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP(+), provides the link between mitochondrial respiration and H2O2 detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H2O2 in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H2O2, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H2O2 catabolism, (b) decreased NADPH and increased NADP(+) levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H2O2 and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H2O2 antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases.

  17. Characterization of human and mouse peroxiredoxin IV: evidence for inhibition by Prx-IV of epidermal growth factor- and p53-induced reactive oxygen species.

    PubMed

    Wong, C M; Chun, A C; Kok, K H; Zhou, Y; Fung, P C; Kung, H F; Jeang, K T; Jin, D Y

    2000-01-01

    The aim of this study was to identify and characterize human and mouse Prx-IV. We identified mouse peroxiredoxin IV (Prx-IV) by virtue of sequence homology to its human ortholog previously called AOE372. Mouse Prx-IV conserves an amino-terminal presequence coding for signal peptide. The amino acid sequences of mature mouse and human Prx-IV share 97.5% identity. Phylogenetic analysis demonstrates that Prx-IV is more closely related to Prx-I/-II/-III than to Prx-V/-VI. Previously, we mapped the mouse Prx-IV gene to chromosome X by analyzing two sets of multiloci genetic crosses. Here we performed further comparative analysis of mouse and human Prx-IV genomic loci. Consistent with the mouse results, human Prx-IV gene localized to chromosome Xp22.135-136, in close proximity to SAT and DXS7178. A bacterial artificial chromosome (BAC) clone containing the complete human Prx-IV locus was identified. The size of 7 exons and the sequences of the splice junctions were confirmed by PCR analysis. We conclude that mouse Prx-IV is abundantly expressed in many tissues. However, we could not detect Prx-IV in the conditioned media of NIH-3T3 and Jurkat cells. Mouse Prx-IV was specifically found in the nucleus-excluded region of cultured mouse cells. Intracellularly, overexpression of mouse Prx-IV prevented the production of reactive oxygen species induced by epidermal growth factor or p53. Taken together, mouse Prx-IV is likely a cytoplasmic or organellar peroxiredoxin involved in intracellular redox signaling.

  18. Regulation of extinction of cocaine-induced place preference by midkine is related to a differential phosphorylation of peroxiredoxin 6 in dorsal striatum.

    PubMed

    Gramage, Esther; Pérez-García, Carmen; Vicente-Rodríguez, Marta; Bollen, Silke; Rojo, Loreto; Herradón, Gonzalo

    2013-09-15

    The neurotrophic factors Midkine (MK) and Pleiotrophin (PTN) have been suggested to modulate drugs of abuse-induced effects. To test this hypothesis, cocaine (10 and 15mg/kg)-induced conditioned place preference (CPP) was rendered in PTN knockout (PTN-/-), MK knockout (MK-/-) and wild type (WT+/+) mice, and then extinguished after repeated saline injections (distributed in 4 extinction sessions). Cocaine induced a similar CPP in all the three genotypes. We found a significantly increased percentage of MK-/- mice that did not extinguish cocaine CPP at the end of the extinction sessions. Particularly, 40% of MK-/- mice did not extinguish cocaine (15mg/kg)-induced CPP compared to WT+/+ and PTN-/- mice (∼0-6%). Interestingly, we found that a greater magnitude of extinction of CPP after the first extinction session (5 days after last administration of cocaine) correlates with increased tyrosine phosphorylation of the enzyme peroxiredoxin 6 in the dorsal striatum of MK-/- mice. On the other hand, a greater magnitude of CPP extinction correlates with increased tyrosine phosphorylation of aconitase 2 in the prefrontal cortex of WT+/+ mice. In contrast, a lower magnitude of CPP extinction correlates with increased phosphorylation of aconitase 2 in the prefrontal cortex of PTN-/- mice, suggesting that the correlation between the tyrosine phosphorylation levels of aconitase 2 and magnitude of CPP extinction depends on the genotype considered. The data demonstrate that MK is a novel genetic factor that plays a role in the extinction of cocaine-induced CPP by mechanisms that may involve specific phosphorylation of striatal peroxiredoxin 6.

  19. Characterization of a 1-cysteine peroxiredoxin from big-belly seahorse (Hippocampus abdominalis); insights into host antioxidant defense, molecular profiling and its expressional response to septic conditions.

    PubMed

    Godahewa, G I; Perera, N C N; Elvitigala, Don Anushka Sandaruwan; Jayasooriya, R G P T; Kim, Gi-Young; Lee, Jehee

    2016-10-01

    1-cysteine peroxiredoxin (Prx6) is an antioxidant enzyme that protects cells by detoxifying multiple peroxide species. This study aimed to describe molecular features, functional assessments and potential immune responses of Prx6 identified from the big-belly seahorse, Hippocampus abdominalis (HaPrx6). The complete ORF (666 bp) of HaPrx6 encodes a polypeptide (24 kDa) of 222 amino acids, and harbors a prominent peroxiredoxin super-family domain, a peroxidatic catalytic center, and a peroxidatic cysteine. The deduced amino acid sequence of HaPrx6 shares a relatively high amino acid sequence similarity and close evolutionary relationship with Oplegnathus fasciatus Prx6. The purified recombinant HaPrx6 protein (rHaPrx6) was shown to protect plasmid DNA in the Metal Catalyzed Oxidation (MCO) assay and, together with 1,4-Dithiothreitol (DTT), protected human leukemia THP-1 cells from extracellular H2O2-mediated cell death. In addition, quantitative real-time PCR revealed that HaPrx6 mRNA was constitutively expressed in 14 different tissues, with the highest expression observed in liver tissue. Inductive transcriptional responses were observed in liver and kidney tissues of fish after treating them with bacterial stimuli, including LPS, Edwardsiella tarda, and Streptococcus iniae. These results suggest that HaPrx6 may play an important role in the immune response of the big-belly seahorse against microbial infection. Collectively, these findings provide structural and functional insights into HaPrx6.

  20. Thiol and sulfenic acid oxidation of AhpE, the one-cysteine peroxiredoxin from Mycobacterium tuberculosis: kinetics, acidity constants, and conformational dynamics.

    PubMed

    Hugo, Martín; Turell, Lucía; Manta, Bruno; Botti, Horacio; Monteiro, Gisele; Netto, Luis E S; Alvarez, Beatriz; Radi, Rafael; Trujillo, Madia

    2009-10-13

    Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen's antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhpE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

  1. Dysregulation of stathmin, a microtubule-destabilizing protein, and up-regulation of Hsp25, Hsp27, and the antioxidant peroxiredoxin 6 in a mouse model of familial amyotrophic lateral sclerosis.

    PubMed

    Strey, Christoph W; Spellman, Daniel; Stieber, Anna; Gonatas, Jacqueline O; Wang, Xiaosong; Lambris, John D; Gonatas, Nicholas K

    2004-11-01

    Gain-of-function mutations of the Cu/Zn superoxide dismutase (SOD1) gene cause dominantly inherited familial amyotrophic lateral sclerosis. The identification of differentially regulated proteins in spinal cords of paralyzed mice expressing SOD1(G93A) may contribute to understanding mechanisms of toxicity by mutant SOD1. Protein profiling showed dysregulation of Stathmin with a marked decrease of its most acidic and phosphorylated isoform, and up-regulation of heat shock proteins 25 and 27, peroxiredoxin 6, phosphatidylinositol transfer protein-alpha, apolipoprotein E, and ferritin heavy chain. Stathmin accumulated in the cytoplasm of 30% of spinal cord motor neurons with fragmented Golgi apparatus. Overexpression of Stathmin in HeLa cells was associated with collapse of microtubule networks and Golgi fragmentation. These results, together with the decrease of one Stathmin isoform, suggest a role of the protein in Golgi fragmentation. Mutant SOD1 co-precipitated and co-localized with Hsp25 in neurons and astrocytes. Mutant SOD1 may thus deprive cells of the anti-apoptotic and other protective activities of Hsp25. Astrocytes contained peroxiredoxin 6, a unique nonredundant antioxidant. The up-regulation of peroxiredoxin 6 probably constitutes a defense to oxidative stress induced by SOD1(G93A). Direct effects of SOD1(G93A) or sequential reactions triggered by the mutant may cause the protein changes.

  2. NTRC and chloroplast-generated reactive oxygen species regulate Pseudomonas syringae pv. tomato disease development in tomato and Arabidopsis.

    PubMed

    Ishiga, Yasuhiro; Ishiga, Takako; Wangdi, Tamding; Mysore, Kirankumar S; Uppalapati, Srinivasa Rao

    2012-03-01

    Coronatine (COR)-producing pathovars of Pseudomonas syringae, including pvs. tomato, maculicola, and glycinea, cause important diseases on tomato, crucifers, and soybean, respectively, and produce symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to COR. However, the significance of COR-induced chlorosis in localized lesion development and the molecular basis of disease-associated cell death is largely unknown. To identify host (chloroplast) genes that play a role in COR-mediated chlorosis, we used a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing and identified a gene which encodes 2-Cys peroxiredoxin (Prxs) that, when silenced, produced a spreading hypersensitive or necrosis-like phenotype instead of chlorosis after COR application in a COI1-dependent manner. Loss-of-function analysis of Prx and NADPH-dependent thioredoxin reductase C (NTRC), the central players of a chloroplast redox detoxification system, resulted in spreading accelerated P. syringae pv. tomato DC3000 disease-associated cell death with enhanced reactive oxygen species (ROS) accumulation in a COR-dependent manner in tomato and Arabidopsis. Consistent with these results, virulent strain DC3000 suppressed the expression of Prx and NTRC in Arabidopsis and tomato during pathogenesis. However, interestingly, authentic COR suppressed the expression of Prx and NTRC in tomato but not in Arabidopsis, suggesting that COR in conjunction with other effectors may modulate ROS and cell death in different host species. Taken together, these results indicated that NTRC or Prx function as a negative regulator of pathogen-induced cell death in the healthy tissues that surround the lesions, and COR-induced chloroplast-localized ROS play a role in enhancing the disease-associated cell death.

  3. Evaluation of hepatic changes and local and systemic immune responses in goats immunized with recombinant Peroxiredoxin (Prx) and challenged with Fasciola hepatica.

    PubMed

    Mendes, Ricardo E; Pérez-Ecija, Rafael A; Zafra, Rafael; Buffoni, Leandro; Martínez-Moreno, Alvaro; Dalton, John P; Mulcahy, Grace; Pérez, José

    2010-04-01

    Protection against Fasciola hepatica in goats immunized with Peroxiredoxin (Prx) was assessed. The experimental trial consisted of three groups of seven animals; group 1 were unimmunized and uninfected, group 2 were immunized with adjuvant only and group 3 were immunized with recombinant Prx in adjuvant (immunized and infected). Immunization with Prx in Quil A adjuvant, group 3, induced a reduction in fluke burden of 33.04% when compared to adjuvant control, group 2, although this difference was not significant. The hepatic gross and microscopical morphometric study revealed lower damage in the Prx-immunized compared to group 2 (p<0.05). Furthermore, immunohistochemical studies revealed that the Prx-immunized group exhibited reduced infiltration of CD4(+), CD8(+), IFN-gamma(+) and TCR(+) (p<0.05); and CD2(+) and IL-4(+) (p<0.001) in hepatic lesions. Levels of anti-Prx serum IgG in group 3 showed a significant increase at the 4th week after challenge infection compared with group 2 (p<0.0001). This is the first report of ruminant immunization with recombinant Prx of F. hepatica. The study shows that this vaccine significantly reduces hepatic damage and encourages further studies to improve the vaccine efficacy.

  4. Molecular identification of 1-Cys peroxiredoxin and anthocyanidin/flavonol 3-O-galactosyltransferase from proanthocyanidin-rich young fruits of persimmon (Diospyros kaki Thunb.).

    PubMed

    Ikegami, Ayako; Akagi, Takashi; Potter, Daniel; Yamada, Masahiko; Sato, Akihiko; Yonemori, Keizo; Kitajima, Akira; Inoue, Kentaro

    2009-09-01

    Fruits of persimmon (Diospyros kaki Thunb.) accumulate large amounts of proanthocyanidins (PAs) in the early stages of development. Astringent (A)-type fruits remain rich in soluble PAs even after they reach full-mature stage, whereas non-astringent (NA)-type fruits lose these compounds before full maturation. As a first step to elucidate the mechanism of PA accumulation in this non-model species, we used suppression subtractive hybridization to identify transcripts accumulating differently in young fruits of A- and NA-type. Interestingly, only a few clones involved in PA biosynthesis were identified in A-NA libraries. Represented by multiple clones were those encoding a novel 1-Cys peroxiredoxin and a new member of family 1 glycosyltransferases. Quantitative RT-PCR analyses confirmed correlation of the amount of PAs and accumulation of transcripts encoding these proteins in young persimmon fruits. Furthermore, the new family 1 glycosyltransferase was produced in Escherichia coli and shown to efficiently catalyze galactosylation at 3-hydroxyl groups of several anthocyanidins and flavonols. These findings suggest a complex mechanism of PA accumulation in persimmon fruits.

  5. Cervical Cancer Cell Line Secretome Highlights the Roles of Transforming Growth Factor-Beta-Induced Protein ig-h3, Peroxiredoxin-2, and NRF2 on Cervical Carcinogenesis

    PubMed Central

    Zoidakis, Jerome; Makridakis, Manousos; Lygirou, Vasiliki; Mermelekas, George; Vougas, Konstantinos; Drakakis, Peter

    2017-01-01

    Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV−), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis. PMID:28261610

  6. Evening and morning peroxiredoxin-2 redox/oligomeric state changes in obstructive sleep apnea red blood cells: Correlation with polysomnographic and metabolic parameters.

    PubMed

    Feliciano, Amélia; Vaz, Fátima; Torres, Vukosava M; Valentim-Coelho, Cristina; Silva, Rita; Prosinecki, Vesna; Alexandre, Bruno M; Carvalho, Ana S; Matthiesen, Rune; Malhotra, Atul; Pinto, Paula; Bárbara, Cristina; Penque, Deborah

    2017-02-01

    We have examined the effects of Obstructive Sleep Apnea (OSA) on red blood cell (RBC) proteome variation at evening/morning day time to uncover new insights into OSA-induced RBC dysfunction that may lead to OSA manifestations. Dysregulated proteins mainly fall in the group of catalytic enzymes, stress response and redox regulators such as peroxiredoxin 2 (PRDX2). Validation assays confirmed that at morning the monomeric/dimeric forms of PRDX2 were more overoxidized in OSA RBC compared to evening samples. Six month of positive airway pressure (PAP) treatment decreased this overoxidation and generated multimeric overoxidized forms associated with chaperone/transduction signaling activity of PRDX2. Morning levels of overoxidized PRDX2 correlated with polysomnographic (PSG)-arousal index and metabolic parameters whereas the evening level of disulfide-linked dimer (associated with peroxidase activity of PRDX2) correlated with PSG parameters. After treatment, morning overoxidized multimer of PRDX2 negatively correlated with fasting glucose and dopamine levels. Overall, these data point toward severe oxidative stress and altered antioxidant homeostasis in OSA RBC occurring mainly at morning time but with consequences till evening. The beneficial effect of PAP involves modulation of the redox/oligomeric state of PRDX2, whose mechanism and associated chaperone/transduction signaling functions deserves further investigation. RBC PRDX2 is a promising candidate biomarker for OSA severity and treatment monitoring, warranting further investigation and validation.

  7. Protein engineering of the quaternary sulfiredoxin.peroxiredoxin enzyme.substrate complex reveals the molecular basis for cysteine sulfinic acid phosphorylation.

    PubMed

    Jönsson, Thomas J; Johnson, Lynnette C; Lowther, W Todd

    2009-11-27

    Oxidative stress can damage the active site cysteine of the antioxidant enzyme peroxiredoxin (Prx) to the sulfinic acid form, Prx-SO(2)(-). This modification leads to inactivation. Sulfiredoxin (Srx) utilizes a unique ATP-Mg(2+)-dependent mechanism to repair the Prx molecule. Using selective protein engineering that involves disulfide bond formation and site-directed mutagenesis, a mimic of the enzyme.substrate complex has been trapped. Here, we present the 2.1 A crystal structure of human Srx in complex with PrxI, ATP, and Mg(2+). The Cys(52) sulfinic acid moiety was substituted by mutating this residue to Asp, leading to a replacement of the sulfur atom with a carbon atom. Because the Srx reaction cannot occur, the structural changes in the Prx active site that lead to the attack on ATP may be visualized. The local unfolding of the helix containing C52D resulted in the packing of Phe(50) in PrxI within a hydrophobic pocket of Srx. Importantly, this structural rearrangement positioned one of the oxygen atoms of Asp(52) within 4.3 A of the gamma-phosphate of ATP bound to Srx. These observations support a mechanism where phosphorylation of Prx-SO(2)(-) is the first chemical step.

  8. Peroxiredoxin 2 is essential for maintaining cancer stem cell-like phenotype through activation of Hedgehog signaling pathway in colon cancer

    PubMed Central

    Wang, Rong; Wei, Jinlai; Zhang, Shouru; Wu, Xingye; Guo, Jinbao; Liu, Maoxi; Du, Kunli; Xu, Jun; Peng, Linglong; Lv, Zhenbing; You, Wenxian; Xiong, Yongfu; Fu, Zhongxue

    2016-01-01

    Cancer stem cells (CSCs) are a key target for reducing tumor growth, metastasis, and recurrence. Redox status is a critical factor in the maintenance of CSCs, and the antioxidant enzyme Peroxiredoxin 2 (Prdx2) plays an important role in the development of colon cancer. Therefore, we investigated the contribution of Prdx2 to the maintenance of stemness of colon CSCs. Here, we used short-hairpin RNAs and a Prdx2-overexpression vector to determine the effects of Prdx2. We demonstrated that knockdown of Prdx2 reduced the self-renewal and sphere formation and resulted in increased 5-FU-induced apoptosis in human colon CSCs. Prdx2 overexpression induced reversion of the self-renewal and sphere formation. Furthermore, the effects of Prdx2 resulted in an altered expression of stemness associated with the Hh/Gli1 signaling pathway. Finally, knockdown of Prdx2 in CD133+ cells reduced the volume of xenograft tumors in BALB/c-nu mice. Taken together, colon CSCs overexpress Prdx2, which promotes their stem cell properties via the Hh/Gli1 signaling pathway. The results suggest that Prdx2 may be an effective therapeutic target for the elimination of CSCs in colorectal cancer. PMID:27894099

  9. Glutathione Peroxidase of Pennisetum glaucum (PgGPx) Is a Functional Cd2+ Dependent Peroxiredoxin that Enhances Tolerance against Salinity and Drought Stress.

    PubMed

    Islam, Tahmina; Manna, Mrinalini; Reddy, Malireddy K

    2015-01-01

    Reactive oxygen species (ROS) arise in the plant system due to inevitable influence of various environmental stimuli. Glutathione peroxidases are one of the important ROS scavengers inside the cell. A glutathione peroxidase (PgGPx) gene was previously found from Pennisetum glauccum abiotic stressed cDNA library. Enzyme kinetics data revealed that PgGPx possessed preference towards thioredoxin rather than glutathione as electron donor and thus belongs to the functional peroxiredoxin group. Moreover, its activity was found to be dependent on divalent cations, especially Cd2+ and homology model showed the presence of Cd2+ binding site in the protein. Site directed mutagenesis study of PgGPx protein revealed the vital role of two conserved Cysteine residues for its enzymatic activity and structural folding. Expression analysis suggested that PgGPx transcript is highly up-regulated in response to salinity and drought stresses. When expressed ectopically, PgGPx showed enhanced tolerance against multiple abiotic stresses in prokaryotic E. coli and model plant, rice. Transgenic rice plants showed lesser accumulation of MDA and H2O2; and higher accumulation of proline as compared to wild type (WT) plants in response to both salinity and drought stresses that clearly indicates suppression of lipid peroxidation and ROS generation in transgenic lines. Moreover, transgenic plants maintained better photosynthesis efficiency and higher level of antioxidant enzyme activity as compared to WT plants under stress conditions. These results clearly indicate the imperative role of PgGPx in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance towards oxidative stress.

  10. Upregulation of peroxiredoxin III in doxorubicin-induced cytotoxicity and the FoxO3a-dependent expression in H9c2 cardiac cells.

    PubMed

    Liu, Mi-Hua; Zhang, Yuan; He, Jun; Tan, Tian-Ping; Wu, Shao-Jian; Fu, Hong-Yun; Chen, Yu-Dan; Liu, Jun; LE, Qun-Fang; Hu, Heng-Jing; Yuan, Cong; Lin, Xiao-Long

    2015-10-01

    Doxorubicin (DOX) is an efficient drug used in cancer therapy; however, it produces reactive oxygen species (ROS) that induce severe cytotoxicity, limiting its clinical application. The aim of the present study was to investigate the role of peroxiredoxin III (Prx III) in DOX-induced H9c2 cell injuries. Following DOX treatment, the expression of phosphorylated-FoxO3a (p-FoxO3a) was decreased and Prx III expression was increased in H9c2 cells. In order to detect whether oxidative stress was involved in the induction of Prx III expression by FoxO3a, exogenous H2O2 was used to induce oxidative stress in the H9c2 cells. Apoptosis of H9c2 cardiomyocytes was assessed using methyl thiazolyl tetrazolium assay and Hoechst staining. The levels of Prx III and p-FoxO3a were evaluated using western blot analysis. As expected, H2O2 was found to mimic the effect of DOX, decreasing the expression of p-FoxO3a and increasing the expression of Prx III. In addition, the study evaluated whether the transcription factor FoxO3a was essential for the expression of Prx III. Pretreatment of H9c2 cells with N-acetyl-L-cysteine (NAC), a scavenger of ROS, prior to exposure to DOX dramatically increased the phosphorylation of FoxO3a and led to a marked reduction in Prx III expression in the H9c2 cells. In conclusion, the results of the current study suggest that FoxO3a mediates the expression of Prx III in DOX-induced injuries.

  11. Close teamwork between Nrf2 and peroxiredoxins 1 and 6 for the regulation of prostaglandin D2 and E2 production in macrophages in acute inflammation.

    PubMed

    Ishii, Tetsuro

    2015-11-01

    Inflammation is a complex biological self-defense reaction triggered by tissue damage or infection by pathogens. Acute inflammation is regulated by the time- and cell type-dependent production of cytokines and small signaling molecules including reactive oxygen species and prostaglandins. Recent studies have unveiled the important role of the transcription factor Nrf2 in the regulation of prostaglandin production through transcriptional regulation of peroxiredoxins 1 and 6 (Prx1 and Prx6) and lipocalin-type prostaglandin D synthase (L-PGDS). Prx1 and Prx6 are multifunctional proteins important for cell protection against oxidative stress, but also work together to facilitate production of prostaglandins E2 and D2 (PGE2 and PGD2). Prx1 secreted from cells under mild oxidative stress binds Toll-like receptor 4 and induces NF-κB activation, important for the expression of cyclooxygenase-2 and microsomal PGE synthase-1 (mPGES-1) expression. The activated MAPKs p38 and ERK phosphorylate Prx6, leading to NADPH oxidase-2 activation, which contributes to production of PGD2 by hematopoietic prostaglandin D synthase (H-PGDS). PGD2 and its end product 15-deoxy-∆(12,14)-prostaglandin J2 (15d-PGJ2) activate Nrf2 thereby forming a positive feedback loop for further production of PGD2 by L-PGDS. Maintenance of cellular glutathione levels is an important role of Nrf2 not only for cell protection but also for the synthesis of prostaglandins, as mPGES-1 and H-PGDS require glutathione for their activities. This review is aimed at describing the functions of Prx1 and Prx6 in the regulation of PGD2 and PGE2 production in acute inflammation in macrophages and the importance of 15d-PGJ2 as an intrinsic Nrf2 activator.

  12. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    PubMed

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

  13. Absence of Peroxiredoxin 6 Amplifies the Effect of Oxidant Stress on Mobility and SCSA/CMA3 Defined Chromatin Quality and Impairs Fertilizing Ability of Mouse Spermatozoa1

    PubMed Central

    Ozkosem, Burak; Feinstein, Sheldon I.; Fisher, Aron B.; O'Flaherty, Cristian

    2016-01-01

    Oxidative stress, the imbalance between reactive oxygen species production and antioxidant defenses, is associated with male infertility. Peroxiredoxins (PRDXs) are antioxidant enzymes with a wide distribution in spermatozoa. PRDX6 is highly abundant and located in all subcellular compartments of the spermatozoon. Infertile men have lower levels of sperm PRDX6 associated with low sperm motility and high DNA damage. In order to better understand the role of PRDX6 in male reproduction, the aim of this study was to elucidate the impact of the lack of PRDX6 on male mouse fertility. Spermatozoa lacking PRDX6 showed significantly increased levels of cellular oxidative damage evidenced by high levels of lipid peroxidation, 8-hydroxy-deoxyguanosine (DNA oxidation), and protein oxidation (S-glutathionylation and carbonylation), lower sperm chromatin quality (high DNA fragmentation and low DNA compaction, due to low levels of protamination and a high percentage of free thiols), along with decreased sperm motility and impairment of capacitation as compared with wild-type (WT) spermatozoa. These manifestations of damage are exacerbated by tert-butyl hydroperoxide treatment in vivo. While WT males partially recovered the quality of their spermatozoa (in terms of motility and sperm DNA integrity), Prdx6−/− males showed higher levels of sperm damage (lower motility and chromatin integrity) 6 mo after the end of treatment. In conclusion, Prdx6−/− males are more vulnerable to oxidative stress than WT males, resulting in impairment of sperm quality and ability to fertilize the oocyte, compatible with the subfertility phenotype observed in these knockout mice. PMID:26792942

  14. Peroxiredoxin 2, glutathione peroxidase, and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress.

    PubMed

    Rocha, S; Gomes, D; Lima, M; Bronze-da-Rocha, E; Santos-Silva, A

    2015-01-01

    Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H2O2-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H2O2 concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H2O2, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H2O2-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton-lipid bilayer, which might lead to membrane destabilization.

  15. Glutathione Peroxidase of Pennisetum glaucum (PgGPx) Is a Functional Cd2+ Dependent Peroxiredoxin that Enhances Tolerance against Salinity and Drought Stress

    PubMed Central

    Islam, Tahmina; Manna, Mrinalini; Reddy, Malireddy K.

    2015-01-01

    Reactive oxygen species (ROS) arise in the plant system due to inevitable influence of various environmental stimuli. Glutathione peroxidases are one of the important ROS scavengers inside the cell. A glutathione peroxidase (PgGPx) gene was previously found from Pennisetum glauccum abiotic stressed cDNA library. Enzyme kinetics data revealed that PgGPx possessed preference towards thioredoxin rather than glutathione as electron donor and thus belongs to the functional peroxiredoxin group. Moreover, its activity was found to be dependent on divalent cations, especially Cd2+ and homology model showed the presence of Cd2+ binding site in the protein. Site directed mutagenesis study of PgGPx protein revealed the vital role of two conserved Cysteine residues for its enzymatic activity and structural folding. Expression analysis suggested that PgGPx transcript is highly up-regulated in response to salinity and drought stresses. When expressed ectopically, PgGPx showed enhanced tolerance against multiple abiotic stresses in prokaryotic E. coli and model plant, rice. Transgenic rice plants showed lesser accumulation of MDA and H2O2; and higher accumulation of proline as compared to wild type (WT) plants in response to both salinity and drought stresses that clearly indicates suppression of lipid peroxidation and ROS generation in transgenic lines. Moreover, transgenic plants maintained better photosynthesis efficiency and higher level of antioxidant enzyme activity as compared to WT plants under stress conditions. These results clearly indicate the imperative role of PgGPx in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance towards oxidative stress. PMID:26600014

  16. Disruption of redox catalytic functions of peroxiredoxin-thioredoxin complex in Mycobacterium tuberculosis H37Rv using small interface binding molecules.

    PubMed

    Gurung, Arun Bahadur; Das, Amit Kumar; Bhattacharjee, Atanu

    2017-04-01

    Mycobacterium tuberculosis has distinctive ability to detoxify various microbicidal superoxides and hydroperoxides via a redox catalytic cycle involving thiol reductants of peroxiredoxin (Prx) and thioredoxin (Trx) systems which has conferred on it resistance against oxidative killing and survivability within host. We have used computational approach to disrupt catalytic functions of Prx-Trx complex which can possibly render the pathogen vulnerable to oxidative killing in the host. Using protein-protein docking method, we have successfully constructed the Prx-Trx complex. Statistics of interface region revealed contact area of each monomer less than 1500Å(2) and enriched in polar amino acids indicating transient interaction between Prx and Trx. We have identified ZINC40139449 as a potent interface binding molecule through virtual screening of drug-like compounds from ZINC database. Molecular dynamics (MD) simulation studies showed differences in structural properties of Prx-Trx complex both in apo and ligand bound states with regard to root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuations (RMSF), solvent accessible surface area (SASA) and number of hydrogen bonds (NHBs). Interestingly, we found stability of two conserved catalytic residues Cys61 and Cys174 of Prx and conserved catalytic motif, WCXXC of Trx upon binding of ZINC40139449. The time dependent displacement study reveals that the compound is quite stable in the interface binding region till 30ns of MD simulation. The structural properties were further validated by principal component analysis (PCA). We report ZINC40139449 as promising lead which can be further evaluated by in vitro or in vivo enzyme inhibition assays.

  17. Differential protein expression of peroxiredoxin I and II by benzo(a)pyrene and quercetin treatment in 22Rv1 and PrEC prostate cell lines

    SciTech Connect

    Chaudhary, Amit; Pechan, Tibor; Willett, Kristine L. . E-mail: kwillett@olemiss.edu

    2007-04-15

    Mechanisms of benzo(a)pyrene (BaP)-mediated toxicity and chemopreventative potential of quercetin in prostate cancer are poorly understood. Two-dimensional gel electrophoresis was used to map the differences in protein expression in BaP (1 {mu}M)- and quercetin (5 {mu}M)-treated 22Rv1 human prostate cancer cells. As compared to DMSO, 26 proteins in BaP and 41 proteins in quercetin were found to be differentially expressed ({+-} 2-fold). Western blots confirmed that BaP increased peroxiredoxin (Prx) Prx I and decreased Prx II in 22Rv1 cells. Similar results were found in PrEC normal prostate epithelial cells. Quercetin (up to 10 {mu}M) upregulated Prx II without altering Prx I levels in 22Rv1 cells whereas in PrEC cells, it did not alter the constitutive protein expression of Prx I or II. The lack of quercetin-mediated changes in Prx expression suggests that quercetin does not interfere with H{sub 2}O{sub 2} levels, and thus may have no deleterious effect in normal prostate cells. Quercetin inhibited both BaP-mediated effects on Prx I and II in 22Rv1 cells. In PrEC cells, quercetin inhibited BaP-mediated upregulation of Prx I and had tendency to neutralize BaP-mediated downregulation of Prx II. Quercetin also inhibited BaP-induced concentrations of reactive oxygen species in both 22Rv1 and PrEC cells. These results suggest that Prx I and II may be involved in BaP-mediated toxicity and the potential chemopreventative mechanisms of quercetin.

  18. p53 status is a major determinant of effects of decreasing peroxiredoxin I expression on tumor growth and response of lung cancer cells to treatment

    SciTech Connect

    Chen, M.-F. . E-mail: miaofen@adm.cgmh.org.tw; Chen, W.-C.; Wu, C.-T.; Lin, P.-Y.; Shau Hungyi; Liao, S.-K.; Yang, C.-T.; Lee, K.-D.

    2006-12-01

    Purpose: The potential roles of peroxiredoxin (Prx) I in carcinogenesis and treatment have been explored. Our previous study revealed differences between A549 (functional p53) and H1299 (null p53) Prx I antisense transfectants. The discrepancy might have resulted from the p53 status. In this study, we further investigated the role of Prx I and p53 on lung cancer growth and the response to treatment in vitro and in vivo. Methods: We established stable A549 and H1299 transfectants with Prx I antisense and p53, respectively. We then examined their characteristics in vitro and used nude mice xenografts of these cell lines to compare their capacity for tumor invasion and spontaneous metastasis and their sensitivity to radiotherapy. Results: Increased reactive oxygen species caused by lower Prx I activity induced p53 expression. In lethal stress, the augmentation of reactive oxygen species was partially reversed by blocking p53 in A549 with Prx I antisense. We demonstrated the potential contribution of p53-dependent mechanisms to inhibit lung tumor growth and increase radiosensitization using H1299 transfected with p53 in vitro and in vivo. An increased p53 level attenuated the capacity of the cells for metastasis by decreasing vascular endothelial growth factor and induced radiosensitization by increased apoptosis and cell senescence and by regulating intracellular reactive oxygen species. Conclusion: These results suggest that p53 status has an important role in the tumor-inhibiting and radiosensitizing effects of decreasing Prx I. Both Prx I and p53 may be powerful prognosticators for lung cancer.

  19. A Redox 2-Cys Mechanism Regulates the Catalytic Activity of Divergent Cyclophilins1[W

    PubMed Central

    Campos, Bruna Medéia; Sforça, Mauricio Luis; Ambrosio, Andre Luis Berteli; Domingues, Mariane Noronha; Brasil de Souza, Tatiana de Arruda Campos; Barbosa, João Alexandre Ribeiro Gonçalvez; Leme, Adriana Franco Paes; Perez, Carlos Alberto; Whittaker, Sara Britt-Marie; Murakami, Mario Tyago; Zeri, Ana Carolina de Matos; Benedetti, Celso Eduardo

    2013-01-01

    The citrus (Citrus sinensis) cyclophilin CsCyp is a target of the Xanthomonas citri transcription activator-like effector PthA, required to elicit cankers on citrus. CsCyp binds the citrus thioredoxin CsTdx and the carboxyl-terminal domain of RNA polymerase II and is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteine (Cys) residues Cys-40 and Cys-168, and the conserved glutamate (Glu) Glu-83. Despite the suggested roles in ATP and metal binding, the functions of these unique structural elements remain unknown. Here, we show that the conserved Cys residues form a disulfide bond that inactivates the enzyme, whereas Glu-83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that Cys-40 and Cys-168 are required for the interaction with CsTdx and that CsCyp binds the citrus carboxyl-terminal domain of RNA polymerase II YSPSAP repeat. Our data support a model where formation of the Cys-40-Cys-168 disulfide bond induces a conformational change that disrupts the interaction of the divergent and catalytic loops, via Glu-83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop-displacement mechanism. PMID:23709667

  20. The Antioxidant Peroxiredoxin 6 (Prdx6) Exhibits Different Profiles in the Livers of Seawater- and Fresh Water-Acclimated Milkfish, Chanos chanos, upon Hypothermal Challenge

    PubMed Central

    Chang, Chia-Hao; Lo, Wan-Yu; Lee, Tsung-Han

    2016-01-01

    A tropical species, the euryhaline milkfish (Chanos chanos), is a crucial economic species in Southeast Asia and is intolerant of water temperature below 12°C. Large numbers of milkfish die during cold periods in winter. Hypothermal environments usually increase oxidative stress in teleosts, and the liver is the major organ for anti-oxidative responses in the body. Peroxiredoxin-6 (Prdx6) in mammals is a multi-functional enzyme and acts as both glutathione peroxidase, phospholipase A2 and acyl-transferase for maintenance of redox status and prevention of cell membrane peroxidation. Prdx6 can protect cells from oxidant-induced membrane damage by translocating the Prdx6 protein from the cytosol to the membrane. Upon cold stress, Ccprdx6 transcript levels were up-regulated after 24 h and 96 h in livers of fresh water (FW)- and seawater (SW)-acclimated milkfish, respectively. In the hypothermal FW group, the Prdx6 protein was up-regulated in the cytosol of hepatocytes with a similar role as glutathione peroxidase to reduce oxidative stress upon hypothermal challenge. Conversely, in hypothermal SW milkfish, total Prdx6 protein was down-regulated. However, cytosolic Prdx6 protein was translocated to the membrane, using the ability of phospholipase A2 to stabilize the membrane redox state. Moreover, H2O2 content was increased in FW-acclimated milkfish livers upon hypothermal challenge. Ex vivo H2O2 treatment of milkfish livers also induced Ccprdx6 transcriptional expression, which provided more evidence of the antioxidant role of milkfish Prdx6. Taken together, upon hypothermal challenge, greater oxidative stress in livers of FW-acclimated milkfish rather than SW-acclimated individuals led to different profiles of hepatic CcPrdx6 expression between the FW and SW group. The results indicated that CcPrdx6 played the role of antioxidant with different mechanisms, i.e., binding to reactive oxygen species and stabilizing membrane fluidity, in livers of hypothermal FW and SW

  1. The Prx Q protein of Arabidopsis thaliana is a member of the luminal chloroplast proteome.

    PubMed

    Petersson, Ulrika A; Kieselbach, Thomas; García-Cerdán, José G; Schröder, Wolfgang P

    2006-11-13

    Peroxiredoxins have been discovered in many organisms ranging from eubacteria to mammals, and their known biological functions include both oxidant defense and signal transduction. The genome of Arabidopsis thaliana encodes for ten individual peroxiredoxins, of which four are located in the chloroplast. The best-characterized member of the chloroplast peroxiredoxins is 2-Cys Prx that is associated with the stroma side of the thylakoid membrane and is considered to participate in antioxidant defense and protection of photosynthesis. This study addressed the chloroplast peroxiredoxin Prx Q and showed that its subcellular location is the lumen of the thylakoid membrane. To get insight in the biological function of the Prx Q protein of Arabidopsis, the protein levels of the Prx Q protein in thylakoid membranes were studied under different light conditions and oxidative stress. A T-DNA knockout mutant of Prx Q did not show any visible phenotype and had normal photosynthetic performance with a slightly increased oxygen evolving activity.

  2. Mutagenesis and modeling of the peroxiredoxin (Prx) complex with the NMR structure of ATP-bound human sulfiredoxin implicate aspartate 187 of Prx I as the catalytic residue in ATP hydrolysis.

    PubMed

    Lee, Duck-Yeon; Park, Sung Jun; Jeong, Woojin; Sung, Ho Jin; Oho, Taena; Wu, Xiongwu; Rhee, Sue Goo; Gruschus, James M

    2006-12-26

    The catalytic cysteine of certain members of the peroxiredoxin (Prx) family can be hyperoxidized to cysteinesulfinic acid during reduction of peroxides. Sulfiredoxin is responsible for the ATP-dependent reduction of cysteinesulfinic acid (SO2H) of hyperoxidized Prx. Here we report the NMR solution structure of human sulfiredoxin (hSrx), both with and without bound ATP, and we model the complex of ATP-bound hSrx with Prx. Binding ATP causes only small changes in the NMR structure of hSrx, and the bound ATP conformation is quite similar to that seen for the previously reported X-ray structure of the ADP-hSrx complex. Although hSrx binds ATP, it does not catalyze hydrolysis by itself and has no catalytic acid residue typical of most ATPase and kinase family proteins. For modeling the complex, the ATP-bound hSrx was docked to hyperoxidized Prx II using EMAP of CHARMM. In the model complex, Asn186 of Prx II (Asp187 of Prx I) is in contact with the hSrx-bound ATP beta- and gamma-phosphate groups. Asp187 of Prx I was mutated to alanine and asparagine, and binding and activity of the mutants with hSrx were compared to those of the wild type. For the D187N mutant, both binding and hydrolysis and reduction activities were comparable to those of the wild type, whereas for D187A, binding was unimpaired but ATP hydrolysis and reduction did not occur. The modeling and mutagenesis analyses strongly implicate Asp187 of Prx I as the catalytic residue responsible for ATP hydrolysis in the cysteinesulfinic acid reduction of Prx by hSrx.

  3. A preliminary study on genotypic differences in transcript abundance of drought-responsive genes in sugar beet.

    PubMed

    Rajabi, Abazar; Ranji, Zabihallah; Griffiths, Zhoward; Ober, Eric S

    2007-10-15

    In this study, four sugar beet genotypes of differing responses to drought were selected from a field experiment conducted under well-watered and water-limited conditions in 2004. In addition, two candidate genes: 2-cysteine peroxiredoxin (2-cys prx) and Nucleoside Diphosphate Kinase (NDPK), thought to be associated with drought tolerance, were chosen from a previous proteomics study in sugar beet. An expression analysis of the two drought-regulated genes using semi-quantitative reverse transcription Polymerase Chain Reaction (RT-PCR) indicated that there were genotypic differences in the transcript abundance of the candidate genes with the differences in the expression level of 2-cys prx being likely associated with the drought responses of the genotypes in a two-year field study. However, the expression analysis of the genes has to be investigated at different stages of the stress period on more genotypes.

  4. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp. PCC7120.

    PubMed

    Sánchez-Riego, Ana M; Mata-Cabana, Alejandro; Galmozzi, Carla V; Florencio, Francisco J

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2-Cys Prx) as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however, nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (ΔntrC), apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species) in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments.

  5. Changes in the alternative electron sinks and antioxidant defence in chloroplasts of the extreme halophyte Eutrema parvulum (Thellungiella parvula) under salinity

    PubMed Central

    Uzilday, Baris; Ozgur, Rengin; Sekmen, A. Hediye; Yildiztugay, Evren; Turkan, Ismail

    2015-01-01

    Background and Aims Eutrema parvulum (synonym, Thellungiella parvula) is an extreme halophyte that thrives in high salt concentrations (100–150 mm) and is closely related to Arabidopsis thaliana. The main aim of this study was to determine how E. parvulum uses reactive oxygen species (ROS) production, antioxidant systems and redox regulation of the electron transport system in chloroplasts to tolerate salinity. Methods Plants of E. parvulum were grown for 30 d and then treated with either 50, 200 or 300 mm NaCl. Physiological parameters including growth and water relationships were measured. Activities of antioxidant enzymes were determined in whole leaves and chloroplasts. In addition, expressions of chloroplastic redox components such as ferrodoxin thioredoxin reductases (FTR), NADPH thioredoxin reductases (NTRC), thioredoxins (TRXs) and peroxiredoxins (PRXs), as well as genes encoding enzymes of the water–water cycle and proline biosynthesis were measured. Key Results Salt treatment affected water relationships negatively and the accumulation of proline was increased by salinity. E. parvulum was able to tolerate 300 mm NaCl over long periods, as evidenced by H2O2 content and lipid peroxidation. While Ca2+ and K+ concentrations were decreased by salinity, Na+ and Cl– concentrations increased. Efficient induction of activities and expressions of water–water cycle enzymes might prevent accumulation of excess ROS in chloroplasts and therefore protect the photosynthetic machinery in E. parvulum. The redox homeostasis in chloroplasts might be achieved by efficient induction of expressions of redox regulatory enzymes such as FTR, NTRC, TRXs and PRXs under salinity. Conclusions E. parvulum was able to adapt to osmotic stress by an efficient osmotic adjustment mechanism involving proline and was able to regulate its ion homeostasis. In addition, efficient induction of water–water cycle enzymes and other redox regulatory components such as TRXs and PRXs in

  6. Thioredoxins, Glutaredoxins, and Peroxiredoxins—Molecular Mechanisms and Health Significance: from Cofactors to Antioxidants to Redox Signaling

    PubMed Central

    Hanschmann, Eva-Maria; Godoy, José Rodrigo; Berndt, Carsten; Hudemann, Christoph

    2013-01-01

    Abstract Thioredoxins (Trxs), glutaredoxins (Grxs), and peroxiredoxins (Prxs) have been characterized as electron donors, guards of the intracellular redox state, and “antioxidants”. Today, these redox catalysts are increasingly recognized for their specific role in redox signaling. The number of publications published on the functions of these proteins continues to increase exponentially. The field is experiencing an exciting transformation, from looking at a general redox homeostasis and the pathological oxidative stress model to realizing redox changes as a part of localized, rapid, specific, and reversible redox-regulated signaling events. This review summarizes the almost 50 years of research on these proteins, focusing primarily on data from vertebrates and mammals. The role of Trx fold proteins in redox signaling is discussed by looking at reaction mechanisms, reversible oxidative post-translational modifications of proteins, and characterized interaction partners. On the basis of this analysis, the specific regulatory functions are exemplified for the cellular processes of apoptosis, proliferation, and iron metabolism. The importance of Trxs, Grxs, and Prxs for human health is addressed in the second part of this review, that is, their potential impact and functions in different cell types, tissues, and various pathological conditions. Antioxid. Redox Signal. 19, 1539–1605. PMID:23397885

  7. Fancy a gene? A surprisingly complex evolutionary history of peroxiredoxins.

    PubMed Central

    Zíková, Alena; Oborník, Miroslav; Lukeš, Julius

    2015-01-01

    While the phylum Apicomplexa includes “only” several thousand described species of obligatory parasites of animals, it may in fact be the most specious group of parasitic protists with over a million species 1. The best known representatives are Plasmodium spp., Toxoplasma gondii and Cryptosporidium spp., which belong to the most important and widespread human parasites exacting an enormous disease burden. On the other hand, dinoflagellates and colpodellids, which are monophyletic with the apicomplexans, are ecologically highly significant, as they belong to the most abundant marine protists 2. As the common ancestor of these groups was most likely a free-living photosynthesizing protist, one wonders, which evolutionary forces contributed to the dramatic transition of some of its descendants into the arguably most successful intracellular parasites? Although a range of various processes and mechanisms contributed to this transition, most likely it also involved an acquisition of genes via horizontal gene transfer (HGT), which might have provided typical characteristics of a parasitic cell, such as immune escape, nutritional dependence and the capacity to invade other cells.

  8. Peroxiredoxins: A Model for a Self-Assembling Nanoscale System

    DTIC Science & Technology

    2014-08-24

    identify if any other similar proteins were known in related bacterial species. However, it was found that the amino acid sequence reported by Logan and...shown in Table 3.1. It is unlikely that any species containing a sulfenic acid (R-SOH in Table 3.1) would be seen, as this species would be expected...that any sulfinic acid groups formed were not present before beginning the assay, as all protein used was from the same source. The sample also shows

  9. Structural Characterisation of Proteins from the Peroxiredoxin Family

    DTIC Science & Technology

    2014-01-01

    discussed in chapter four). Although a vaccine and multiple drugs are available, tuberculosis (TB) still kills around 1.3 million and infects over 8...Mycobacterium tuberculosis infections depend on the Thiol Peroxidase TPx. PLoS ONE 4, e5150. Hugo, M., Turell, L., Manta, B., Botti, H., Monteiro, G., Netto...dormant phase Mycobacterium tuberculosis infections . BioMed Central Infectious Diseases 7, 84. Muthuramalingam, M., Seidel, T., Laxa, M., Nunes de

  10. A functionally conserved Zn2 Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat.

    PubMed

    Rybak, Kasia; See, Pao Theen; Phan, Huyen T T; Syme, Robert A; Moffat, Caroline S; Oliver, Richard P; Tan, Kar-Chun

    2017-04-01

    The fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch of wheat (Triticum aestivum). The interaction is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three best-characterized effector-sensitivity gene systems are SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. These effector genes are highly expressed during early infection, but expression decreases as the infection progresses to tissue necrosis and sporulation. However, the mechanism of regulation is unknown. We have identified and functionally characterized a gene, referred to as PnPf2, which encodes a putative zinc finger transcription factor. PnPf2 deletion resulted in the down-regulation of SnToxA and SnTox3 expression. Virulence on Tsn1 and Snn3 wheat cultivars was strongly reduced. The SnTox1-Snn1 interaction remained unaffected. Furthermore, we have also identified and deleted an orthologous PtrPf2 from the tan spot fungus Pyrenophora tritici-repentis which possesses a near-identical ToxA that was acquired from P. nodorum via horizontal gene transfer. PtrPf2 deletion also resulted in the down-regulation of PtrToxA expression and a near-complete loss of virulence on Tsn1 wheat. We have demonstrated, for the first time, evidence for a functionally conserved signalling component that plays a role in the regulation of a common/horizontally transferred effector found in two major fungal pathogens of wheat.

  11. Overexpression of the pepper antimicrobial protein CaAMP1 gene regulates the oxidative stress- and disease-related proteome in Arabidopsis.

    PubMed

    Lee, Sung Chul; Hwang, In Sun; Hwang, Byung Kook

    2011-12-01

    Proteomics facilitates our understanding of cellular processes and network functions in the plant defense response during abiotic and biotic stresses. Here, we demonstrate that the ectopic expression of the Capsicum annuum antimicrobial protein CaAMP1 gene in Arabidopsis thaliana confers enhanced tolerance to methyl viologen (MV)-induced oxidative stress, which is accompanied by lower levels of lipid peroxidation. Quantitative comparative proteome analyses using two-dimensional gel electrophoresis coupled with mass spectrometry identified some of the oxidative stress- and disease-related proteins that are differentially regulated by CaAMP1 overexpression in Arabidopsis leaves. Antioxidant- and defense-related proteins, such as 2-cys peroxiredoxin, L-ascorbate peroxidase, peroxiredoxin, glutathione S-transferase and copper homeostasis factor, were up-regulated in the CaAMP1 transgenic leaf tissues. In contrast, GSH-dependent dehydroascorbate reductase and WD-40 repeat family protein were down-regulated by CaAMP1 overexpression. In addition, CaAMP1 overexpression enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and also H(2)O(2) accumulation in Arabidopsis. The identified antioxidant- and defense-related genes were differentially expressed during MV-induced oxidative stress and Pst DC3000 infection. Taken together, we conclude that CaAMP1 overexpression can regulate the differential expression of defense-related proteins in response to environmental stresses to maintain reactive oxygen species (ROS) homeostasis.

  12. PGD2 and PGE2 regulate gene expression of Prx 6 in primary macrophages via Nrf2.

    PubMed

    Erttmann, Saskia F; Bast, Antje; Seidel, Julia; Breitbach, Katrin; Walther, Reinhard; Steinmetz, Ivo

    2011-08-01

    Peroxiredoxin 6 (Prx 6) is a bifunctional enzyme with both glutathione peroxidase and acidic Ca(2+)-independent phospholipase A(2) activities. We have recently shown that exposure of murine bone marrow-derived macrophages to LPS and IFN-γ leads to induction of COX-2 expression and secretion of PGE(2), up-regulating Prx 6 mRNA levels. This study was designed to investigate various prostaglandins (PGs) for their ability to induce gene expression of Prxs, in particular Prx 6, and to determine the underlying regulatory mechanisms. We provide evidence that both conventional and cyclopentenone PGs enhance Prx 6 mRNA expression. Treatment with either activators or inhibitors of adenylate cyclase as well as cAMP analogs indicated that Prx 6 gene expression is regulated by adenylate cyclase in response to PGD(2) or PGE(2). Furthermore, our study revealed that JAK2, PI3K, PKC, and p38 MAPK contribute to the PGD(2)- or PGE(2)-dependent Prx 6 induction. Using stimulated macrophages from Nrf2-deficient mice or activators of Nrf2 and PPARγ, we found that Nrf2, but not PPARγ, is involved in the PG-dependent increase in Prx 6 mRNA expression. In summary, our data suggest multiple signaling pathways of Prx 6 regulation by PGs and identified Nrf2 as a critical player mediating transcriptional induction.

  13. Thioredoxin-1/peroxiredoxin-1 as sensors of oxidative stress mediated by NADPH oxidase activity in atherosclerosis.

    PubMed

    Madrigal-Matute, Julio; Fernandez-Garcia, Carlos-Ernesto; Blanco-Colio, Luis Miguel; Burillo, Elena; Fortuño, Ana; Martinez-Pinna, Roxana; Llamas-Granda, Patricia; Beloqui, Oscar; Egido, Jesus; Zalba, Guillermo; Martin-Ventura, José Luis

    2015-09-01

    To assess the potential association between TRX-1/PRX-1 and NADPH oxidase (Nox) activity in vivo and in vitro, TRX-1/PRX-1 levels were assessed by ELISA in 84 asymptomatic subjects with known phagocytic NADPH oxidase activity and carotid intima-media thickness (IMT). We found a positive correlation between TRX-1/PRX-1 and NADPH oxidase-dependent superoxide production (r=0.48 and 0.47; p<0.001 for both) and IMT (r=0.31 and 0.36; p<0.01 for both) adjusted by age and sex. Moreover, asymptomatic subjects with plaques have higher PRX-1 and TRX plasma levels (p<0.01 for both). These data were confirmed in a second study in which patients with carotid atherosclerosis showed higher PRX-1 and TRX plasma levels than healthy subjects (p<0.001 for both). In human atherosclerotic plaques, the NADPH oxidase subunit p22phox colocalized with TRX-1/PRX-1 in macrophages (immunohistochemistry). In monocytes and macrophages, phorbol 12-myristate 13-acetate (PMA) induced NADPH activation and TRX-1/PRX-1 release to the extracellular medium, with a concomitant decrease in their intracellular levels, which was reversed by the NADPH inhibitor apocynin (Western blot). In loss-of-function experiments, genetic silencing of the NADPH oxidase subunit Nox2 blocked PMA-induced intracellular TRX-1/PRX-1 downregulation in macrophages. Furthermore, the PMA-induced release of TRX-1/PRX-1 involves the modulation of their redox status and exosome-like vesicles. TRX-1/PRX-1 levels are associated with NADPH oxidase-activity in vivo and in vitro. These data could suggest a coordinated antioxidant response to oxidative stress in atherothrombosis.

  14. Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice

    PubMed Central

    Ozkosem, Burak; Feinstein, Sheldon I.; Fisher, Aron B.; O’Flaherty, Cristian

    2015-01-01

    Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6−/− mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6−/− males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6−/− males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6−/− males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice. PMID:25796034

  15. Peroxiredoxin 1 interacts with and blocks the redox factor APE1 from activating interleukin-8 expression

    PubMed Central

    Nassour, Hassan; Wang, Zhiqiang; Saad, Amine; Papaluca, Arturo; Brosseau, Nicolas; Affar, El Bachir; Alaoui-Jamali, Moulay A.; Ramotar, Dindial

    2016-01-01

    APE1 is an essential DNA repair protein that also possesses the ability to regulate transcription. It has a unique cysteine residue C65, which maintains the reduce state of several transcriptional activators such as NF-κB. How APE1 is being recruited to execute the various biological functions remains unknown. Herein, we show that APE1 interacts with a novel partner PRDX1, a peroxidase that can also prevent oxidative damage to proteins by serving as a chaperone. PRDX1 knockdown did not interfere with APE1 expression level or its DNA repair activities. However, PRDX1 knockdown greatly facilitates APE1 detection within the nucleus by indirect immunofluorescence analysis, even though APE1 level was unchanged. The loss of APE1 interaction with PRDX1 promotes APE1 redox function to activate binding of the transcription factor NF-κB onto the promoter of a target gene, the proinflammatory chemokine IL-8 involved in cancer invasion and metastasis, resulting in its upregulation. Depletion of APE1 blocked the upregulation of IL-8 in the PRDX1 knockdown cells. Our findings suggest that the interaction of PRDX1 with APE1 represents a novel anti-inflammatory function of PRDX1, whereby the association safeguards APE1 from reducing transcription factors and activating superfluous gene expression, which otherwise could trigger cancer invasion and metastasis. PMID:27388124

  16. Structural, Evolutionary, and Functional Analysis of the Class III Peroxidase Gene Family in Chinese Pear (Pyrus bretschneideri)

    PubMed Central

    Cao, Yunpeng; Han, Yahui; Meng, Dandan; Li, Dahui; Jin, Qing; Lin, Yi; Cai, Yongping

    2016-01-01

    Peroxidases (PRXs) are widely existed in various organisms and could be divided into different types according to their structures and functions. Specifically, the Class III Peroxidase, a plant-specific multi-gene family, involves in many physiological processes, such as the metabolism of auxin, the extension and thickening of cell wall, as well as the formation of lignin. By searching the pear genome database, 94 non-redundant PRXs from Pyrus bretschneideri (PbPRXs) were identified. Subsequently, analysis of phylogenetic relationships, gene structures, conserved motifs, and microsynteny was performed. These PbPRXs were unevenly distributed among 17 chromosomes of pear. In addition, 26 segmental duplication events but only one tandem duplication were occurred in these PbPRXs, implying segmental duplication was the main contributor to the expansion of the PbPRX family. By the Ka/Ks analysis, 26 out of 27 duplicated PbPRXs has experienced purifying selection. Twenty motifs were identified in PbPRXs based on the MEME analysis, 11 of which were enriched in pear. A total of 41 expressed genes were identified from ESTs of pear fruit. According to qRT-PCR, the expression trends of five PbPRXs in subgroup C were consistent with the change of lignin content during pear fruit development. So we inferred that the five PbPRXs were candidate genes involved in the lignin synthesis pathway. These results provided useful information for further researches of PRX genes in pear. PMID:28018406

  17. Structural, Evolutionary, and Functional Analysis of the Class III Peroxidase Gene Family in Chinese Pear (Pyrus bretschneideri).

    PubMed

    Cao, Yunpeng; Han, Yahui; Meng, Dandan; Li, Dahui; Jin, Qing; Lin, Yi; Cai, Yongping

    2016-01-01

    Peroxidases (PRXs) are widely existed in various organisms and could be divided into different types according to their structures and functions. Specifically, the Class III Peroxidase, a plant-specific multi-gene family, involves in many physiological processes, such as the metabolism of auxin, the extension and thickening of cell wall, as well as the formation of lignin. By searching the pear genome database, 94 non-redundant PRXs from Pyrus bretschneideri (PbPRXs) were identified. Subsequently, analysis of phylogenetic relationships, gene structures, conserved motifs, and microsynteny was performed. These PbPRXs were unevenly distributed among 17 chromosomes of pear. In addition, 26 segmental duplication events but only one tandem duplication were occurred in these PbPRXs, implying segmental duplication was the main contributor to the expansion of the PbPRX family. By the Ka/Ks analysis, 26 out of 27 duplicated PbPRXs has experienced purifying selection. Twenty motifs were identified in PbPRXs based on the MEME analysis, 11 of which were enriched in pear. A total of 41 expressed genes were identified from ESTs of pear fruit. According to qRT-PCR, the expression trends of five PbPRXs in subgroup C were consistent with the change of lignin content during pear fruit development. So we inferred that the five PbPRXs were candidate genes involved in the lignin synthesis pathway. These results provided useful information for further researches of PRX genes in pear.

  18. Lysosomal pH-inducible supramolecular dissociation of polyrotaxanes possessing acid-labile N-triphenylmethyl end groups and their therapeutic potential for Niemann-Pick type C disease

    PubMed Central

    Tamura, Atsushi; Nishida, Kei; Yui, Nobuhiko

    2016-01-01

    Abstract Niemann-Pick type C (NPC) disease is characterized by the accumulation of cholesterol in lysosomes. We have previously reported that biocleavable polyrotaxanes (PRXs) composed of β-cyclodextrins (β-CDs) threaded onto a linear polymer capped with bulky stopper molecules via intracellularly cleavable linkers show remarkable cholesterol reducing effects in NPC disease patient-derived fibroblasts owing to the stimuli-responsive intracellular dissociation of PRXs and subsequent β-CD release from the PRXs. Herein, we describe a series of novel acid-labile 2-(2-hydroxyethoxy)ethyl group-modified PRXs (HEE-PRXs) bearing terminal N-triphenylmethyl (N-Trt) groups as a cleavable component for the treatment of NPC disease. The N-Trt end groups of the HEE-PRXs underwent acidic pH-induced cleavage and led to the dissociation of their supramolecular structure. A kinetic study revealed that the number of HEE groups on the PRX did not affect the cleavage kinetics of the N-Trt end groups of the HEE-PRXs. The effect of the number of HEE groups of the HEE-PRXs, which was modified to impart water solubility to the PRXs, on cellular internalization efficiency, lysosomal localization efficiency, and cholesterol reduction ability in NPC disease-derived fibroblasts (NPC1 fibroblasts) was also investigated. The cellular uptake and lysosomal localization efficiency were almost equivalent for HEE-PRXs with different numbers of HEE groups. However, the cholesterol reducing ability of the HEE-PRXs in NPC1 fibroblasts was affected by the number of HEE groups, and HEE-PRXs with a high number of HEE groups were unable to reduce lysosomal cholesterol accumulation. This deficiency is most likely due to the cholesterol-solubilizing ability of HEE-modified β-CDs released from the HEE-PRXs. We conclude that the N-Trt group acts as a cleavable component to induce the lysosomal dissociation of HEE-PRXs, and acid-labile HEE-PRXs with an optimal number of HEE groups (4.1 to 5.4 HEE groups per

  19. Calredoxin represents a novel type of calcium-dependent sensor-responder connected to redox regulation in the chloroplast

    PubMed Central

    Hochmal, Ana Karina; Zinzius, Karen; Charoenwattanasatien, Ratana; Gäbelein, Philipp; Mutoh, Risa; Tanaka, Hideaki; Schulze, Stefan; Liu, Gai; Scholz, Martin; Nordhues, André; Offenborn, Jan Niklas; Petroutsos, Dimitris; Finazzi, Giovanni; Fufezan, Christian; Huang, Kaiyao; Kurisu, Genji; Hippler, Michael

    2016-01-01

    Calcium (Ca2+) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca2+- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca2+-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca2+-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca2+-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca2+-dependent ‘sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation. PMID:27297041

  20. Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli

    PubMed Central

    Lee, Jae Taek; Lee, Seung Sik; Mondal, Suvendu; Tripathi, Bhumi Nath; Kim, Siu; Lee, Keun Woo; Hong, Sung Hyun; Bai, Hyoung-Woo; Cho, Jae-Young; Chung, Byung Yeoup

    2016-01-01

    Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of Ser78 to Cys78 resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of Cys78 in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone. PMID:27457208

  1. Papain-Based Vaccination Modulates Schistosoma mansoni Infection-Induced Cytokine Signals.

    PubMed

    Abdel Aziz, N; Tallima, H; Hafez, E A; El Ridi, R

    2016-02-01

    We have previously shown that immunization of outbred rodents with cysteine peptidases-based vaccine elicited type 2-biased immune responses associated with consistent and reproducible protection against challenge Schistosoma mansoni. We herein start to elucidate the molecular basis of C57BL/6 mouse resistance to S. mansoni following treatment with the cysteine peptidase, papain. We evaluated the early cytokine signals delivered by epidermal, dermal, and draining lymph node cells of naïve, and S. mansoni -infected mice treated 1 day earlier with 0 or 50 μg papain, or immunized twice with papain only (10 μg/mouse), papain-free recombinant S. mansoni glyceraldehyde 3-phosphate dehydrogenase and 2-Cys peroxiredoxin peptide (10 and 15 μg/mouse, respectively = antigen Mix), or papain-adjuvanted antigen Mix. Schistosoma mansoni infection induced epidermal and lymph node cells to release type 1, type 2 and type 17 cytokines, known to counteract each other. Expectedly, humoral immune responses were negligible until patency. Papain pretreatment or papain-based vaccination diminished or shut off S. mansoni infection early induction of type 1, type 17 and type 2 cytokines except for thymic stromal lymphopoietin and programmed the immune system towards a polarized type 2 immune milieu, associated with highly significant (P < 0.005 - <0.0001) resistance to S. mansoni infection.

  2. Sestrin 2 protein regulates platelet-derived growth factor receptor β (Pdgfrβ) expression by modulating proteasomal and Nrf2 transcription factor functions.

    PubMed

    Tomasovic, Ana; Kurrle, Nina; Sürün, Duran; Heidler, Juliana; Husnjak, Koraljka; Poser, Ina; Schnütgen, Frank; Scheibe, Susan; Seimetz, Michael; Jaksch, Peter; Hyman, Anthony; Weissmann, Norbert; von Melchner, Harald

    2015-04-10

    We recently identified the antioxidant protein Sestrin 2 (Sesn2) as a suppressor of platelet-derived growth factor receptor β (Pdgfrβ) signaling and Pdgfrβ signaling as an inducer of lung regeneration and injury repair. Here, we identified Sesn2 and the antioxidant gene inducer nuclear factor erythroid 2-related factor 2 (Nrf2) as positive regulators of proteasomal function. Inactivation of Sesn2 or Nrf2 induced reactive oxygen species-mediated proteasomal inhibition and Pdgfrβ accumulation. Using bacterial artificial chromosome (BAC) transgenic HeLa and mouse embryonic stem cells stably expressing enhanced green fluorescent protein-tagged Sesn2 at nearly endogenous levels, we also showed that Sesn2 physically interacts with 2-Cys peroxiredoxins and Nrf2 albeit under different reductive conditions. Overall, we characterized a novel, redox-sensitive Sesn2/Pdgfrβ suppressor pathway that negatively interferes with lung regeneration and is up-regulated in the emphysematous lungs of patients with chronic obstructive pulmonary disease (COPD).

  3. Involvement of glutathione peroxidase 1 in growth and peroxisome formation in Saccharomyces cerevisiae in oleic acid medium.

    PubMed

    Ohdate, Takumi; Inoue, Yoshiharu

    2012-09-01

    Saccharomyces cerevisiae is able to use some fatty acids, such as oleic acid, as a sole source of carbon. β-oxidation, which occurs in a single membrane-enveloped organelle or peroxisome, is responsible for the assimilation of fatty acids. In S. cerevisiae, β-oxidation occurs only in peroxisomes, and H(2)O(2) is generated during this fatty acid-metabolizing pathway. S. cerevisiae has three GPX genes (GPX1, GPX2, and GPX3) encoding atypical 2-Cys peroxiredoxins. Here we show that expression of GPX1 was induced in medium containing oleic acid as a carbon source in an Msn2/Msn4-dependent manner. We found that Gpx1 was located in the peroxisomal matrix. The peroxisomal Gpx1 showed peroxidase activity using thioredoxin or glutathione as a reducing power. Peroxisome biogenesis was induced when cells were cultured with oleic acid. Peroxisome biogenesis was impaired in gpx1∆ cells, and subsequently, the growth of gpx1∆ cells was lowered in oleic acid-containing medium. Gpx1 contains six cysteine residues. Of the cysteine-substituted mutants of Gpx1, Gpx1(C36S) was not able to restore growth and peroxisome formation in oleic acid-containing medium, therefore, redox regulation of Gpx1 seems to be involved in the mechanism of peroxisome formation.

  4. Calredoxin represents a novel type of calcium-dependent sensor-responder connected to redox regulation in the chloroplast.

    PubMed

    Hochmal, Ana Karina; Zinzius, Karen; Charoenwattanasatien, Ratana; Gäbelein, Philipp; Mutoh, Risa; Tanaka, Hideaki; Schulze, Stefan; Liu, Gai; Scholz, Martin; Nordhues, André; Offenborn, Jan Niklas; Petroutsos, Dimitris; Finazzi, Giovanni; Fufezan, Christian; Huang, Kaiyao; Kurisu, Genji; Hippler, Michael

    2016-06-14

    Calcium (Ca(2+)) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca(2+)- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca(2+)-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca(2+)-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca(2+)-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca(2+)-dependent 'sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation.

  5. The Tumor Suppressor Mst1 Promotes Changes in the Cellular Redox State by Phosphorylation and Inactivation of Peroxiredoxin-1 Protein*

    PubMed Central

    Rawat, Sonali Jalan; Creasy, Caretha L.; Peterson, Jeffrey R.; Chernoff, Jonathan

    2013-01-01

    The serine/threonine protein kinases Mst1 and Mst2 can be activated by cellular stressors including hydrogen peroxide. Using two independent protein interaction screens, we show that these kinases associate, in an oxidation-dependent manner, with Prdx1, an enzyme that regulates the cellular redox state by reducing hydrogen peroxide to water and oxygen. Mst1 inactivates Prdx1 by phosphorylating it at Thr-90 and Thr-183, leading to accumulation of hydrogen peroxide in cells. These results suggest that hydrogen peroxide-stimulated Mst1 activates a positive feedback loop to sustain an oxidizing cellular state. PMID:23386615

  6. Screening and Functional Analysis of the Peroxiredoxin Specifically Expressed in Bursaphelenchus xylophilus—The Causative Agent of Pine Wilt Disease

    PubMed Central

    Fu, Han-Yu; Ren, Jia-Hong; Huang, Lin; Li, Hao; Ye, Jian-Ren

    2014-01-01

    The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. Accurately differentiating B. xylophilus from other nematodes species, especially its related species B. mucronatus, is important for pine wood nematode detection. Thus, we attempted to identify a specific protein in the pine wood nematode using proteomics technology. Here, we compared the proteomes of B. xylophilus and B. mucronatus using Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF-MS) technologies. In total, 15 highly expressed proteins were identified in B. xylophilus compared with B. mucronatus. Subsequently, the specificity of the proteins identified was confirmed by PCR using the genomic DNA of other nematode species. Finally, a gene encoding a specific protein (Bx-Prx) was obtained. This gene was cloned and expressed in E. coli. The in situ hybridisation pattern of Bx-Prx showed that it was expressed strongly in the tail of B. xylophilus. RNAi was used to assess the function of Bx-Prx, the results indicated that the gene was associated with the reproduction and pathogenicity of B. xylophilus. This discovery provides fundamental information for identifying B. xylophilus via a molecular approach. Moreover, the purified recombinant protein has potential as a candidate diagnostic antigen of pine wilt disease, which may lead to a new immunological detection method for the pine wood nematode. PMID:24918285

  7. Peroxiredoxin IV regulates pro-inflammatory responses in large yellow croaker (Pseudosciaena crocea) and protects against bacterial challenge.

    PubMed

    Yu, Suhong; Mu, Yinnan; Ao, Jingqun; Chen, Xinhua

    2010-03-05

    In this study, we applied a comparative proteomic approach to the analysis of differentially expressed proteins in the spleens of large yellow croaker following treatment with an inactivated trivalent bacterial vaccine. Twenty-four altered proteins were identified by MALDI-TOF or MALDI-TOF-TOF, including immune-related proteins, antioxidant proteins, signal transducers, protein biosynthesis and catabolism modulators, and carbonic anhydrases. Three Prx family members, namely, Prx I, Prx II, and Prx IV, were upregulated after treatment with the vaccine, indicating potentially important roles for these antioxidant proteins in the antibacterial immune response. Large yellow croaker Prx IV (LycPrxIV), which has thiol-dependent peroxidase activity, was constitutively expressed in all tissues examined. Immunoelectron microscopy showed that LycPrxIV was primarily localized to the rER or peroxisome in spleen cells of healthy fish, and its synthesis on the rER increased following treatment with bacterial vaccine. Suppression of LycPrxIV by siRNA resulted in an increase in NF-kappaB activity in spleen tissues, while in vivo administration of recombinant LycPrxIV (rLycPrxIV) caused a decrease in NF-kappaB activity, indicating that LycPrxIV negatively regulates NF-kappaB activation. Likewise, siRNA-mediated knockdown of LycPrxIV increased the expression of TNF-alpha and CC chemokine, and downregulated the expression of IL-10. However, injection of fish with rLycPrxIV induced the opposite expression pattern of these cytokines, suggesting a role for LycPrxIV in regulating pro-inflammatory responses. Bacterial challenge experiments showed that suppression of LycPrxIV expression by siRNA significantly increased fish mortality as compared to controls, whereas rLycPrxIV provided a protective effect. Together, our data suggest that LycPrxIV may regulate pro-inflammatory responses to protect large yellow croaker from bacterial challenge, revealing a novel antibacterial mechanism in fish.

  8. On the Connectivity and Multihop Delay of Ad Hoc Cognitive Radio Networks

    DTIC Science & Technology

    2010-05-01

    depends on the density of SUs and the traffic load of PUs. 9 Connectivity: Static Case When PTxs/ PRxs are static over time: PSfrag replacements λ P T...Connectivity: Dynamic Case When temporal dynamics of PTxs/ PRxs are sufficiently rich: PSfrag replacements λ P T (D en si ty of P T xs ) λS (Density of SUs)λ

  9. Diverse functions and reactions of class III peroxidases.

    PubMed

    Shigeto, Jun; Tsutsumi, Yuji

    2016-03-01

    Higher plants contain plant-specific peroxidases (class III peroxidase; Prxs) that exist as large multigene families. Reverse genetic studies to characterize the function of each Prx have revealed that Prxs are involved in lignification, cell elongation, stress defense and seed germination. However, the underlying mechanisms associated with plant phenotypes following genetic engineering of Prx genes are not fully understood. This is because Prxs can function as catalytic enzymes that oxidize phenolic compounds while consuming hydrogen peroxide and/or as generators of reactive oxygen species. Moreover, biochemical efforts to characterize Prxs responsible for lignin polymerization have revealed specialized activities of Prxs. In conclusion, not only spatiotemporal regulation of gene expression and protein distribution, but also differentiated oxidation properties of each Prx define the function of this class of peroxidases.

  10. Threaded macromolecules as a versatile framework for biomaterials.

    PubMed

    Tamura, Atsushi; Yui, Nobuhiko

    2014-11-14

    Polyrotaxanes (PRXs) are a class of supramolecular threaded macromolecules, in which cyclic molecules are threaded onto the main- or side-chain of polymers. To date, various studies have been conducted on the synthesis of PRXs, and various combinations of cyclic molecules and polymers that can form a PRX have been discovered. Among these combinations, PRXs composed of cyclodextrins (CDs) and a linear polymer have attracted much attention and have been investigated by many researchers. Because of the non-covalently associated characteristic of PRXs, these supermolecules exhibit unique properties, such as the dynamic motion of the threaded cyclic molecules along a polymer axle and complete dissociation of the supramolecular structure, that are never observed in other synthetic polymers. These inherent properties of PRXs are of interest in the design of novel biomaterials, such as hydrogels, scaffolds in tissue engineering, drug delivery carriers, and polymer-drug conjugates. Thus, various studies have been conducted to utilize PRXs as a framework for biomaterials. In this review, we describe the recent progress in biomaterial application of PRXs such as drug delivery and gene delivery.

  11. Exploring the venom proteome of the western diamondback rattlesnake, Crotalus atrox, via snake venomics and combinatorial peptide ligand library approaches.

    PubMed

    Calvete, Juan J; Fasoli, Elisa; Sanz, Libia; Boschetti, Egisto; Righetti, Pier Giorgio

    2009-06-01

    We report the proteomic characterization of the venom of the medically important North American western diamondback rattlesnake, Crotalus atrox, using two complementary approaches: snake venomics (to gain an insight of the overall venom proteome), and two solid-phase combinatorial peptide ligand libraries (CPLL), followed by 2D electrophoresis and mass spectrometric characterization of in-gel digested protein bands (to capture and "amplify" low-abundance proteins). The venomics approach revealed approximately 24 distinct proteins belonging to 2 major protein families (snake venom metalloproteinases, SVMP, and serine proteinases), which represent 69.5% of the total venom proteins, 4 medium abundance families (medium-size disintegrin, PLA(2), cysteine-rich secretory protein, and l-amino acid oxidase) amounting to 25.8% of the venom proteins, and 3 minor protein families (vasoactive peptides, endogenous inhibitor of SVMP, and C-type lectin-like). This toxin profile potentially explains the cytotoxic, myotoxic, hemotoxic, and hemorrhagic effects evoked by C. atrox envenomation. Further, our results showing that C. atrox exhibits a similar level of venom variation as Sistrurus miliarius points to a "diversity gain" scenario in the lineage leading to the Sistrurus catenatus taxa. On the other hand, the two combinatorial hexapeptide libraries captured distinct sets of proteins. Although the CPLL-treated samples did not retain a representative venom proteome, protein spots barely, or not at all, detectable in the whole venom were enriched in the two CPLL-treated samples. The amplified low copy number C. atrox venom proteins comprised a C-type lectin-like protein, several PLA(2) molecules, PIII-SVMP isoforms, glutaminyl cyclase isoforms, and a 2-cys peroxiredoxin highly conserved across the animal kingdom. Peroxiredoxin and glutaminyl cyclase may participate, respectively, in redox processes leading to the structural/functional diversification of toxins, and in the N

  12. Proteomic analysis of chromium stress and sulfur deficiency responses in leaves of two canola (Brassica napus L.) cultivars differing in Cr(VI) tolerance.

    PubMed

    Yıldız, Mustafa; Terzi, Hakan

    2016-02-01

    Sulfur (S) is an essential macronutrient for plant growth and development, and it plays an essential role in response to environmental stresses. Plants suffer with combined stress of S deficiency and hexavalent chromium [Cr(VI)] in the rhizosphere. Little is known about the impact of S deficiency on leaf metabolism of canola (Brassica napus L.) under Cr(VI) stress. Therefore, this study is the first to examine the effects of Cr(VI) stress and S deficiency in canola at a molecular level. A comparative proteomic approach was used to investigate the differences in protein abundance between Cr-tolerant NK Petrol and Cr-sensitive Sary cultivars. The germinated seeds were grown hydroponically in S-sufficient (+S) nutrient solution for 7 days and then subjected to S-deficiency (-S) for 7 days. S-deficient and +S seedlings were then exposed to 100μM Cr(VI) for 3 days. Protein patterns analyzed by two-dimensional electrophoresis (2-DE) revealed that 58 protein spots were differentially regulated by Cr(VI) stress (+S/+Cr), S-deficiency (-S/-Cr) and combined stress (-S/+Cr). Of these, 39 protein spots were identified by MALDI-TOF/TOF mass spectrometry. Differentially regulated proteins predominantly had functions not only in photosynthesis, but also in energy metabolism, stress defense, protein folding and stabilization, signal transduction, redox regulation and sulfur metabolism. Six stress defense related proteins including 2-Cys peroxiredoxin BAS1, glutathione S-transferase, ferritin-1, l-ascorbate peroxidase, thiazole biosynthetic enzyme and myrosinase-binding protein-like At3g16470 exhibited a greater increase in NK Petrol. The stress-related proteins play an important role in the detoxification of Cr(VI) and maintaining cellular homeostasis under variable S nutrition.

  13. Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

    PubMed

    Chow-Shi-Yée, Mélanie; Briard, Jennie G; Grondin, Mélanie; Averill-Bates, Diana A; Ben, Robert N; Ouellet, François

    2016-05-01

    Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells.

  14. Characterisation and analysis of thioredoxin peroxidase as a potential antigen for the serodiagnosis of sarcoptic mange in rabbits by dot-ELISA

    PubMed Central

    2013-01-01

    Background Scabies caused by Sarcoptes scabiei is a widespread but a neglected tropical zoonosis. In this study, we characterised a S. scabiei thioredoxin peroxidase (SsTPx) and evaluated a recombinant SsTPx as a diagnostic antigen in rabbits. Methods The open reading frame of the gene encoding SsTPx-2 was amplified and the recombinant protein was expressed in Escherichia coli cells and purified. SsTPx was localized in mite tissue by immunolocalisation using the purified recombinant protein. Serodiagnosis assays were carried out in 203 New Zealand White rabbit serum samples by dot-ELISA. Result The open reading frame (489 bp) of the gene encodes an 18.11 kDa protein, which showed highly homology to that of Psoroptes cuniculi (98.77% identity) and belongs to the 2-Cys family of peroxiredoxins. SsTPx was mainly distributed in muscle tissues of mites, integument of the epidermis and the anterior end of S. scabiei. Although SsTPx cross-reactivity with psoroptic mites was observed, the SsTPx dot-ELISA showed excellent diagnostic ability, with 95.3% sensitivity and 93.8% specificity in mange-infected and uninfected groups. Conclusions This study showed that the purified SsTPx is a highly sensitive antigen for the diagnosis of mange infection by dot-ELISA. This technique is a rapid and convenient method that can be used worldwide for the clinical diagnosis of sarcoptic mange in rabbits, and is especially useful in developing regions. PMID:23875925

  15. The Physcomitrella patens Chloroplast Proteome Changes in Response to Protoplastation

    PubMed Central

    Fesenko, Igor; Seredina, Anna; Arapidi, Georgij; Ptushenko, Vasily; Urban, Anatoly; Butenko, Ivan; Kovalchuk, Sergey; Babalyan, Konstantin; Knyazev, Andrey; Khazigaleeva, Regina; Pushkova, Elena; Anikanov, Nikolai; Ivanov, Vadim; Govorun, Vadim M.

    2016-01-01

    Plant protoplasts are widely used for genetic manipulation and functional studies in transient expression systems. However, little is known about the molecular pathways involved in a cell response to the combined stress factors resulted from protoplast generation. Plants often face more than one type of stress at a time, and how plants respond to combined stress factors is therefore of great interest. Here, we used protoplasts of the moss Physcomitrella patens as a model to study the effects of short-term stress on the chloroplast proteome. Using label-free comparative quantitative proteomic analysis (SWATH-MS), we quantified 479 chloroplast proteins, 219 of which showed a more than 1.4-fold change in abundance in protoplasts. We additionally quantified 1451 chloroplast proteins using emPAI. We observed degradation of a significant portion of the chloroplast proteome following the first hour of stress imposed by the protoplast isolation process. Electron-transport chain (ETC) components underwent the heaviest degradation, resulting in the decline of photosynthetic activity. We also compared the proteome changes to those in the transcriptional level of nuclear-encoded chloroplast genes. Globally, the levels of the quantified proteins and their corresponding mRNAs showed limited correlation. Genes involved in the biosynthesis of chlorophyll and components of the outer chloroplast membrane showed decreases in both transcript and protein abundance. However, proteins like dehydroascorbate reductase 1 and 2-cys peroxiredoxin B responsible for ROS detoxification increased in abundance. Further, genes such as thylakoid ascorbate peroxidase were induced at the transcriptional level but down-regulated at the proteomic level. Together, our results demonstrate that the initial chloroplast reaction to stress is due changes at the proteomic level. PMID:27867392

  16. Characterization of gene expression associated with drought avoidance and tolerance traits in a perennial grass species.

    PubMed

    Zhou, Peng; An, Yuan; Wang, Zhaolong; Du, Hongmei; Huang, Bingru

    2014-01-01

    To understand molecular mechanisms of perennial grass adaptation to drought stress, genes associated with drought avoidance or tolerance traits were identified and their expression patterns were characterized in C4 hybrid bermudagrass [Cynodon dactylon (L.) Pers.×C. transvaalensis Burtt Davy, cv. Tifway] and common bermudagrass (C. dactylon, cv. C299). Plants of drought-tolerant 'Tifway' and drought-sensitive 'C299' were exposed to drought for 5 d (mild stress) and 10 d (severe stress) by withholding irrigation in a growth chamber. 'Tifway' maintained significantly lower electrolyte leakage and higher relative water content than 'C299' at both 5 and 10 d of drought stress. Four cDNA libraries via suppression subtractive hybridization analysis were constructed and identified 277 drought-responsive genes in the two genotypes at 5 and 10 d of drought stress, which were mainly classified into the functional categories of stress defense, metabolism, osmoregulation, membrane system, signal and regulator, structural protein, protein synthesis and degradation, and energy metabolism. Quantitative-PCR analysis confirmed the expression of 36 drought up-regulated genes that were more highly expressed in drought-tolerant 'Tifway' than drought-sensitive 'C299', including those for drought avoidance traits, such as cuticle wax formation (CER1 and sterol desaturase), for drought tolerance traits, such as dehydration-protective proteins (dehydrins, HVA-22-like protein) and oxidative stress defense (superoxide dismutase, dehydroascorbate reductase, 2-Cys peroxiredoxins), and for stress signaling (EREBP-4 like protein and WRKY transcription factor). The results suggest that the expression of genes for stress signaling, cuticle wax accumulation, antioxidant defense, and dehydration-protective protein accumulation could be critically important for warm-season perennial grass adaptation to long-term drought stress.

  17. OxyR2 Functions as a Three-state Redox Switch to Tightly Regulate Production of Prx2, a Peroxiredoxin of Vibrio vulnificus.

    PubMed

    Bang, Ye-Ji; Lee, Zee-Won; Kim, Dukyun; Jo, Inseong; Ha, Nam-Chul; Choi, Sang Ho

    2016-07-29

    The bacterial transcriptional regulator OxyR is known to function as a two-state redox switch. OxyR senses cellular levels of H2O2 via a "sensing cysteine" that switches from the reduced to a disulfide state upon H2O2 exposure, inducing the expression of antioxidant genes. The reduced and disulfide states of OxyR, respectively, bind to extended and compact regions of DNA, where the reduced state blocks and the oxidized state allows transcription and further induces target gene expression by interacting with RNA polymerase. Vibrio vulnificus OxyR2 senses H2O2 with high sensitivity and induces the gene encoding the antioxidant Prx2. In this study, we used mass spectrometry to identify a third redox state of OxyR2, in which the sensing cysteine was overoxidized to S-sulfonated cysteine (Cys-SO3H) by high H2O2 in vitro and in vivo, where the modification deterred the transcription of prx2 The DNA binding preferences of OxyR25CA-C206D, which mimics overoxidized OxyR2, suggested that overoxidized OxyR2 binds to the extended DNA site, masking the -35 region of the prx2 promoter. These combined results demonstrate that OxyR2 functions as a three-state redox switch to tightly regulate the expression of prx2, preventing futile production of Prx2 in cells exposed to high levels of H2O2 sufficient to inactivate Prx2. We further provide evidence that another OxyR homolog, OxyR1, displays similar three-state behavior, inviting further exploration of this phenomenon as a potentially general regulatory mechanism.

  18. Induction of Protective Immune Responses Against Schistosomiasis haematobium in Hamsters and Mice Using Cysteine Peptidase-Based Vaccine

    PubMed Central

    Tallima, Hatem; Dalton, John P.; El Ridi, Rashika

    2015-01-01

    One of the major lessons we learned from the radiation-attenuated cercariae vaccine studies is that protective immunity against schistosomiasis is dependent on the induction of T helper (Th)1-/Th2-related immune responses. Since most schistosome larval and adult-worm-derived molecules used for vaccination uniformly induce a polarized Th1 response, it was essential to include a type 2 immune response-inducing molecule, such as cysteine peptidases, in the vaccine formula. Here, we demonstrate that a single subcutaneous injection of Syrian hamsters with 200 μg active papain, 1 h before percutaneous exposure to 150 cercariae of Schistosoma haematobium, led to highly significant (P < 0.005) reduction of >50% in worm burden and worm egg counts in intestine. Immunization of hamsters with 20 μg recombinant glyceraldehyde 3-phosphate dehydrogenase (rSG3PDH) and 20 μg 2-cys peroxiredoxin-derived peptide in a multiple antigen peptide construct (PRX MAP) together with papain (20 μg/hamster), as adjuvant led to considerable (64%) protection against challenge S. haematobium infection, similar to the levels reported with irradiated cercariae. Cysteine peptidases-based vaccination was also effective in protecting outbred mice against a percutaneous challenge infection with S. haematobium cercariae. In two experiments, a mixture of Schistosoma mansoni cathepsin B1 (SmCB1) and Fasciola hepatica cathepsin L1 (FhCL1) led to highly significant (P < 0.005) reduction of 70% in challenge S. haematobium worm burden and 60% reduction in liver egg counts. Mice vaccinated with SmCB1/FhCL1/rSG3PDH mixture and challenged with S. haematobium cercariae 3 weeks after the second immunization displayed highly significant (P < 0.005) reduction of 72% in challenge worm burden and no eggs in liver of 8–10 mice/group, as compared to unimmunized mice, associated with production of a mixture of type 1- and type 2-related cytokines and antibody responses. PMID:25852696

  19. Investigation of the Credibility of In-Situ Measurements of Radial and Tangential Stress in a Salt Test Bed.

    DTIC Science & Technology

    1986-05-31

    STEWS-TE-N K CUMMINGS APPLIED RESEARCH ASSOCIATES, INC ATTN: J D’ARCY APPLIED RESEARCH ASSOCIATES, INC ATTN: R FRANK Dist-1 IIIIIIII DNATR46.169 (DL...LIBRARY/B. KINSLOW 2 CYS ATTN: N LIPNER KAMAN TEMPO ATTN: TECH INFO CTR,DOC ACQ ATTN: DASIAC WASHINGTON STATE UNIVERSITY KAMAN TEMPO 2 CYS ATTN: PROF. Y

  20. 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.

    PubMed

    Munhoz, Daniela Cristina; Netto, Luis Eduardo Soares

    2004-08-20

    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 DeltacTPxII strain; these cells were highly sensitive to tert-butylhydroperoxide (TBHP) but presented resistance to H(2)O(2) in fermentative and respiratory conditions. In contrast, DeltacTPxI strain was very sensitive to both TBHP and H(2)O(2), 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 (K(cat) /K(m)) in the removals of both kinds of peroxide. This specific sensitivity of DeltacTPxII 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 H(2)O(2) and TBHP when cells were grown in different conditions. Finally, peroxide-removing assays were performed and showed that catalase activity increased significantly only in DeltacTPxII cells, which appear to be related with the resistance of this strain to H(2)O(2). 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 H(2)O(2), catalases (peroxisomal and/or cytosolic) and cTPxII seemed to cooperate with cTPxI in the defense of yeast against this oxidant.

  1. Versatile synthesis of end-reactive polyrotaxanes applicable to fabrication of supramolecular biomaterials.

    PubMed

    Tamura, Atsushi; Tonegawa, Asato; Arisaka, Yoshinori; Yui, Nobuhiko

    2016-01-01

    Cyclodextrin (CD)-threaded polyrotaxanes (PRXs) with reactive functional groups at the terminals of the axle polymers are attractive candidates for the design of supramolecular materials. Herein, we describe a novel and simple synthetic method for end-reactive PRXs using bis(2-amino-3-phenylpropyl) poly(ethylene glycol) (PEG-Ph-NH2) as an axle polymer and commercially available 4-substituted benzoic acids as capping reagents. The terminal 2-amino-3-phenylpropyl groups of PEG-Ph-NH2 block the dethreading of the α-CDs after capping with 4-substituted benzoic acids. By this method, two series of azide group-terminated polyrotaxanes (benzylazide: PRX-Bn-N3, phenylazide: PRX-Ph-N3,) were synthesized for functionalization via click reactions. The PRX-Bn-N3 and PRX-Ph-N3 reacted quickly and efficiently with p-(tert-butyl)phenylacetylene via copper-catalyzed click reactions. Additionally, the terminal azide groups of the PRX-Bn-N3 could be modified with dibenzylcyclooctyne (DBCO)-conjugated fluorescent molecules via a copper-free click reaction; this fluorescently labeled PRX was utilized for intracellular fluorescence imaging. The method of synthesizing end-reactive PRXs described herein is simple and versatile for the design of diverse functional PRXs and can be applied to the fabrication of PRX-based supramolecular biomaterials.

  2. Versatile synthesis of end-reactive polyrotaxanes applicable to fabrication of supramolecular biomaterials

    PubMed Central

    Tamura, Atsushi; Tonegawa, Asato; Arisaka, Yoshinori

    2016-01-01

    Cyclodextrin (CD)-threaded polyrotaxanes (PRXs) with reactive functional groups at the terminals of the axle polymers are attractive candidates for the design of supramolecular materials. Herein, we describe a novel and simple synthetic method for end-reactive PRXs using bis(2-amino-3-phenylpropyl) poly(ethylene glycol) (PEG-Ph-NH2) as an axle polymer and commercially available 4-substituted benzoic acids as capping reagents. The terminal 2-amino-3-phenylpropyl groups of PEG-Ph-NH2 block the dethreading of the α-CDs after capping with 4-substituted benzoic acids. By this method, two series of azide group-terminated polyrotaxanes (benzylazide: PRX-Bn-N3, phenylazide: PRX-Ph-N3,) were synthesized for functionalization via click reactions. The PRX-Bn-N3 and PRX-Ph-N3 reacted quickly and efficiently with p-(tert-butyl)phenylacetylene via copper-catalyzed click reactions. Additionally, the terminal azide groups of the PRX-Bn-N3 could be modified with dibenzylcyclooctyne (DBCO)-conjugated fluorescent molecules via a copper-free click reaction; this fluorescently labeled PRX was utilized for intracellular fluorescence imaging. The method of synthesizing end-reactive PRXs described herein is simple and versatile for the design of diverse functional PRXs and can be applied to the fabrication of PRX-based supramolecular biomaterials. PMID:28144361

  3. Subthreshold Technique for Fixed and Interface Trapped Charge Separation in Irradiated MOSFETs

    DTIC Science & Technology

    1990-04-01

    in Irradiated MOSFETs Edward W. Enlow Ronald L. Pease David R. Alexander Mission Research Corporation 1720 Randolph Road SE Albuquerque, NM 87106-4245...Edward W. Enlow , Ronald L. Pease, and David R. Alexander TA WU 7 PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFURMING ORGANIZATION Mission...CYS ATTN: D R ALEXANDER ATTN: DIR SCIENCE & TECH DIV 2 CYS ATTN: E W ENLOW ATTN: J ERSKINE 2 CYS ATTN: R L PEASE KAMAN SCIENCES CORP MISSION RESEARCH

  4. PRDX4 — EDRN Public Portal

    Cancer.gov

    From NCBI Gene: The protein encoded by this gene is an antioxidant enzyme and belongs to the peroxiredoxin family. The protein is localized to the cytoplasm. Peroxidases of the peroxiredoxin family reduce hydrogen peroxide and alkyl hydroperoxides to water and alcohol with the use of reducing equivalents derived from thiol-containing donor molecules. This protein has been found to play a regulatory role in the activation of the transcription factor NF-kappaB. [provided by RefSeq, Jul 2008

  5. Differential expression and immunolocalization of antioxidant enzymes in Entamoeba histolytica isolates during metronidazole stress.

    PubMed

    Iyer, Lakshmi Rani; Singh, Nishant; Verma, Anil Kumar; Paul, Jaishree

    2014-01-01

    Entamoeba histolytica infections are endemic in the Indian subcontinent. Five to eight percent of urban population residing under poor sanitary conditions suffers from Entamoeba infections. Metronidazole is the most widely prescribed drug used for amoebiasis. In order to understand the impact of metronidazole stress on the parasite, we evaluated the expression of two antioxidant enzymes, peroxiredoxin and FeSOD, in Entamoeba histolytica isolates during metronidazole stress. The results reveal that, under metronidazole stress, the mRNA expression levels of these enzymes did not undergo any significant change. Interestingly, immunolocalization studies with antibodies targeting peroxiredoxin indicate differential localization of the protein in the cell during metronidazole stress. In normal conditions, all the Entamoeba isolates exhibit presence of peroxiredoxin in the nucleus as well as in the membrane; however with metronidazole stress the protein localized mostly to the membrane. The change in the localization pattern was more pronounced when the cells were subjected to short term metronidazole stress compared to cells adapted to metronidazole. The protein localization to the cell membrane could be the stress response mechanism in these isolates. Colocalization pattern of peroxiredoxin with CaBp1, a cytosolic protein, revealed that the membrane and nuclear localization was specific to peroxiredoxin during metronidazole stress.

  6. Beneficial Roles of Melatonin on Redox Regulation of Photosynthetic Electron Transport and Synthesis of D1 Protein in Tomato Seedlings under Salt Stress

    PubMed Central

    Zhou, Xiaoting; Zhao, Hailiang; Cao, Kai; Hu, Lipan; Du, Tianhao; Baluška, František; Zou, Zhirong

    2016-01-01

    Melatonin is important in the protection of plants suffering various forms of abiotic stress. The molecular mechanisms underlying the melatonin-mediated protection of their photosynthetic machinery are not completely resolved. This study investigates the effects of exogenous melatonin applications on salt-induced damage to the light reaction components of the photosynthetic machinery of tomato seedlings. The results showed that melatonin pretreatments can help maintain growth and net photosynthetic rate (PN) under salt stress conditions. Pretreatment with melatonin increased the effective quantum yield of photosystem II (ΦPSII), the photochemical quenching coefficient (qP) and the proportion of PSII centers that are “open” (qL) under saline conditions. In this way, damage to the photosynthetic electron transport chain (PET) in photosystem II (PSII) was mitigated. In addition, melatonin pretreatment facilitated the repair of PSII by maintaining the availability of D1 protein that was otherwise reduced by salinity. The ROS levels and the gene expressions of the chloroplast TRXs and PRXs were also investigated. Salt stress resulted in increased levels of reactive oxygen species (ROS), which were mitigated by melatonin. In tomato leaves under salt stress, the expressions of PRXs and TRXf declined but the expressions of TRXm1/4 and TRXm2 increased. Melatonin pretreatment promoted the expression of TRXf and the abundances of TRXf and TRXm gene products but had no effects on the expressions of PRXs. In summary, melatonin improves the photosynthetic activities of tomato seedlings under salt stress. The mechanism could be that: (1) Melatonin controls ROS levels and prevents damaging elevations of ROS caused by salt stress. (2) Melatonin facilitates the recovery of PET and D1 protein synthesis, thus enhancing the tolerance of photosynthetic activities to salinity. (3) Melatonin induces the expression of TRXf and regulates the abundance of TRXf and TRXm gene products

  7. Beneficial Roles of Melatonin on Redox Regulation of Photosynthetic Electron Transport and Synthesis of D1 Protein in Tomato Seedlings under Salt Stress.

    PubMed

    Zhou, Xiaoting; Zhao, Hailiang; Cao, Kai; Hu, Lipan; Du, Tianhao; Baluška, František; Zou, Zhirong

    2016-01-01

    Melatonin is important in the protection of plants suffering various forms of abiotic stress. The molecular mechanisms underlying the melatonin-mediated protection of their photosynthetic machinery are not completely resolved. This study investigates the effects of exogenous melatonin applications on salt-induced damage to the light reaction components of the photosynthetic machinery of tomato seedlings. The results showed that melatonin pretreatments can help maintain growth and net photosynthetic rate (PN) under salt stress conditions. Pretreatment with melatonin increased the effective quantum yield of photosystem II (ΦPSII), the photochemical quenching coefficient (qP) and the proportion of PSII centers that are "open" (qL) under saline conditions. In this way, damage to the photosynthetic electron transport chain (PET) in photosystem II (PSII) was mitigated. In addition, melatonin pretreatment facilitated the repair of PSII by maintaining the availability of D1 protein that was otherwise reduced by salinity. The ROS levels and the gene expressions of the chloroplast TRXs and PRXs were also investigated. Salt stress resulted in increased levels of reactive oxygen species (ROS), which were mitigated by melatonin. In tomato leaves under salt stress, the expressions of PRXs and TRXf declined but the expressions of TRXm1/4 and TRXm2 increased. Melatonin pretreatment promoted the expression of TRXf and the abundances of TRXf and TRXm gene products but had no effects on the expressions of PRXs. In summary, melatonin improves the photosynthetic activities of tomato seedlings under salt stress. The mechanism could be that: (1) Melatonin controls ROS levels and prevents damaging elevations of ROS caused by salt stress. (2) Melatonin facilitates the recovery of PET and D1 protein synthesis, thus enhancing the tolerance of photosynthetic activities to salinity. (3) Melatonin induces the expression of TRXf and regulates the abundance of TRXf and TRXm gene products, which

  8. Axisymmetric Compression of a Mohr-Coulomb Medium with Arbitrary Dilatancy

    DTIC Science & Technology

    1991-11-01

    MROED-S H GAUBE ATTN: R FRANK U S ARMY ENGR WATERWAYS EXPER STATION BDM ENGINEERING SERVICES CO ATTN: C NORMAN 2 CYS ATTN: D BURGESS ATTN: CEWES J K... KINSLOW ATTN: TECHNICAL REPORT SYSTEM ATTN: RICHARD KEEFFE SCIENCE APPLICATIONS INTL CORP KAMAN SCIENCES CORP ATTN: W LAYSON ATTN: DASIAC SOUTHWEST RESEARCH

  9. Involvement of ZFR1 of Fusarium verticilliodes in kernel colonization and the regulation of FST1, a putative sugar transporter gene required for fumonisin biosynthesis on maize kernels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fumonisins comprise a class of carcinogenic mycotoxins produced by Fusarium verticillioides during colonization of maize kernels. In previous work, we identified ZFR1, which is predicted to encode a Zn(II)2Cys6 zinc finger transcription factor required for fumonisin B1 (FB1) production during growt...

  10. Regulation of the aflatoxin-like toxin dothistromin by AflJ

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biosynthesis by Aspergillus parasiticus of aflatoxin, one of the most potent known naturally occurring carcinogens, requires the activity of two regulatory proteins, AflR and AflJ, which are encoded by divergently transcribed genes within the aflatoxin gene cluster. Although the Zn2Cys6 transcriptio...

  11. Approximations of Surface Roughness Effects for Airblast Calculations

    DTIC Science & Technology

    1985-11-01

    burst might reasonably suffice for a measurement of Zo. * At the ground range for which R = Ro, the overpressure is AP 1.4 P1. 13 SECTION 5 ROUGHNESS...ESTIMATES) STRATEGIC AND THEATER NUCLEAR FORCES ATTN: DI-5 ATTN: DR WOODRUFF ATTN: DIA/VPA-2 (FED RES DIV) 2 CYS ATTN: RTS-2B U S EURL . EAN COMMAND ATTN

  12. Construction of a Fusion Enzyme Exhibiting Superoxide Dismutase and Peroxidase Activity.

    PubMed

    Sharapov, M G; Novoselov, V I; Ravin, V K

    2016-04-01

    A chimeric gene construct encoding human peroxiredoxin 6 and Mn-superoxide dismutase from Escherichia coli was developed. Conditions for expression of the fusion protein in E. coli cell were optimized. Fusing of the enzymes into a single polypeptide chain with peroxiredoxin 6 at the N-terminus (PSH) did not affect their activities. On the contrary, the chimeric protein with reverse order of enzymes (SPH) was not obtained in a water-soluble active form. The active chimeric protein (PSH) exhibiting both peroxidase and superoxide dismutase activities was prepared and its physicochemical properties were characterized.

  13. Formation of Hg(II) tetrathiolate complexes with cysteine at neutral pH

    DOE PAGES

    Warner, Thomas; Jalilehvand, Farideh

    2016-01-04

    Mercury(II) ions precipitate from aqueous cysteine (H2Cys) solutions containing H2Cys/Hg(II) mole ratio ≥ 2.0 as Hg(S-HCys)2. In absence of additional cysteine, the precipitate dissolves at pH ~12 with the [Hg(S,N-Cys)2]2- complex dominating. With excess cysteine (H2Cys/Hg(II) mole ratio ≥ 4.0), higher complexes form and the precipitate dissolves at lower pH values. Previously, we found that tetrathiolate [Hg(S-Cys)4]6- complexes form at pH = 11.0; in this work we extend the investigation to pH values of physiological interest. We examined two series of Hg(II)-cysteine solutions in which CHg(II) varied between 8 – 9 mM and 80 – 100 mM, respectively, with H2Cys/Hg(II)more » mole ratios from 4 to ~20. The solutions were prepared in the pH range 7.1 – 8.8, at the pH at which the initial Hg(S-HCys)2 precipitate dissolved. The variations in the Hg(II) speciation were followed by 199Hg NMR, X-ray absorption and Raman spectroscopic techniques. Our results show that in the dilute solutions (CHg(II) = 8 – 9 mM), mixtures of di-, tri- (major) and tetrathiolate complexes exist at moderate cysteine excess (CH2Cys ~ 0.16 M) at pH 7.1. In the more concentrated solutions (CHg(II) = 80 – 100 mM) with high cysteine excess (CH2Cys > 0.9 M), tetrathiolate [Hg(S-cysteinate)4]m-6 (m = 0 – 4) complexes dominate in the pH range 7.3 – 7.8, with lower charge than for the [Hg(S-Cys)4]6- complex due to protonation of some (m) of the amino groups of the coordinated cysteine ligands. In conclusion, the results of this investigation could provide a key to the mechanism of biosorption and accumulation of Hg(II) ions in biological / environmental systems.« less

  14. KatP contributes to OxyR-regulated hydrogen peroxide resistance in Escherichia coli serotype O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli K12 defends against peroxide mediated oxidative damage using two catalases, hydroperoxidase I (katG) and hydroperoxidase II (katE) and the peroxiredoxin, alkyl hydroperoxide reductase (ahpC). In E. coli O157:H7 strain ATCC 43895 (EDL933), plasmid pO157 encodes for an additional cata...

  15. Using PCR-RFLP Technology to Teach Single Nucleotide Polymorphism for Undergraduates

    ERIC Educational Resources Information Center

    Zhang, Bo; Wang, Yan; Xu, Xiaofeng; Guan, Xingying; Bai, Yun

    2013-01-01

    Recent studies indicated that the aberrant gene expression of peroxiredoxin-6 (prdx6) was found in various kinds of cancers. Because of its biochemical function and gene expression pattern in cancer cells, the association between genetic polymorphism of Prdx6 and cancer onset is interesting. In this report, we have developed and implemented a…

  16. A novel L-arabinose-responsive regulator discovered in the rice-blast fungus Pyricularia oryzae (Magnaporthe oryzae).

    PubMed

    Klaubauf, Sylvia; Zhou, Miaomiao; Lebrun, Marc-Henri; de Vries, Ronald P; Battaglia, Evy

    2016-02-01

    In this study we identified the L-arabinose-responsive regulator of Pyricularia oryzae that regulates L-arabinose release and catabolism. Previously we identified the Zn2Cys6 transcription factor (TF), AraR, that has this role in the Trichocomaceae family (Eurotiales), but is absent in other fungi. Candidate Zn2Cys6 TF genes were selected according to their transcript profiles on L-arabinose. Deletion mutants of these genes were screened for their growth phenotype on L-arabinose. One mutant, named Δara1, was further analyzed. Our analysis demonstrated that Ara1 from P. oryzae is the functional analog of AraR from A. niger, while there is no significant sequence similarity between them.

  17. In Silico Analysis for Transcription Factors With Zn(II)2C6 Binuclear Cluster DNA-Binding Domains in Candida albicans

    PubMed Central

    Maicas, Sergi; Moreno, Inmaculada; Nieto, Almudena; Gómez, Micaela; Sentandreu, Rafael

    2005-01-01

    A total of 6047 open reading frames in the Candida albicans genome were screened for Zn(II)2C6-type zinc cluster proteins (or binuclear cluster proteins) involved in DNA recognition. These fungal proteins are transcription regulators of genes involved in a wide range of cellular processes, including metabolism of different compounds such as sugars or amino acids, as well as multi-drug resistance, control of meiosis, cell wall architecture, etc. The selection criteria used in the sequence analysis were the presence of the CysX2CysX6CysX5-16CysX2CysX6-8Cys motif and a putative nuclear localization signal. Using this approach, 70 putative Zn(II)2C6 transcription factors have been found in the genome of C. albicans. PMID:18629206

  18. Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom.

    PubMed

    Yamaguchi, Yoko; Peigneur, Steve; Liu, Junyi; Uemura, Shiho; Nose, Takeru; Nirthanan, Selvanayagam; Gopalakrishnakone, Ponnampalam; Tytgat, Jan; Sato, Kazuki

    2016-11-01

    Spinoxin (SPX; α-KTx6.13), isolated from venom of the scorpion Heterometrus spinifer, is a K(+) channel-specific peptide toxin (KTx), which adopts a cysteine-stabilized α/β scaffold that is cross-linked by four disulfide bridges (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7, and Cys4-Cys8). To investigate the role of the individual disulfide bonds in the structure-activity relationship of SPX, we synthesized four SPX analogs in which each pair of cysteine residues was replaced by alanine residues. The analysis of circular dichroism spectra and inhibitory activity against Kv1.3 channels showed that the SPX analogs lacking any of three specific disulfide bonds (Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7) were unable to form the native secondary structure and completely lost inhibitory activities. Thus, we conclude that Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 are required for the inhibition of the Kv1.3 channel by SPX. In contrast, the analog lacking Cys4-Cys8 retained both native secondary structure and inhibitory activity. Interestingly, one of the isomers of the analog lacking Cys1-Cys5 also showed inhibitory activities, although its inhibition was ∼18-fold weaker than native SPX. This isomer had an atypical disulfide bond pairing (Cys3-Cys4 and Cys7-Cys8) that corresponds to that of maurotoxin (MTX), another α-KTx6 family member. These results indicate that the Cys1-Cys5 and Cys2-Cys6 bonds are important for restricting the toxin from forming an atypical (MTX-type) disulfide bond pairing among the remaining four cysteine residues (Cys3, Cys4, Cys7, and Cys8) in native SPX.

  19. Oxygen resuscitation after hypoxia ischemia stimulates prostaglandin pathway in rat cortex

    PubMed Central

    Perez-Polo, J. Regino; Reilly, Conor B.; Rea, Harriet C.

    2011-01-01

    Exposure to hypoxia and hyperoxia in a rodent model of perinatal ischemia results in delayed cell death and and inflammation. Hyperoxia increases oxidative stress that can trigger inflammatory cascades, neutrophil activation, and brain microvascular injury. Here we show that 100% oxygen resuscitation in our rodent model of perinatal ischemia increases cortical COX-2 protein levels, S-nitrosylated COX-2cys526, PGE2, iNOS and 5-LOX, all components of the prostaglandin and leukotriene inflammatory pathway. PMID:21514373

  20. Inactivated Influenza Vaccine That Provides Rapid, Innate-Immune-System-Mediated Protection and Subsequent Long-Term Adaptive Immunity

    PubMed Central

    Wong, Chinn Yi; Mifsud, Edin J.; Edenborough, Kathryn M.; Sekiya, Toshiki; Tan, Amabel C. L.; Mercuri, Francesca; Rockman, Steve; Chen, Weisan; Turner, Stephen J.; Doherty, Peter C.; Kelso, Anne; Brown, Lorena E.; Jackson, David C.

    2015-01-01

    ABSTRACT The continual threat to global health posed by influenza has led to increased efforts to improve the effectiveness of influenza vaccines for use in epidemics and pandemics. We show in this study that formulation of a low dose of inactivated detergent-split influenza vaccine with a Toll-like receptor 2 (TLR2) agonist-based lipopeptide adjuvant (R4Pam2Cys) provides (i) immediate, antigen-independent immunity mediated by the innate immune system and (ii) significant enhancement of antigen-dependent immunity which exhibits an increased breadth of effector function. Intranasal administration of mice with vaccine formulated with R4Pam2Cys but not vaccine alone provides protection against both homologous and serologically distinct (heterologous) viral strains within a day of administration. Vaccination in the presence of R4Pam2Cys subsequently also induces high levels of systemic IgM, IgG1, and IgG2b antibodies and pulmonary IgA antibodies that inhibit hemagglutination (HA) and neuraminidase (NA) activities of homologous but not heterologous virus. Improved primary virus nucleoprotein (NP)-specific CD8+ T cell responses are also induced by the use of R4Pam2Cys and are associated with robust recall responses to provide heterologous protection. These protective effects are demonstrated in wild-type and antibody-deficient animals but not in those depleted of CD8+ T cells. Using a contact-dependent virus transmission model, we also found that heterologous virus transmission from vaccinated mice to naive mice is significantly reduced. These results demonstrate the potential of adding a TLR2 agonist to an existing seasonal influenza vaccine to improve its utility by inducing immediate short-term nonspecific antiviral protection and also antigen-specific responses to provide homologous and heterologous immunity. PMID:26507227

  1. Commercial Parts Radiation Testing

    DTIC Science & Technology

    2015-01-13

    AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVSE/Keith Avery 1 cy ... AFRL -RV-PS- AFRL -RV-PS- TR-2014-0172 TR-2014-0172 COMMERCIAL PARTS RADIATION TESTING Craig J. Kief COSMIAC at UNM 2350 Alamo Avenue SE Suite 300...Vehicles Directorate 3550 Aberdeen Ave SE AIR FORCE MATERIEL COMMAND KIRTLAND AIR FORCE BASE, NM 87117-5776 DTIC COPY NOTICE AND SIGNATURE

  2. An Investigation of the Physical Properties of Erupting Solar Prominences, Phase II

    DTIC Science & Technology

    2014-12-30

    1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVBXS/Dr. Richard Altrock 1 cy 40 Approved for Public Release; Distribution is Unlimited. ... AFRL -RV-PS- AFRL -RV-PS- TR-2014-0195 TR-2014-0195 AN INVESTIGATION OF THE PHYSICAL PROPERTIES OF ERUPTING SOLAR PROMINENCES, PHASE II...AIR FORCE RESEARCH LABORATORY Space Vehicles Directorate 3550 Aberdeen Ave SE AIR FORCE MATERIEL COMMAND KIRTLAND AIR FORCE BASE, NM

  3. Advanced Space-Based Detectors

    DTIC Science & Technology

    2014-07-17

    Research Laboratory 8. PERFORMING ORGANIZATION REPORT NUMBER Space Vehicles Directorate 3550 Aberdeen Ave., SE Kirtland AFB, NM 87117-5776 AFRL -RV...Suite 0944 Ft Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official... AFRL -RV-PS- AFRL -RV-PS- TR-2014-0010 TR-2014-0010 ADVANCED SPACE-BASED DETECTORS David Cardimona 17 Jul 2014 Final Report APPROVED FOR PUBLIC

  4. Growth And Characterization Studies Of Advanced Infrared Heterostructures

    DTIC Science & Technology

    2015-06-30

    Research Laboratory AFRL /RVSS Space Vehicles Directorate 3550 Aberdeen Ave., SE 11. SPONSOR/MONITOR’S REPORT Kirtland AFB, NM 87117-5776 NUMBER(S... Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVSS/Christian Morath 1 cy ... AFRL -RV-PS- TR-2015-0126 AFRL -RV-PS- TR-2015-0126 GROWTH AND CHARACTERIZATION STUDIES OF ADVANCED INFRARED HETEROSTRUCTURES Sanjay Krishna

  5. Mutual Neutralization of Atomic Rare-Gas Cations (Ne+, Ar+, Kr+, Xe+) with Atomic Halide Anions (Cl-, Br-, I-)

    DTIC Science & Technology

    2015-01-07

    Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVBXT/Dr. Albert Viggiano 1 cy... AFRL -RV-PS- TP-2015-0001 AFRL -RV-PS- TP-2015-0001 MUTUAL NEUTRALIZATION OF ATOMIC RARE- GAS CATIONS (Ne+, Ar+, Kr+, Xe+) WITH ATOMIC HALIDE...RESEARCH LABORATORY Space Vehicles Directorate 3550 Aberdeen Ave SE AIR FORCE MATERIEL COMMAND KIRTLAND AIR FORCE BASE, NM 87117-5776 REPORT

  6. Damping Models for Shear-Deformable Beam with Applications to Spacecraft Wiring Harness

    DTIC Science & Technology

    2014-10-28

    Air Force Research Laboratory AFRL /RVSV Space Vehicles Directorate 3550 Aberdeen Ave., SE 11. SPONSOR/MONITOR’S REPORT Kirtland AFB, NM 87117...Kingman Rd, Suite 0944 Ft Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVSV/Derek... AFRL -RV-PS- TR-2014-0189 AFRL -RV-PS- TR-2014-0189 DAMPING MODELS FOR SHEAR-DEFORMABLE BEAM WITH APPLICATIONS TO SPACECRAFT WIRING HARNESS

  7. Robust Satellite Communications Under Hostile Interference

    DTIC Science & Technology

    2015-01-08

    AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVSV/Steven A. Lane 1 cy ... AFRL -RV-PS- AFRL -RV-PS- TR-2014-0207 TR-2014-0207 ROBUST SATELLITE COMMUNICATIONS UNDER HOSTILE INTERFERENCE Marc Lichtman and Jeffrey Reed...FORCE RESEARCH LABORATORY Space Vehicles Directorate 3550 Aberdeen Ave SE AIR FORCE MATERIEL COMMAND KIRTLAND AIR FORCE BASE, NM 87117-5776

  8. Ion Mobility-Mass Spectrometry as a Tool for the Structural Characterization of Peptides Bearing Intramolecular Disulfide Bond(s)

    NASA Astrophysics Data System (ADS)

    Massonnet, Philippe; Haler, Jean R. N.; Upert, Gregory; Degueldre, Michel; Morsa, Denis; Smargiasso, Nicolas; Mourier, Gilles; Gilles, Nicolas; Quinton, Loïc; De Pauw, Edwin

    2016-10-01

    Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated.

  9. Transcriptional profiling of Medicago truncatula during Erysiphe pisi infection

    PubMed Central

    Curto, Miguel; Krajinski, Franziska; Schlereth, Armin; Rubiales, Diego

    2015-01-01

    Resistance to powdery mildew has been studied in a number of plant species, yet the molecular mechanisms remain largely unknown. Transcription factors (TFs) play a critical role in the plant defense response by regulating the transcriptional machinery which coordinates the expression of a large group of genes involved in plant defense. Using high-throughput quantitative real-time PCR (qPCR) technology more than 1000 Medicago truncatula TFs were screened in a pair of susceptible and resistant genotypes of M. truncatula after 4 h of Erysiphe pisi infection. Seventy nine TF genes, belonging to 33 families showed a significant transcriptional change in response to E. pisi infection. Forty eight TF genes were differentially expressed in the resistant genotypes compared to the susceptible one in response to E. pisi infection, including pathogenesis-related transcriptional factors, AP2/EREBP (APETALA2/ETHYLENE-RESPONSIVE ELEMENT BINDING FACTORS), WRKY (highly conserved WRKYGQK amino-acid sequence), MYB (Myeloblastoma), homeodomain (HD) and zinc finger C2C2 (CYS2-CYS2), C2H2, (CYS2-HIS2), LIM (Lin-11, Isl-1, Mec-3) gene families, which are involved in known defense responses. Our results suggest that these TF genes are among the E. pisi responsive genes in resistant M. truncatula that may constitute a regulatory network which controls the transcriptional changes in defense genes involved in resistance to E. pisi. PMID:26217367

  10. Looking for Arabidopsis thaliana peroxidases involved in lignin biosynthesis.

    PubMed

    Herrero, Joaquín; Esteban-Carrasco, Alberto; Zapata, José Miguel

    2013-06-01

    Monolignol polymerization into lignin is catalyzed by peroxidases or laccases. Recently, a Zinnia elegans peroxidase (ZePrx) that is considered responsible for monolignol polymerization in this plant has been molecularly and functionally characterized. Nevertheless, Arabidopsis thaliana has become an alternative model plant for studies of lignification, filling the gaps that may occur with Z. elegans. The arabidopsis genome offers the possibility of performing bioinformatic analyses and data mining that are not yet feasible with other plant species, in order to obtain preliminary evidence on the role of genes and proteins. In our search for arabidopsis homologs to the ZePrx, we performed an exhaustive in silico characterization of everything from the protein to the transcript of Arabidopsis thaliana peroxidases (AtPrxs) homologous to ZePrx, with the aim of identifying one or more peroxidases that may be involved in monolignol polymerization. Nine peroxidases (AtPrx 4, 5, 52, 68, 67, 36, 14, 49 and 72) with an E-value greater than 1e-80 with ZePrx were selected for this study. The results demonstrate that a high level of 1D, 2D and 3D homology between these AtPrxs and ZePrx are not always accompanied by the presence of the same electrostatic and mRNA properties that indicate a peroxidase is involved in lignin biosynthesis. In summary, we can confirm that the peroxidases involved in lignification are among AtPrx 4, 52, 49 and 72. Their structural and mRNA features indicate that exert their action in the cell wall similar to ZePrx.

  11. Self-construction of supramolecular polyrotaxane films by an electrotriggered morphogen-driven process.

    PubMed

    Rydzek, Gaulthier; Garnier, Tony; Schaaf, Pierre; Voegel, Jean-Claude; Senger, Bernard; Frisch, Benoît; Haikel, Youssef; Petit, Corinne; Schlatter, Guy; Jierry, Loïc; Boulmedais, Fouzia

    2013-08-27

    The design of films using a one-pot process has recently attracted increasing interest in the field of polymer thin film formation. Herein we describe the preparation of one-pot supramolecular polyrotaxane (PRX) films using the morphogen-driven self-construction process. This one-pot buildup strategy where the film growth is triggered by the electrochemical formation and diffusion of a catalyst in close vicinity of the substrate has recently been introduced by our group. A one-pot mixture was used that contained (i) poly(acrylic acid) (PAA) functionalized by azide groups grafted on the polymer chain through oligo(ethylene glycol) (EG) arms, leading to PAA-EG13-N3, (ii) cyclodextrins (α and β CD), as macrocycles that can be threaded along EG arms, (iii) alkyne-functionalized stoppers (ferrocene or adamantane), to cap the PRX assembly by click chemistry, and (iv) copper sulfate. The one-pot mixture solution was brought into contact with a gold electrode. Cu(I), the morphogen, was generated electrochemically from Cu(II) at the electrode/one-pot solution interface. This electrotriggered click reaction leads to the capping of polypseudorotaxane yielding to PRXs. The PRXs can self-assemble through lateral supramolecular interactions to form aggregates and ensure the cohesion of the film. The film buildup was investigated using different types of CD and alkyne functionalized stoppers. Supramolecular PRX aggregates were characterized by X-ray diffraction measurements. The film topographies were imaged by atomic force microscopy. The influence of the concentration in CD and the presence of a competitor were studied as well. The stability of the resulting film was tested in contact with 8 M urea and during the electrochemical oxidation of ferrocene.

  12. Thiol-Based Peroxidases and Ascorbate Peroxidases: Why Plants Rely on Multiple Peroxidase Systems in the Photosynthesizing Chloroplast?

    PubMed

    Dietz, Karl-Josef

    2016-01-01

    Photosynthesis is a highly robust process allowing for rapid adjustment to changing environmental conditions. The efficient acclimation depends on balanced redox metabolism and control of reactive oxygen species release which triggers signaling cascades and potentially detrimental oxidation reactions. Thiol peroxidases of the peroxiredoxin and glutathione peroxidase type, and ascorbate peroxidases are the main peroxide detoxifying enzymes of the chloroplast. They use different electron donors and are linked to distinct redox networks. In addition, the peroxiredoxins serve functions in redox regulation and retrograde signaling. The complexity of plastid peroxidases is discussed in context of suborganellar localization, substrate preference, metabolic coupling, protein abundance, activity regulation, interactions, signaling functions, and the conditional requirement for high antioxidant capacity. Thus the review provides an opinion on the advantage of linking detoxification of peroxides to different enzymatic systems and implementing mechanisms for their inactivation to enforce signal propagation within and from the chloroplast.

  13. Thiol-Based Peroxidases and Ascorbate Peroxidases: Why Plants Rely on Multiple Peroxidase Systems in the Photosynthesizing Chloroplast?

    PubMed Central

    Dietz, Karl-Josef

    2016-01-01

    Photosynthesis is a highly robust process allowing for rapid adjustment to changing environmental conditions. The efficient acclimation depends on balanced redox metabolism and control of reactive oxygen species release which triggers signaling cascades and potentially detrimental oxidation reactions. Thiol peroxidases of the peroxiredoxin and glutathione peroxidase type, and ascorbate peroxidases are the main peroxide detoxifying enzymes of the chloroplast. They use different electron donors and are linked to distinct redox networks. In addition, the peroxiredoxins serve functions in redox regulation and retrograde signaling. The complexity of plastid peroxidases is discussed in context of suborganellar localization, substrate preference, metabolic coupling, protein abundance, activity regulation, interactions, signaling functions, and the conditional requirement for high antioxidant capacity. Thus the review provides an opinion on the advantage of linking detoxification of peroxides to different enzymatic systems and implementing mechanisms for their inactivation to enforce signal propagation within and from the chloroplast. PMID:26810073

  14. Oxidative stress management in the filamentous, heterocystous, diazotrophic cyanobacterium, Anabaena PCC7120.

    PubMed

    Banerjee, Manisha; Raghavan, Prashanth S; Ballal, Anand; Rajaram, Hema; Apte, S K

    2013-10-10

    Reactive oxygen species (ROS) are inevitably generated as by-products of respiratory/photosynthetic electron transport in oxygenic photoautotrophs. Unless effectively scavenged, these ROS can damage all cellular components. The filamentous, heterocystous, nitrogen-fixing strains of the cyanobacterium, Anabaena, serve as naturally abundant contributors of nitrogen biofertilizers in tropical rice paddy fields. Anabaena strains are known to tolerate several abiotic stresses, such as heat, UV, gamma radiation, desiccation, etc., that are known to generate ROS. ROS are detoxified by specific antioxidant enzymes like superoxide dismutases (SOD), catalases and peroxiredoxins. The genome of Anabaena PCC7120 encodes two SODs, two catalases and seven peroxiredoxins, indicating the presence of an elaborate antioxidant enzymatic machinery to defend its cellular components from ROS. This article summarizes recent findings and depicts important perspectives in oxidative stress management in Anabaena PCC7120.

  15. Understanding the role of the catalase/peroxide genes in H2O2 resistance of E. coli serotype O157:H7 biofilms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Escherichia coli serotype O157:H7 defenses against H2O2 include the peroxiredoxin AhpC and three catalases: KatG (catalase-peroxidase), KatE (catalase), and the plasmid-encoded KatP (catalase/peroxidase). AhpC, KatG, and KatP are induced by OxyR in exponential phase, while KatE is indu...

  16. Peroxide resistance in Escherichia coli serotype O157:H7 biofilms is regulated by both RpoS dependent and independent mechanisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In many Escherichia coli serotype O157:H7 strains, defenses against peroxide damage include the peroxiredoxin AhpCF and three catalases: KatG (catalase-peroxidase), KatE (catalase), and the plasmid-encoded KatP (catalase/peroxidase). AhpC, KatG, and KatP are induced by OxyR /s70 in exponential phase...

  17. Distribution of Prx-linked hydroperoxide reductase activity among microorganisms.

    PubMed

    Takeda, Kouji; Nishiyama, Yoshitaka; Yoda, Koji; Watanabe, Toshihiro; Nimura-Matsune, Kaori; Mura, Kiyoshi; Tokue, Chiyoko; Katoh, Tetzuya; Kawasaki, Shinji; Niimura, Youichi

    2004-01-01

    Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

  18. Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State

    NASA Astrophysics Data System (ADS)

    Folda, Alessandra; Citta, Anna; Scalcon, Valeria; Calì, Tito; Zonta, Francesco; Scutari, Guido; Bindoli, Alberto; Rigobello, Maria Pia

    2016-03-01

    The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release.

  19. Differential proteomics and functional research following gene therapy in a mouse model of Leber congenital amaurosis.

    PubMed

    Zheng, Qinxiang; Ren, Yueping; Tzekov, Radouil; Zhang, Yuanping; Chen, Bo; Hou, Jiangping; Zhao, Chunhui; Zhu, Jiali; Zhang, Ying; Dai, Xufeng; Ma, Shan; Li, Jia; Pang, Jijing; Qu, Jia; Li, Wensheng

    2012-01-01

    Leber congenital amaurosis (LCA) is one of the most severe forms of inherited retinal degeneration and can be caused by mutations in at least 15 different genes. To clarify the proteomic differences in LCA eyes, a cohort of retinal degeneration 12 (rd12) mice, an LCA2 model caused by a mutation in the RPE65 gene, were injected subretinally with an AAV vector (scAAV5-smCBA-hRPE65) in one eye, while the contralateral eye served as a control. Proteomics were compared between untreated rd12 and normal control retinas on P14 and P21, and among treated and untreated rd12 retinas and control retinas on P42. Gene therapy in rd12 mice restored retinal function in treated eyes, which was demonstrated by electroretinography (ERG). Proteomic analysis successfully identified 39 proteins expressed differently among the 3 groups. The expression of 3 proteins involved in regulation of apoptosis and neuroptotection (alpha A crystallin, heat shock protein 70 and peroxiredoxin 6) were investigated further. Immunofluorescence, Western blot and real-time PCR confirmed the quantitative changes in their expression. Furthermore, cell culture studies suggested that peroxiredoxin 6 could act in an antioxidant role in rd12 mice. Our findings support the feasibility of gene therapy in LCA2 patients and support a role for alpha A crystallin, heat shock protein 70 and peroxiredoxin 6 in the pathogenetic mechanisms involved in LCA2 disease process.

  20. Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State

    PubMed Central

    Folda, Alessandra; Citta, Anna; Scalcon, Valeria; Calì, Tito; Zonta, Francesco; Scutari, Guido; Bindoli, Alberto; Rigobello, Maria Pia

    2016-01-01

    The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release. PMID:26975474

  1. Adaptation in Multi-Satellite Constellation Cooperation

    DTIC Science & Technology

    2014-08-01

    Directorate 3550 Aberdeen Ave., SE 11. SPONSOR/MONITOR’S REPORT Kirtland AFB, NM 87117-5776 NUMBER(S) AFRL -RV-PS-TR-2014-0113 12. DISTRIBUTION...Kingman Rd, Suite 0944 Ft Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVSV/Khanh Pham... AFRL -RV-PS- AFRL -RV-PS- TR-2014-0113 TR-2014-0113 ADAPTATION IN MULTI-SATELLITE CONSTELLATION COOPERATION Chengyu Cao University of Connecticut

  2. Computer Prediction of Store Aerodynamic Loading during Separation.

    DTIC Science & Technology

    1982-12-01

    to be taken. 120 .°- XBAR=O x positive forward of store CG, relative to moment center YBAR =O y positive right of store CG, relative to moment center...Sequence 2, C m Mach=0.6, YRefo3 Feet .. .. ......74 19 Run Sequence 2, C n Mach=0.6, Y ~ef=3 Feet .. .. ......75 20 Run Sequence 2, CYS Mach=0.6. YRef=3...sectional area SR reference area, d2/4 t time, seconds u’v,w incompressible perturbation velocities in x, y ,z directions of figure 1; compressible

  3. The DMSP Space Weather Sensors Data Archive Listing (1982-2013) and File Formats Descriptions

    DTIC Science & Technology

    2014-08-01

    6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVBXP/Dr. Gordon Wilson 1 cy 63 Approved for public release... AFRL -RV-PS- AFRL -RV-PS- TR-2014-0174 TR-2014-0174 THE DMSP SPACE WEATHER SENSORS DATA ARCHIVE LISTING (1982-2013) AND FILE FORMATS DESCRIPTIONS...Vehicles Directorate 3550 Aberdeen Ave SE AIR FORCE MATERIEL COMMAND KIRTLAND AIR FORCE BASE, NM 87117-5776 DTIC COPY NOTICE AND SIGNATURE PAGE

  4. Estimate of Solar Maximum Using the 1-8 Angstrom Geostationary Operational Environmental Satellites X-Ray Measurements

    DTIC Science & Technology

    2014-12-12

    Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVBXS/Dr. K. S..Balasubramaniam 1 cy 5... AFRL -RV-PS- AFRL -RV-PS- TR-2015-0005 TR-2015-0005 ESTIMATE OF SOLAR MAXIMUM USING THE 1–8 Å GEOSTATIONARY OPERATIONAL ENVIRONMENTAL SATELLITES X...AIR FORCE RESEARCH LABORATORY Space Vehicles Directorate 3550 Aberdeen Ave SE AIR FORCE MATERIEL COMMAND KIRTLAND AIR FORCE BASE, NM 87117-5776

  5. Biological Nanoplatforms for Self-Assembled Electronics

    DTIC Science & Technology

    2015-03-24

    Kirtland AFB, NM 87117-5776 NUMBER(S) AFRL -RV-PS-TR-2015-0024 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for public release; distribution is...LIST DTIC/OCP 8725 John J. Kingman Rd, Suite 0944 Ft Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official... AFRL -RV-PS- AFRL -RV-PS- TR-2015-0024 TR-2015-0024 BIOLOGICAL NANOPLATFORMS FOR SELF- ASSEMBLED ELECTRONICS Stephen Jett University of New Mexico 1

  6. Nanoscale Semiconductor Electronics

    DTIC Science & Technology

    2015-02-25

    MONITOR’S REPORT Kirtland AFB, NM 87117-5776 NUMBER(S) AFRL -RV-PS-TR-2014-0202 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for public release...Kingman Rd, Suite 0944 Ft Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVSE/Jesse Mee 1 cy ... AFRL -RV-PS- AFRL -RV-PS- TR-2014-0202 TR-2014-0202 NANOSCALE SEMICONDUCTOR ELECTRONICS Steven R. J. Brueck and Ganesh Balakrishnan University of New

  7. Data Association Using a Minimum-Fuel Metric

    DTIC Science & Technology

    2014-08-26

    Directorate 3550 Aberdeen Ave. SE Kirtland AFB, NM 87117-5776 NUMBER(S) AFRL -RV-PS-TR-2014-0143 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved...DISTRIBUTION LIST DTIC/OCP 8725 John J. Kingman Rd, Suite 0944 Ft Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys... AFRL -RV-PS- AFRL -RV-PS- TR-2014-0143 TR-2014-0143 DATA ASSOCIATION USING A MINIMUM-FUEL METRIC Andris D. Jaunzemis and Marcus J. Holzinger Georgia

  8. Generation of High-Frequency P and S Wave Energy by Rock Fracture During a Buried Explosion

    DTIC Science & Technology

    2015-07-20

    MONITOR’S ACRONYM(S) Air Force Research Laboratory Space Vehicles Directorate 3550 Aberdeen Avenue SE Kirtland AFB, NM 87117-5776 AFRL /RVBYE 11...DISTRIBUTION LIST DTIC/OCP 8725 John J. Kingman Rd, Suite 0944 Ft Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official... AFRL -RV -PS- TR-2015-0145 AFRL -RV -PS- TR-2015-0145 GENERATION OF HIGH-FREQUENCY P AND S WAVE ENERGY BY ROCK FRACTURE DURING A BURIED EXPLOSION

  9. Cyclic and Long-Term Variation of Sunspot Magnetic Fields

    DTIC Science & Technology

    2014-10-15

    Vehicles Directorate 3550 Aberdeen Avenue SE Kirtland AFB, NM 87117-5776 8. PERFORMING ORGANIZATION REPORT NUMBER AFRL -RV-PS-TP-2014-0009 9...1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVBXS/Dr. Carl Henney 1 cy Approved for public release; distribution... AFRL -RV-PS- TP-2014-0009 AFRL -RV-PS- TP-2014-0009 CYCLIC AND LONG-TERM VARIATION OF SUNSPOT MAGNETIC FIELDS Alexei A. Pevtsov, et al. 15

  10. Computational Study of Collisions Between O(3P) and NO(2Pi) at Temperatures Relevant to the Hypersonic Flight Regime

    DTIC Science & Technology

    2014-10-29

    LIST DTIC/OCP 8725 John J. Kingman Rd, Suite 0944 Ft Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record... AFRL -RV-PS- AFRL -RV-PS- TR-2015-0072 TR-2015-0072 COMPUTATIONAL STUDY OF COLLISIONS BETWEEN O(3P) AND NO(2Π) AT TEMPERATURES RELEVANT TO THE...UNLIMITED. AIR FORCE RESEARCH LABORATORY Space Vehicles Directorate 3550 Aberdeen Ave SE AIR FORCE MATERIEL COMMAND KIRTLAND AIR FORCE BASE, NM

  11. A Trade Study of Thermosphere Empirical Neutral Density Models

    DTIC Science & Technology

    2014-08-01

    OCP 8725 John J. Kingman Rd, Suite 0944 Ft Belvoir, VA 22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL ... AFRL -RV-PS- TR-2016-0008 AFRL -RV-PS- TR-2016-0008 A TRADE STUDY OF THERMOSPHERE EMPIRICAL NEUTRAL DENSITY MODELS Chin S. Lin, et al. 1 August...Ave SE AIR FORCE MATERIEL COMMAND KIRTLAND AIR FORCE BASE, NM 87117-5776 DTIC COPY NOTICE AND SIGNATURE PAGE Using Government drawings

  12. Robust Satellite Communications Under Hostile Interference

    DTIC Science & Technology

    2016-05-20

    Kirtland AFB, NM 87117-5776 AFRL /RVSW 11. SPONSOR/MONITOR’S REPORT NUMBER(S) AFRL -RV-PS-TR-2016-0079 12. DISTRIBUTION / AVAILABILITY STATEMENT...22060-6218 1 cy AFRL /RVIL Kirtland AFB, NM 87117-5776 2 cys Official Record Copy AFRL /RVSW/Khanh Pham 1 cy 28 Approved for public release... AFRL -RV-PS- AFRL -RV-PS- TR-2016-0079 TR-2016-0079 ROBUST SATELLITE COMMUNICATIONS UNDER HOSTILE INTERFERENCE Marc Lichtman and Jeffrey Reed

  13. Pichia pastoris Fep1 is a [2Fe-2S] protein with a Zn finger that displays an unusual oxygen-dependent role in cluster binding

    PubMed Central

    Cutone, Antimo; Howes, Barry D.; Miele, Adriana E.; Miele, Rossella; Giorgi, Alessandra; Battistoni, Andrea; Smulevich, Giulietta; Musci, Giovanni; di Patti, Maria Carmela Bonaccorsi

    2016-01-01

    Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys2-Cys2 type zinc fingers and a set of four conserved cysteines arranged in a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster. PMID:27546548

  14. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei

    PubMed Central

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-01-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. PMID:25044501

  15. The leukotriene E4 puzzle: finding the missing pieces and revealing the pathobiologic implications.

    PubMed

    Austen, K Frank; Maekawa, Akiko; Kanaoka, Yoshihide; Boyce, Joshua A

    2009-09-01

    The intracellular parent of the cysteinyl leukotrienes (cysLTs), leukotriene (LT) C(4), is formed by conjugation of LTA(4) and reduced glutathione by LTC(4) synthase in mast cells, eosinophils, basophils, and macrophages. After extracellular export, LTC(4) is converted to LTD(4) and LTE(4) through sequential enzymatic removal of glutamic acid and then glycine. Only LTE(4) is sufficiently stable to be prominent in biologic fluids, such as urine or bronchoalveolar lavage fluid, of asthmatic individuals and at sites of inflammation in animal models. LTE(4) has received little attention because it binds poorly to the classical type 1 and 2 cysLT receptors and is much less active on normal airways than LTC(4) or LTD(4). However, early studies indicated that LTE(4) caused skin swelling in human subjects as potently as LTC(4) and LTD(4), that airways of asthmatic subjects (particularly those that were aspirin sensitive) were selectively hyperresponsive to LTE(4), and that a potential distinct LTE(4) receptor was present in guinea pig trachea. Recent studies have begun to uncover receptors selective for LTE(4): P2Y(12), an adenosine diphosphate receptor, and CysLT(E)R, which was observed functionally in the skin of mice lacking the type 1 and 2 cysLT receptors. These findings prompt a renewed focus on LTE(4) receptors as therapeutic targets that are not currently addressed by available receptor antagonists.

  16. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

    PubMed

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-10-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.

  17. Shared weapons of blood- and plant-feeding insects: Surprising commonalities for manipulating hosts.

    PubMed

    Guiguet, Antoine; Dubreuil, Géraldine; Harris, Marion O; Appel, Heidi M; Schultz, Jack C; Pereira, Marcos H; Giron, David

    2016-01-01

    Insects that reprogram host plants during colonization remind us that the insect side of plant-insect story is just as interesting as the plant side. Insect effectors secreted by the salivary glands play an important role in plant reprogramming. Recent discoveries point to large numbers of salivary effectors being produced by a single herbivore species. Since genetic and functional characterization of effectors is an arduous task, narrowing the field of candidates is useful. We present ideas about types and functions of effectors from research on blood-feeding parasites and their mammalian hosts. Because of their importance for human health, blood-feeding parasites have more tools from genomics and other - omics than plant-feeding parasites. Four themes have emerged: (1) mechanical damage resulting from attack by blood-feeding parasites triggers "early danger signals" in mammalian hosts, which are mediated by eATP, calcium, and hydrogen peroxide, (2) mammalian hosts need to modulate their immune responses to the three "early danger signals" and use apyrases, calreticulins, and peroxiredoxins, respectively, to achieve this, (3) blood-feeding parasites, like their mammalian hosts, rely on some of the same "early danger signals" and modulate their immune responses using the same proteins, and (4) blood-feeding parasites deploy apyrases, calreticulins, and peroxiredoxins in their saliva to manipulate the "danger signals" of their mammalian hosts. We review emerging evidence that plant-feeding insects also interfere with "early danger signals" of their hosts by deploying apyrases, calreticulins and peroxiredoxins in saliva. Given emerging links between these molecules, and plant growth and defense, we propose that these effectors interfere with phytohormone signaling, and therefore have a special importance for gall-inducing and leaf-mining insects, which manipulate host-plants to create better food and shelter.

  18. PrxQ B from Mycobacterium tuberculosis is a monomeric, thioredoxin-dependent and highly efficient fatty acid hydroperoxide reductase.

    PubMed

    Reyes, Aníbal M; Vazquez, Diego S; Zeida, Ari; Hugo, Martín; Piñeyro, M Dolores; De Armas, María Inés; Estrin, Darío; Radi, Rafael; Santos, Javier; Trujillo, Madia

    2016-12-01

    Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M. tuberculosis or MtPrxQ B). The protein has been reported to be essential for M. tuberculosis growth in cholesterol-rich medium. We demonstrated the M. tuberculosis thioredoxin B/C-dependent peroxidase activity of MtPrxQ B, which acted as a two-cysteine peroxiredoxin that could function, although less efficiently, using a one-cysteine mechanism. Through steady-state and competition kinetic analysis, we proved that the net forward rate constant of MtPrxQ B reaction was 3 orders of magnitude faster for fatty acid hydroperoxides than for hydrogen peroxide (3×10(6)vs 6×10(3)M(-)(1)s(-)(1), respectively), while the rate constant of peroxynitrite reduction was (0.6-1.4) ×10(6)M(-)(1)s(-)(1) at pH 7.4. The enzyme lacked activity towards cholesterol hydroperoxides solubilized in sodium deoxycholate. Both thioredoxin B and C rapidly reduced the oxidized form of MtPrxQ B, with rates constants of 0.5×10(6) and 1×10(6)M(-)(1)s(-)(1), respectively. Our data indicated that MtPrxQ B is monomeric in solution both under reduced and oxidized states. In spite of the similar hydrodynamic behavior the reduced and oxidized forms of the protein showed important structural differences that were reflected in the protein circular dichroism spectra.

  19. Comparative proteomic analysis of drug sodium iron chlorophyllin addition to Hep 3B cell line.

    PubMed

    Zhang, Jun; Wang, Wenhai; Yang, Fengying; Zhou, Xinwen; Jin, Hong; Yang, Peng-yuan

    2012-09-21

    The human hepatoma 3B cell line was chosen as an experimental model for in vitro test of drug screening. The drugs included chlorophyllin and its derivatives such as fluo-chlorophyllin, sodium copper chlorophyllin, and sodium iron chlorophyllin. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method was used in this study to obtain the primary screening results. The results showed that sodium iron chlorophyllin had the best LC(50) value. Proteomic analysis was then performed for further investigation of the effect of sodium iron chlorophyllin addition to the Hep 3B cell line. The proteins identified from a total protein extract of Hep 3B before and after the drug addition were compared by two-dimensional-gel-electrophoresis. Then 32 three-fold differentially expressed proteins were successfully identified by MALDI-TOF-TOF-MS. There are 29 unique proteins among those identified proteins. These proteins include proliferating cell nuclear antigen (PCNA), T-complex protein, heterogeneous nuclear protein, nucleophosmin, heat shock protein A5 (HspA5) and peroxiredoxin. HspA5 is one of the proteins which are involved in protecting cancer cells against stress-induced apoptosis in cultured cells, protecting them against apoptosis through various mechanisms. Peroxiredoxin has anti-oxidant function and is related to cell proliferation, and signal transduction. It can protect the oxidation of other proteins. Peroxiredoxin has a close relationship with cancer and can eventually become a disease biomarker. This might help to develop a novel treatment method for carcinoma cancer.

  20. Metabolic Cycles in Yeast Share Features Conserved among Circadian Rhythms.

    PubMed

    Causton, Helen C; Feeney, Kevin A; Ziegler, Christine A; O'Neill, John S

    2015-04-20

    Cell-autonomous circadian rhythms allow organisms to temporally orchestrate their internal state to anticipate and/or resonate with the external environment. Although ∼24-hr periodicity is observed across aerobic eukaryotes, the central mechanism has been hard to dissect because few simple models exist, and known clock proteins are not conserved across phylogenetic kingdoms. In contrast, contributions to circadian rhythmicity made by a handful of post-translational mechanisms, such as phosphorylation of clock proteins by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3), appear conserved among phyla. These kinases have many other essential cellular functions and are better conserved in their contribution to timekeeping than any of the clock proteins they phosphorylate. Rhythmic oscillations in cellular redox state are another universal feature of circadian timekeeping, e.g., over-oxidation cycles of abundant peroxiredoxin proteins. Here, we use comparative chronobiology to distinguish fundamental clock mechanisms from species and/or tissue-specific adaptations and thereby identify features shared between circadian rhythms in mammalian cells and non-circadian temperature-compensated respiratory oscillations in budding yeast. We find that both types of oscillations are coupled with the cell division cycle, exhibit period determination by CK1 and GSK3, and have peroxiredoxin over-oxidation cycles. We also explore how peroxiredoxins contribute to YROs. Our data point to common mechanisms underlying both YROs and circadian rhythms and suggest two interpretations: either certain biochemical systems are simply permissive for cellular oscillations (with frequencies from hours to days) or this commonality arose via divergence from an ancestral cellular clock.

  1. Characterization of Odin, a Novel Inhibitory Molecule, in EGF Receptor Signaling

    DTIC Science & Technology

    2007-04-01

    2 isoform a 2.7 SLC25A6 solute carrier family 25, member A6 2.6 PRDX4 thioredoxin peroxidase (peroxiredoxin 4) 2.59 EEF2 eukaryotic translation elongation factor 2 [ Homo sapiens ] 2.58 ...ABSTRACT: Protein phosphorylation plays a key role in the regulation of the function of the proteins and the control of wild range of cellular process...Introduction Tyrosine kinase mediated signaling events are important for controlling a diverse range of cellular processes ranging from proliferation and

  2. Proteins as Supramolecular Building Blocks for Responsive Materials and Nanodevices

    DTIC Science & Technology

    2015-12-20

    the date of this printing . List the papers, including journal references, in the following categories: 08/11/2013 08/24/2014 12/20/2015 12/20/2015...Microscopy Structure of Human Peroxiredoxin-3 Filament Reveals the Assembly of a Putative Chaperone, Structure, (05 2015): 0. doi: 10.1016/j.str...military relevance.  Highlights for the programme:   Publication of a paper describing the first  3D  structures of a toroid, double toroid, and

  3. The potential biomarkers of drug addiction: proteomic and metabolomics challenges.

    PubMed

    Wang, Lv; Wu, Ning; Zhao, Tai-Yun; Li, Jin

    2016-07-28

    Drug addiction places a significant burden on society and individuals. Proteomics and metabolomics approaches pave the road for searching potential biomarkers to assist the diagnosis and treatment. This review summarized putative drug addiction-related biomarkers in proteomics and metabolomics studies and discussed challenges and prospects in future studies. Alterations of several hundred proteins and metabolites were reported when exposure to abused drug, which enriched in energy metabolism, oxidative stress response, protein modification and degradation, synaptic function and neurotrasmission, etc. Hsp70, peroxiredoxin-6 and α- and β-synuclein, as well as n-methylserotonin and purine metabolites, were promising as potential biomarker for drug addiction.

  4. Volta phase plate cryo-EM of the small protein complex Prx3

    NASA Astrophysics Data System (ADS)

    Khoshouei, Maryam; Radjainia, Mazdak; Phillips, Amy J.; Gerrard, Juliet A.; Mitra, Alok K.; Plitzko, Jürgen M.; Baumeister, Wolfgang; Danev, Radostin

    2016-01-01

    Cryo-EM of large, macromolecular assemblies has seen a significant increase in the numbers of high-resolution structures since the arrival of direct electron detectors. However, sub-nanometre resolution cryo-EM structures are rare compared with crystal structure depositions, particularly for relatively small particles (<400 kDa). Here we demonstrate the benefits of Volta phase plates for single-particle analysis by time-efficient cryo-EM structure determination of 257 kDa human peroxiredoxin-3 dodecamers at 4.4 Å resolution. The Volta phase plate improves the applicability of cryo-EM for small molecules and accelerates structure determination.

  5. The serpentine path to a novel mechanism-based inhibitor of acute inflammatory lung injury

    PubMed Central

    2014-01-01

    The Comroe lecture on which this review is based described my research path during the past 45 years, beginning with studies of oxidant stress (hyperoxia) and eventuating in the discovery of a synthetic inhibitor of phospholipase A2 activity (called MJ33) that prevents acute lung injury in mice exposed to lipopolysaccharide. In between were studies of lung ischemia, lung surfactant metabolism, the protein peroxiredoxin 6 and its phospholipase A2 activity, and mechanisms for NADPH oxidase activation. These seemingly unrelated research activities provided the nexus for identification of a novel target and a potentially novel therapeutic agent for prevention or treatment of acute lung injury. PMID:24744383

  6. The class III peroxidase PRX17 is a direct target of the MADS-box transcription factor AGAMOUS-LIKE15 (AGL15) and participates in lignified tissue formation.

    PubMed

    Cosio, Claudia; Ranocha, Philippe; Francoz, Edith; Burlat, Vincent; Zheng, Yumei; Perry, Sharyn E; Ripoll, Juan-Jose; Yanofsky, Martin; Dunand, Christophe

    2017-01-01

    Several physiological functions have been attributed to class III peroxidases (PRXs) in plants, but the in planta role of most members of this family still remains undetermined. Here, we report the first functional characterization of PRX17 (At2g22420), one of the 73 members of this family in Arabidopsis thaliana. Localization of PRX17 was examined by transient expression in Nicotiana benthamiana. Loss- and gain-of-function mutants in A. thaliana were studied. Regulation at the gene and protein levels was analyzed using β-glucuronidase (GUS) activity, quantitative reverse transcriptase (qRT)-PCR, zymography, and chromatin immunoprecipitation. Phenotypes were characterized including lignin and xylan contents. PRX17 was expressed in various tissues, including vascular tissues, and PRX17 was localized to the cell wall. In prx17, the lignin content was reduced in the stem and siliques and bolting was delayed, while the opposite phenotype was observed in 35S:PRX17 plants, together with a significant increase of lignin and xylan immunofluorescence signal. Finally, we demonstrated that the transcription factor AGAMOUS-LIKE15 (AGL15) binds to the PRX17 promoter and regulates PRX17 expression level. This converging set of structural, transcriptomic and physiological data suggests that PRX17, under the control of AGL15, contributes to developmental programs by playing an essential role in regulating age-dependent lignified tissue formation, including changes in cell wall properties.

  7. CaPrx, a Coffea arabica gene encoding a putative class III peroxidase induced by root-knot nematode infection.

    PubMed

    Severino, Fábio E; Brandalise, Marcos; Costa, Carolina S; Wilcken, Sílvia R S; Maluf, Mirian P; Gonçalves, Wallace; Maia, Ivan G

    2012-08-01

    Class III peroxidases (Prxs) are enzymes involved in a multitude of physiological and stress-related processes in plants. Here, we report on the characterization of a putative peroxidase-encoding gene from Coffea arabica (CaPrx) that is expressed in early stages of root-knot nematode (RKN) infection. CaPrx showed enhanced expression in coffee roots inoculated with RKN (at 12 h post-inoculation), but no significant difference in expression was observed between susceptible and resistant plants. Assays using transgenic tobacco plants harboring a promoter-β-glucuronidase (GUS) fusion revealed that the CaPrx promoter was exclusively active in the galls induced by RKN. In cross sections of galls, GUS staining was predominantly localized in giant cells. Up-regulation of GUS expression in roots of transgenic plants following RKN inoculation was observed within 16 h. Moreover, no increase in GUS expression after treatment with jasmonic acid was detected. Altogether, these results point to a putative role of this peroxidase in the general coffee response to RKN infection.

  8. Lead(II) complex formation with l-cysteine in aqueous solution

    DOE PAGES

    Jalilehvand, Farideh; Sisombath, Natalie S.; Schell, Adam C.; ...

    2015-02-19

    The lead(II) complexes formed with the multidentate chelator l-cysteine (H2Cys) in an alkaline aqueous solution were studied using 207Pb, 13C, and 1H NMR, Pb LIII-edge X-ray absorption, and UV–vis spectroscopic techniques, complemented by electrospray ion mass spectrometry (ESI-MS). The H2Cys/PbII mole ratios were varied from 2.1 to 10.0 for two sets of solutions with CPbII = 0.01 and 0.1 M, respectively, prepared at pH values (9.1–10.4) for which precipitates of lead(II) cysteine dissolved. At low H2Cys/PbII mole ratios (2.1–3.0), a mixture of the dithiolate [Pb(S,N-Cys)2]2– and [Pb(S,N,O-Cys)(S-HCys)]– complexes with average Pb–(N/O) and Pb–S distances of 2.42 ± 0.04 and 2.64more » ± 0.04 Å, respectively, was found to dominate. At high concentration of free cysteinate (>0.7 M), a significant amount converts to the trithiolate [Pb(S,N-Cys)(S-HCys)2]2–, including a minor amount of a PbS3-coordinated [Pb(S-HCys)3]– complex. The coordination mode was evaluated by fitting linear combinations of EXAFS oscillations to the experimental spectra and by examining the 207Pb NMR signals in the chemical shift range δPb = 2006–2507 ppm, which became increasingly deshielded with increasing free cysteinate concentration. One-pulse magic-angle-spinning (MAS) 207Pb NMR spectra of crystalline Pb(aet)2 (Haet = 2-aminoethanethiol or cysteamine) with PbS2N2 coordination were measured for comparison (δiso = 2105 ppm). The UV–vis spectra displayed absorption maxima at 298–300 nm (S– → PbII charge transfer) for the dithiolate PbS2N(N/O) species; with increasing ligand excess, a shoulder appeared at ~330 nm for the trithiolate PbS3N and PbS3 (minor) complexes. Finally, the results provide spectroscopic fingerprints for structural models for lead(II) coordination modes to proteins and enzymes.« less

  9. Albumin-bound fatty acids but not albumin itself alter redox balance in tubular epithelial cells and induce a peroxide-mediated redox-sensitive apoptosis.

    PubMed

    Ruggiero, Christine; Elks, Carrie M; Kruger, Claudia; Cleland, Ellen; Addison, Kaity; Noland, Robert C; Stadler, Krisztian

    2014-04-15

    Albuminuria is associated with metabolic syndrome and diabetes. It correlates with the progression of chronic kidney disease, particularly with tubular atrophy. The fatty acid load on albumin significantly increases in obesity, presenting a proinflammatory environment to the proximal tubules. However, little is known about changes in the redox milieu during fatty acid overload and how redox-sensitive mechanisms mediate cell death. Here, we show that albumin with fatty acid impurities or conjugated with palmitate but not albumin itself compromised mitochondrial and cell viability, membrane potential and respiration. Fatty acid overload led to a redox imbalance which deactivated the antioxidant protein peroxiredoxin 2 and caused a peroxide-mediated apoptosis through the redox-sensitive pJNK/caspase-3 pathway. Transfection of tubular cells with peroxiredoxin 2 was protective and mitigated apoptosis. Mitochondrial fatty acid entry and ceramide synthesis modulators suggested that mitochondrial β oxidation but not ceramide synthesis may modulate lipotoxic effects on tubular cell survival. These results suggest that albumin overloaded with fatty acids but not albumin itself changes the redox environment in the tubules, inducing a peroxide-mediated redox-sensitive apoptosis. Thus, mitigating circulating fatty acid levels may be an important factor in both preserving redox balance and preventing tubular cell damage in proteinuric diseases.

  10. Cytoprotective Effect of Makgeolli Lees on Paraquat Induced Oxidative Stress in A549 Cells via Activation of NRF2 and Antioxidant Genes.

    PubMed

    Jeon, Miso; Rahman, Naimur; Kim, Yong-Sik

    2016-02-01

    Makgeolli lees (ML) has several physiological effects such as antioxidant, antidiabetic, and anticancer properties, but its biological functions have not been determined definitively. Here, we tested whether ML has a cytoprotective effect on paraquat (PQ)-induced oxidative stress in the human lung carcinoma cell line A549. At 0.1 mg/ml ML, viability of PQ-exposed A549 cells was restored by 12.4%, 18.5%, and 48.6% after 24, 48, and 72 h, respectively. ML also reduced production of the intracellular reactive oxygen species (ROS) that were generated by PQ treatment. Further experiments revealed that ML treatment enhanced the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2) as well as ARE-GFP reporter activity. ML treatment also effectively increased the expression of NRF2's target genes NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase 1 (HO-1). Moreover, we found that expression of cytoprotective genes, including glutathione peroxidases (GPXs), superoxide dismutase (SOD1), catalase (CAT), peroxiredoxin 3 (PRDX3), and peroxiredoxin 4 (PRDX4), was greatly enhanced by treatment with ML during PQ exposure. Taken together, the data suggest that treatment of PQ-exposed A549 cells with ML ameliorates cytotoxicity through induction of NRF2 expression and its target genes HO-1, NQO1, and other antioxidant genes. Thus, ML may serve as a functional food applicable to ROS-mediated human diseases.

  11. Peroxides and peroxide-degrading enzymes in the thyroid.

    PubMed

    Schweizer, Ulrich; Chiu, Jazmin; Köhrle, Josef

    2008-09-01

    Iodination of thyroglobulin is the key step of thyroid hormone biosynthesis. It is catalyzed by thyroid peroxidase and occurs within the follicular space at the apical plasma membrane. Hydrogen peroxide produced by thyrocytes as an oxidant for iodide may compromise cellular and genomic integrity of the surrounding cells, unless these are sufficiently protected by peroxidases. Thus, peroxidases play two opposing roles in thyroid biology. Both aspects of peroxide biology in the thyroid are separated in space and time and respond to the different physiological states of the thyrocytes. Redox-protective peroxidases in the thyroid are peroxiredoxins, glutathione peroxidases, and catalase. Glutathione peroxidases are selenoenzymes, whereas selenium-independent peroxiredoxins are functionally linked to the selenoenzymes of the thioredoxin reductase family through their thioredoxin cofactors. Thus, selenium impacts directly and indirectly on protective enzymes in the thyroid, a link that has been supported by animal experiments and clinical observations. In view of this relationship, it is remarkable that rather little is known about selenoprotein expression and their potential functional roles in the thyroid. Moreover, selenium-dependent and -independent peroxidases have rarely been examined in the same studies. Therefore, we review the relevant literature and present expression data of both selenium-dependent and -independent peroxidases in the murine thyroid.

  12. The Expression of Porcine Prdx6 Gene Is Up-Regulated by C/EBPβ and CREB.

    PubMed

    Wu, Xinyu; Ji, Panlong; Zhang, Liang; Bu, Guowei; Gu, Hao; Wang, Xiaojing; Xiong, Yuanzhu; Zuo, Bo

    2015-01-01

    Peroxiredoxin6 (Prdx6) is one of the peroxiredoxin (Prdxs) family members that play an important role in maintaining cell homeostasis. Our previous studies demonstrated that Prdx6 was significantly associated with pig meat quality, especially meat tenderness. However, the transcriptional regulation of porcine Prdx6 remains unclear. In this study, we determined the transcription start site (TSS) of porcine Prdx6 gene by 5' rapid-amplification of cDNA ends (5' RACE). Several regulatory elements including CCAAT/enhancer-binding proteinβ (C/EBPβ), Myogenic Differentiation (MyoD), cAMP response element binding protein (CREB), stimulating protein1 (Sp1) and heat shock factor (HSF) binding sites were found by computational analyses together with luciferase reporter system. Overexpression and RNA interference experiments showed that C/EBPβ or CREB could up-regulate the expression of porcine Prdx6 gene at both mRNA and protein level. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP) confirmed that C/EBPβ and CREB could interact with Prdx6 promoter. Immuoprecipitation results also showed that C/EBPβ could interact with Prdx6 in vivo. Taken together, our findings identified C/EBPβ and CREB as the important regulators of porcine Prdx6 gene expression, and offered clues for further investigation of Prdx6 gene function.

  13. Modulation of proteomic profile in H295R adrenocortical cell line induced by mitotane.

    PubMed

    Stigliano, A; Cerquetti, L; Borro, M; Gentile, G; Bucci, B; Misiti, S; Piergrossi, P; Brunetti, E; Simmaco, M; Toscano, V

    2008-03-01

    Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p'-DDD), is a compound that represents the effective agent in the treatment of the adrenocortical carcinoma (ACC), able to block cortisol synthesis. In this type of cancer, the biological mechanism induced by this treatment remains still unknown. In this study, we have already shown a greater impairment in the first steps of the steroidogenesis and recognized a little effect on cell cycle. We also evaluated the variation of proteomic profile of the H295R ACC cell line, either in total cell extract or in mitochondria-enriched fraction after treatment with mitotane. In total cell extracts, triose phosphate isomerase, alpha-enolase, D-3-phosphoglycerate dehydrogenase, peroxiredoxin II and VI, heat shock protein 27, prohibitin, histidine triad nucleotide-binding protein, and profilin-1 showed a different expression. In the mitochondrial fraction, the following proteins appeared to be down regulated: aldolase A, peroxiredoxin I, heterogenous nuclear ribonucleoprotein A2/B1, tubulin-beta isoform II, heat shock cognate 71 kDa protein, and nucleotide diphosphate kinase, whereas adrenodoxin reductase, cathepsin D, and heat shock 70 kDa protein 1A were positively up-regulated. This study represents the first proteomic study on the mitotane effects on ACC. It permits to identify some protein classes affected by the drug involved in energetic metabolism, stress response, cytoskeleton structure, and tumorigenesis.

  14. Proteome of the early embryo-maternal dialogue in the cattle uterus.

    PubMed

    Muñoz, Marta; Corrales, Fernando J; Caamaño, José N; Díez, Carmen; Trigal, Beatriz; Mora, María I; Martín, David; Carrocera, Susana; Gómez, Enrique

    2012-02-03

    We analyzed embryo-maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development.

  15. Functional and Structural Characterization of a Thiol Peroxidase from Mycobacterium tuberculosis

    SciTech Connect

    Rho,B.; Hung, L.; Holton, J.; Vigil, D.; Kim, S.; Park, M.; Terwilliger, T.; Pedelacq, j.

    2006-01-01

    A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H{sub 2}O{sub 2}, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 Angstroms shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Ca atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.

  16. Function of antioxidant enzymes and metabolites during maturation of pea fruits.

    PubMed

    Matamoros, Manuel A; Loscos, Jorge; Dietz, Karl-Josef; Aparicio-Tejo, Pedro M; Becana, Manuel

    2010-01-01

    In plant cells, antioxidants keep reactive oxygen species at low concentrations, avoiding oxidative damage while allowing them to play crucial functions in signal transduction. However, little is known about the role of antioxidants during fruit maturation, especially in legumes. Snap pea (Pisum sativum) plants, which have edible fruits, were grown under nodulating and non-nodulating conditions. Fruits were classified in three maturity stages and antioxidants were determined in the seeds and seedless pods. Maturation or prolonged storage of fruits at 25 degrees C led to a decline in antioxidant activities and metabolites and in gamma-glutamylcysteine synthetase protein. Notable exceptions were superoxide dismutase activity and glutathione peroxidase protein, which increased in one or both of these processes. During maturation, cytosolic peroxiredoxin decreased in seeds but increased in pods, and ascorbate oxidase activity was largely reduced in seeds. In stored fruits, ascorbate oxidase activity was nearly abolished in seeds but doubled in pods. It is concluded that symbiotic nitrogen fixation is as effective as nitrogen fertilization in maintaining the antioxidant capacity of pea fruits and that, contrary to climacteric fruits, a general decrease in antioxidants during maturation does not involve oxidative stress. Results underscore the importance of the antioxidant system in reproductive organs and point to ascorbate-glutathione metabolism and cytosolic peroxiredoxin as key players in pea fruit development.

  17. Influence of high temperature during grain filling on the accumulation of storage proteins and grain quality in rice (Oryza sativa L.).

    PubMed

    Lin, Chin-Ju; Li, Chia-Yu; Lin, Shao-Kai; Yang, Fan-Hsuan; Huang, Ji-Jwo; Liu, Yun-Hua; Lur, Huu-Sheng

    2010-10-13

    The present study was performed to understand the effects of high temperature (HT) during filling on the expression of storage proteins and the quality of rice grains. HT (35/30 °C day/night) reduced the weight, amylose content, and flour gel consistency of grains. It increased the accumulation of all classes of storage proteins at early filling stage but decreased the accumulation of prolamins at maturation. For albumins, the expressions of cyclophilin 2, peroxiredoxin, and HSP16.9 were differentially enhanced by HT. For globulins, HT decreased the accumulation of globulin but increased that of glyoxalase I and peroxiredoxin. HT enhanced the transcription of genes for glutelins, prolamins, globulins, and protein disulfide isomerase at early filling stage but decreased the expression of these genes at a later stage. Low amounts of prolamins and globulins, as well as low pH value, were found in sound, immature, and dead kernels grown under HT. The relationships among HT, storage proteins, and grain quality are discussed.

  18. A short report: PAMM, a novel antioxidant protein, induced by oxidative stress

    PubMed Central

    Xu, Yan; Morse, Leslie R.; da Silva, Raquel Assed Bezerra; Wang, Dianhua; Battaglino, Ricardo A.

    2015-01-01

    Reactive oxygen species (ROS) play a central role in estrogen deficiency-induced bone loss. We previously identified and characterized a novel member of the Peroxiredoxin (PRX) like 2 family that we called PAMM: Peroxiredoxin Activated in M-CSF stimulated Monocytes, a redox regulatory protein that modulates osteoclast differentiation in vitro. In this study, we report increased PAMM expression in H2O2-treated cells and in bones from ovariectomized (OVX) mice 4 weeks after surgery, models for oxidative stress in vitro and in vivo, respectively. We also detected increased PAMM abundance and phosphorylated Akt in OVX mice treated with estrogen. In addition, Wortmannin, a specific PI3Kinase inhibitor and Rapamycin, an inhibitor of the PI3Kinase/Akt pathway, blocked Akt phosphorylation and stimulation of PAMM expression by M-CSF. These results indicate that M-CSF-induced PAMM expression is mediated by Akt phosphorylation. Our data also suggest that estrogen-induced PAMM expression is mediated by phosphorylation of Akt. These findings point to PAMM as a potential candidate for Akt-mediated protection against oxidative stress. PMID:26402163

  19. Bifunctional Electrophiles Cross-Link Thioredoxins with Redox Relay Partners in Cells

    PubMed Central

    Naticchia, Matthew R.; Brown, Haley A.; Garcia, Francisco J.; Lamade, Andrew M.; Justice, Samantha L.; Herrin, Rachelle P.; Morano, Kevin A.; West, James D.

    2013-01-01

    Thioredoxin protects cells against oxidative damage by reducing disulfide bonds in improperly oxidized proteins. Previously, we found that the baker's yeast cytosolic thioredoxin Trx2 undergoes cross-linking to form several protein-protein complexes in cells treated with the bifunctional electrophile divinyl sulfone (DVSF). Here, we report that the peroxiredoxin Tsa1 and the thioredoxin reductase Trr1, both of which function in a redox relay network with thioredoxin, become cross-linked in complexes with Trx2 upon DVSF treatment. Treatment of yeast with other bifunctional electrophiles, including diethyl acetylenedicarboxylate (DAD), mechlorethamine (HN2), and 1,2,3,4-diepoxybutane (DEB), resulted in the formation of similar cross-linked complexes. Cross-linking of Trx2 and Tsa1 to other proteins by DVSF and DAD is dependent on modification of the active site Cys residues within these proteins. In addition, the human cytosolic thioredoxin, cytosolic thioredoxin reductase, and peroxiredoxin 2 form cross-linked complexes to other proteins in the presence of DVSF, although each protein shows different susceptibilities to modification by DAD, HN2, and DEB. Taken together, our results indicate that bifunctional electrophiles potentially disrupt redox homeostasis in yeast and human cells by forming cross-linked complexes between thioredoxins and their redox partners. PMID:23414292

  20. Soft modelling for the resolution of highly overlapped voltammetric peaks: application to some Pb-phytochelatin systems.

    PubMed

    Alberich, Arístides; Ariño, Cristina; Díaz-Cruz, José Manuel; Esteban, Miquel

    2007-01-15

    A differential pulse polarographic (DPP) study of the Pb(2+)/Cys-Gly, Pb(2+)/gamma-Glu-Cys, Pb(2+)/PC(2) and Pb(2+)/PC(3) systems is performed, being PC(2) and PC(3) the phytochelatins of general structure (gamma-Glu-Cys)(n)-Gly, with n=2 and 3, respectively. Analysis of DPP data is assisted by multivariate curve resolution with alternating least squares (MCR-ALS) method in order to establish the complexes formation sequence and their final stoichiometries. DPP signals of these systems present, besides overlapping of peaks due to free metal ion and metal complexes, interference of mercury anodic signals. Despite these complications, MCR-ALS allows us to propose a model of complexation for each system, and some tentative structures for the complexes.

  1. The fnr Gene of Bacillus licheniformis and the Cysteine Ligands of the C-Terminal FeS Cluster

    PubMed Central

    Klinger, Anette; Schirawski, Jan; Glaser, Philippe; Unden, Gottfried

    1998-01-01

    In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from Bacillus subtilis, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from B. subtilis, this cluster (Cys226-X2-Cys229-X4-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the B. licheniformis gene to an fnr mutant of B. subtilis complemented the ability for synthesis of nitrate reductase during anaerobic growth. PMID:9642208

  2. The fnr gene of Bacillus licheniformis and the cysteine ligands of the C-terminal FeS cluster.

    PubMed

    Klinger, A; Schirawski, J; Glaser, P; Unden, G

    1998-07-01

    In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from Bacillus subtilis, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from B. subtilis, this cluster (Cys226-X2-Cys229-X4-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the B. licheniformis gene to an fnr mutant of B. subtilis complemented the ability for synthesis of nitrate reductase during anaerobic growth.

  3. Regulatory Genes Controlling Fatty Acid Catabolism and Peroxisomal Functions in the Filamentous Fungus Aspergillus nidulans†

    PubMed Central

    Hynes, Michael J.; Murray, Sandra L.; Duncan, Anna; Khew, Gillian S.; Davis, Meryl A.

    2006-01-01

    The catabolism of fatty acids is important in the lifestyle of many fungi, including plant and animal pathogens. This has been investigated in Aspergillus nidulans, which can grow on acetate and fatty acids as sources of carbon, resulting in the production of acetyl coenzyme A (CoA). Acetyl-CoA is metabolized via the glyoxalate bypass, located in peroxisomes, enabling gluconeogenesis. Acetate induction of enzymes specific for acetate utilization as well as glyoxalate bypass enzymes is via the Zn2-Cys6 binuclear cluster activator FacB. However, enzymes of the glyoxalate bypass as well as fatty acid beta-oxidation and peroxisomal proteins are also inducible by fatty acids. We have isolated mutants that cannot grow on fatty acids. Two of the corresponding genes, farA and farB, encode two highly conserved families of related Zn2-Cys6 binuclear proteins present in filamentous ascomycetes, including plant pathogens. A single ortholog is found in the yeasts Candida albicans, Debaryomyces hansenii, and Yarrowia lipolytica, but not in the Ashbya, Kluyveromyces, Saccharomyces lineage. Northern blot analysis has shown that deletion of the farA gene eliminates induction of a number of genes by both short- and long-chain fatty acids, while deletion of the farB gene eliminates short-chain induction. An identical core 6-bp in vitro binding site for each protein has been identified in genes encoding glyoxalate bypass, beta-oxidation, and peroxisomal functions. This sequence is overrepresented in the 5′ region of genes predicted to be fatty acid induced in other filamentous ascomycetes, C. albicans, D. hansenii, and Y. lipolytica, but not in the corresponding genes in Saccharomyces cerevisiae. PMID:16682457

  4. cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells.

    PubMed

    Lusson, J; Vieau, D; Hamelin, J; Day, R; Chrétien, M; Seidah, N G

    1993-07-15

    By using reverse transcriptase/PCR and oligonucleotide sequences derived from conserved segments (including the conserved RRGDL sequence) of the known proprotein convertases (PCs) PC1, PC2, furin, and PC4, we identified a subtilisin/kexin-like PC called PC5 in both mouse and rat tissues. The composite structure (2.85 kb) was deduced from the analysis of the reverse transcription/PCR products combined with the sequence from a clone isolated from a cDNA library made from corticotropin-activated mouse adrenocortical Y1 cells. The deduced cDNA structures of mouse PC5 and rat PC5 showed that the closest homologue is PACE4. Furthermore, like furin, Drosophila melanogaster (d) dfurin2, and PACE4, PC5 shows the presence of a C-terminal Cys-rich domain containing either 5 (PC5 and PACE4) or 10 (dfurin2) repeats of the consensus motif Cys-Xaa2-Cys-Xaa3-Cys-Xaa(5-7)-Cys-Xaa2-Cys-Xaa (8-15)-Cys-Xaa3-Cys-Xaa(9-16). The richest sources of rat PC5 mRNA (3.8 kb) are the adrenal and gut, but it can also be detected in many endocrine and nonendocrine tissues. Corticotropin-stimulated adrenocortical Y1 cells showed an increased expression of PC5 mRNA, suggesting an upregulation by cAMP. In situ hybridization of rat brain sections demonstrated a unique distribution of PC5 compared to PC1, PC2, and furin.

  5. cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells.

    PubMed Central

    Lusson, J; Vieau, D; Hamelin, J; Day, R; Chrétien, M; Seidah, N G

    1993-01-01

    By using reverse transcriptase/PCR and oligonucleotide sequences derived from conserved segments (including the conserved RRGDL sequence) of the known proprotein convertases (PCs) PC1, PC2, furin, and PC4, we identified a subtilisin/kexin-like PC called PC5 in both mouse and rat tissues. The composite structure (2.85 kb) was deduced from the analysis of the reverse transcription/PCR products combined with the sequence from a clone isolated from a cDNA library made from corticotropin-activated mouse adrenocortical Y1 cells. The deduced cDNA structures of mouse PC5 and rat PC5 showed that the closest homologue is PACE4. Furthermore, like furin, Drosophila melanogaster (d) dfurin2, and PACE4, PC5 shows the presence of a C-terminal Cys-rich domain containing either 5 (PC5 and PACE4) or 10 (dfurin2) repeats of the consensus motif Cys-Xaa2-Cys-Xaa3-Cys-Xaa(5-7)-Cys-Xaa2-Cys-Xaa (8-15)-Cys-Xaa3-Cys-Xaa(9-16). The richest sources of rat PC5 mRNA (3.8 kb) are the adrenal and gut, but it can also be detected in many endocrine and nonendocrine tissues. Corticotropin-stimulated adrenocortical Y1 cells showed an increased expression of PC5 mRNA, suggesting an upregulation by cAMP. In situ hybridization of rat brain sections demonstrated a unique distribution of PC5 compared to PC1, PC2, and furin. Images Fig. 3 Fig. 4 PMID:8341687

  6. Solvation of Co(III)-cysteinato complexes in water: a DFT-based molecular dynamics study.

    PubMed

    Spezia, Riccardo; Bresson, Carole; Den Auwer, Christophe; Gaigeot, Marie-Pierre

    2008-05-22

    Structural, dynamical, and vibrational properties of complexes made of metal cobalt(III) coordinated to different amounts of cysteine molecules were investigated with DFT-based Car-Parrinello molecular dynamics (CPMD) simulations in liquid water solution. The systems are composed of Co(III):3Cys and Co(III):2Cys immersed in liquid water which are modeled by about 110 explicit water molecules, thus one of the biggest molecular systems studied with ab initio molecular simulations so far. In such a way, we were able to investigate structural and dynamical properties of a model of a typical metal binding site used by several proteins. Cobalt, mainly a toxicological agent, can replace the natural binding metal and thus modify the biochemical activity. The structure of the surrounding solvent around the metal-ligands complexes is reported in detail, as well as the metal-ligands coordination bonds, using radial distribution functions and electronic analyses with Mayer bond orders. Structures of the Cocysteine complexes are found in very good agreement with EXAFS experimental data, stressing the importance of considering the surrounding solvent in the modeling. A vibrational analysis is also conducted and compared to experiment, which strengthens the reliability of the solvent interactions with the Cocysteine complexes from our molecular dynamics simulations, as well as the dynamics of the systems. From this preliminary analysis, we could suggest a vibrational fingerprint able to distinguish Co(III):2Cys from Co(III):3Cys. Our simulations also show the importance of considering a quantum explicit solvent, as solute-to-solvent proton transfer events have been observed.

  7. Revisiting and re-engineering the classical zinc finger peptide: consensus peptide-1 (CP-1).

    PubMed

    Besold, Angelique N; Widger, Leland R; Namuswe, Frances; Michalek, Jamie L; Michel, Sarah L J; Goldberg, David P

    2016-04-01

    Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(II). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(II)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(II) and Co(II) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(II) and Zn(II) and a pentacoordinate geometry for Co(II)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(II)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(II)-CP-1(CAHH) and Co(II)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(II) complexes.

  8. Conformational analysis by quantitative NOE measurements of the β-proton pairs across individual disulfide bonds in proteins.

    PubMed

    Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune

    2012-02-01

    NOEs between the β-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U-(13)C, (15)N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal β-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[β-(13)C; α,β-(2)H(2)] Cys and (2R, 3R)-[β-(13)C; α,β-(2)H(2)] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely β2 (proS) or β3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the β-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the β2 and β3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ(2) and χ(3), can be determined based on the precise proton distances of the four β-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.

  9. A zinc finger protein from Candida albicans is involved in sucrose utilization.

    PubMed Central

    Kelly, R; Kwon-Chung, K J

    1992-01-01

    A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans. Images PMID:1729210

  10. Functional and molecular effects of mercury compounds on the human OCTN1 cation transporter: C50 and C136 are the targets for potent inhibition.

    PubMed

    Galluccio, Michele; Pochini, Lorena; Peta, Valentina; Iannì, Maria; Scalise, Mariafrancesca; Indiveri, Cesare

    2015-03-01

    The effect of mercury compounds has been tested on the organic cation transporter, hOCTN1. MeHg(+), Hg(2+), or Cd(2+) caused strong inhibition of transport. 1,4-Dithioerythritol (DTE), cysteine (Cys), and N-acetyl-l-cysteine reversed (NAC) the inhibition at different extents. 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), a prototype SH reagent, exerted inhibition of transport similar to that observed for the mercurial agents. To investigate the mechanism of action of mercurials, mutants of hOCTN1 in which each of the Cys residues was substituted by Ala have been constructed, over-expressed in Escherichia coli, and purified. Tetraethylammonium chloride (TEA) uptake mediated by each mutant in proteoliposomes was comparable to that of wild type (WT). IC50 values of the WT and mutants for the mercury compounds were derived from dose-response analyses. The mutants C50A and C136A showed significant increase of IC50 indicating that the 2 Cys residues were involved in the interaction with the mercury compounds and inhibition of the transporter. The double mutant C50A/C136A was constructed; the lack of inhibition confirmed that the 2 Cys residues are the targets of mercury compounds. MTSEA showed similar behavior with respect to the mercurial reagents with the difference that increased IC50 was observed also in the C81A mutant. Similar results were obtained when transport was measured as acetylcholine uptake. Ethyl mercury (Thimerosal) inhibited hOCTN1 as well. C50A, C50A/C136A and, at very lower extent, C136A showed increased IC50 indicating that C50 was the major target of this mercury compound. The homology model of hOCTN1 was built using as template PiPT and validated by the experimental data on mutant proteins.

  11. Proteomic analysis of membrane-associated proteins from rat liver autophagosomes.

    PubMed

    Øverbye, Anders; Fengsrud, Monica; Seglen, Per O

    2007-01-01

    Proteins associated with membranes from purified rat liver autophagosomes were separated by two-dimensional (2D) gel electrophoresis (zoom gels, pl 4-7 and 6-9), silver-stained and identified by MALDI-TOF mass spectrometry. Among >1,500 detectable protein spots, 58 (derived from 39 different known proteins) were at least twofold (and significantly) enriched in autophagosomal membranes relative to cytoplasmic membranes. All of these membrane-associated proteins were also present in the cytosol, many of them being truncated enzyme variants that would be expected to serve a binding rather than an enzymatic function. Eleven proteins were highly enriched (consistent with the theoretical maximum of 25x), corresponding to an exclusive membrane localization in the delimiting membrane of the autophagosome. Three of these were methyltransferases: betaine:homocysteine methyltransferase (five variants); catechol O-methyltransferase (one phosphorylated and one unphosphorylated variant) and methionine adenosyltransferase, perhaps indicating that methylation/demethylation of membrane components could play a role in autophagy. A fourth highly enriched autophagosomal protein, phosphatidylethanolamine binding protein, is particularly interesting considering that the autophagic marker protein, LC3/ Atg8, is linked to autophagosomal membranes through its covalent conjugation with phosphatidylethanolamine (as the form LC3-II). LC3-II was not detectable on silver-stained 2D-gels, but could be shown by immunoblotting to be highly enriched in autophagosomal membranes. Other highly enriched proteins were heat shock cognate protein Hsc70 (one short and one long variant), peroxiredoxin 2, peroxiredoxin 6 (two variants), fructose 1,6-bisphosphatase (one phosphorylated and one unphosphorylated variant), adenosine kinase, inorganic pyrophosphatase and selenium-binding protein 2. Hsc70, a chaperonin that plays an important role in the recognition and proteasomal degradation of aggregated

  12. Differential proteome analysis of human embryonic kidney cell line (HEK-293) following mycophenolic acid treatment

    PubMed Central

    2011-01-01

    Background Mycophenolic acid (MPA) is widely used as a post transplantation medicine to prevent acute organ rejection. In the present study we used proteomics approach to identify proteome alterations in human embryonic kidney cells (HEK-293) after treatment with therapeutic dose of MPA. Following 72 hours MPA treatment, total protein lysates were prepared, resolved by two dimensional gel electrophoresis and differentially expressed proteins were identified by QTOF-MS/MS analysis. Expressional regulations of selected proteins were further validated by real time PCR and Western blotting. Results The proliferation assay demonstrated that therapeutic MPA concentration causes a dose dependent inhibition of HEK-293 cell proliferation. A significant apoptosis was observed after MPA treatment, as revealed by caspase 3 activity. Proteome analysis showed a total of 12 protein spots exhibiting differential expression after incubation with MPA, of which 7 proteins (complement component 1 Q subcomponent-binding protein, electron transfer flavoprotein subunit beta, cytochrome b-c1 complex subunit, peroxiredoxin 1, thioredoxin domain-containing protein 12, myosin regulatory light chain 2, and profilin 1) showed significant increase in their expression. The expression of 5 proteins (protein SET, stathmin, 40S ribosomal protein S12, histone H2B type 1 A, and histone H2B type 1-C/E/F/G/I) were down-regulated. MPA mainly altered the proteins associated with the cytoskeleton (26%), chromatin structure/dynamics (17%) and energy production/conversion (17%). Both real time PCR and Western blotting confirmed the regulation of myosin regulatory light chain 2 and peroxiredoxin 1 by MPA treatment. Furthermore, HT-29 cells treated with MPA and total kidney cell lysate from MMF treated rats showed similar increased expression of myosin regulatory light chain 2. Conclusion The emerging use of MPA in diverse pathophysiological conditions demands in-depth studies to understand molecular basis of

  13. Catalytic Profile of Arabidopsis Peroxidases, AtPrx-2, 25 and 71, Contributing to Stem Lignification

    PubMed Central

    Shigeto, Jun; Nagano, Mariko; Fujita, Koki; Tsutsumi, Yuji

    2014-01-01

    Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors. PMID:25137070

  14. Catalytic profile of Arabidopsis peroxidases, AtPrx-2, 25 and 71, contributing to stem lignification.

    PubMed

    Shigeto, Jun; Nagano, Mariko; Fujita, Koki; Tsutsumi, Yuji

    2014-01-01

    Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.

  15. Prograde hbl- and bio-breakdown reactions at high temperatures: A natural example from the Wilmington Complex, PA-DE

    SciTech Connect

    Srogi, L.; Bosbyshell, H. . Dept. of Geology and Astronomy)

    1992-01-01

    Breakdown of hornblende (hbl) and biotite (bio) in the deep crust can be a source of fluids and silicic melts. In the Wilmington Complex, prograde reaction stages at P = 600 MPa are preserved in granulite-facies gneisses near the margin of an intrusive gabbro stock. Away from intrusive plutons, the low-K, mafic and felsic gneisses contain opx-cpx-hbl-plag(An40-55)-mt [+-] qtz [+-] bio [+-] ilm. At 50--100 m from the contact with the intrusive gabbro, hbl is partly rimmed by symplectic intergrowths of opx-cpx-plag; individual grains are several [mu]ms in any dimension. At 15 m from the contact, the reaction texture is less obvious, but small grains of opx and cpx occur along hbl grain boundaries. Modal hbl and qtz decrease toward the contact; samples within a few m of the contact contain no hbl or bio. Plag composition is zoned in the matrix and is variable within symplectites. An overall reaction can be written using compositions of matrix hbl and symplectite prxs: 1 Hbl + 2.3 Qtz = 1.6 Opx + 1.2 Cpx + 1.3 Plag (An58), (+fluid+K). Any supercritical fluid or silicate melt produced migrated out of the volume of gneiss samples. Two-prx thermometry indicates that maximum temperatures reached at least 850 C, 50 m from the contact. The preservation of fine-scale disequilibrium features suggests that high-T metamorphism was not followed by retrogression or deformation. This work supports previous work on associated aluminous gneisses, in which relict bio and sill co-exist in the same thin section with restite assemblages of cordierite, spinel, corundum, opx, and gar. Dehydration partial melting of bio produced low-K silicic melts.

  16. Photosynthesis-dependent physiological and genetic crosstalk between cold acclimation and cold-induced resistance to fungal pathogens in triticale (Triticosecale Wittm.).

    PubMed

    Szechyńska-Hebda, Magdalena; Wąsek, Iwona; Gołębiowska-Pikania, Gabriela; Dubas, Ewa; Żur, Iwona; Wędzony, Maria

    2015-04-01

    The breeding for resistance against fungal pathogens in winter triticale (Triticosecale Wittm.) continues to be hindered by a complexity of the resistance mechanisms, strong interaction with environmental conditions, and dependence on the plant genotype. We showed, that temperature below 4 °C induced the plant genotype-dependent resistance against the fungal pathogen Microdochium nivale. The mechanism involved, at least, the adjustment of the reactions in the PSII proximity and photoprotection, followed by an improvement of the growth and development. The genotypes capable to develop the cold-induced resistance, showed a higher maximum quantum yield of PSII and a more efficient integration of the primary photochemistry of light reactions with the dark reactions. Moreover, induction of the photoprotective mechanism, involving at least the peroxidases scavenging hydrogen peroxide, was observed for such genotypes. Adjustment of the photosynthesis and stress acclimation has enabled fast plant growth and avoidance of the developmental stages sensitive to fungal infection. The same mechanisms allowed the quick regrow of plants during the post-disease period. In contrast, genotypes that were unable to develop resistance despite cold hardening had less flexible balancing of the photoprotection and photoinhibition processes. Traits related to: photosynthesis-dependent cold-acclimation and cold-induced resistance; biomass accumulation and growth; as well as protection system involving peroxidases; were integrated also at a genetic level. Analysing 95 lines of the mapping population SaKa3006×Modus we determined region on chromosomes 5B and 7R shared within all tested traits. Moreover, similar expression pattern of a set of the genes related to PSII was determined with the metaanalysis of the multiple microarray experiments. Comparable results for peroxidases, involving APXs and GPXs and followed by PRXs, indicated a similar function during cold acclimation and defense

  17. Optimized NSAIDs for Breast Cancer Prevention

    DTIC Science & Technology

    2007-04-01

    Drugs 26µM 52µM 104µM 208µM 416µM R-etodolac 96.3±4.5 85.2±1.2 76.2±2.4 58.5±3.1 35.6±1.1 Acetylsalicylic acid 101.8±0.8 96.9±1.4 96.3±0.6 92.3±0.4...beta isoform 8 - Peroxiredoxin 1 7 - Bcas1 7 - Acid phosphatase 1, soluble Fold Induction 17.5 - Major urinary protein 1 11 - Collagenous repeat...Acrp30) 6 - Carbonic anhydrase 3 6 - Fatty acid binding protein 4 Principal Investigator: Carson, Dennis A. Award Number: W81XWH-04-1-0453 8 b. Optimize

  18. The role of methylglyoxal-modified proteins in gastric ulcer healing.

    PubMed

    Takagi, T; Naito, Y; Oya-Ito, T; Yoshikawa, T

    2012-01-01

    Methylglyoxal is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts nonenzymatically with proteins to form products such as argpyrimidine at arginine residues. Abnormal accumulation of methylglyoxal and methylglyoxal-derived advanced glycation end products (AGEs) occurs under hyperglycemic conditions and has been implicated in endothelium dysfunction, arterial stiffening, and microvascular complications in diabetes. However, the role of methylglyoxal in the healing process of diabetic gastric ulcers has not been fully investigated. Recently, methylglyoxal modification of peroxiredoxin-VI was found to be associated with delayed healing of diabetic gastric ulcers. Thus, inhibition of methylglyoxal modification might have therapeutic potential for the treatment of such ulcers. In this review, we present what is currently known regarding the role of methylglyoxal in the healing of diabetic gastric ulcers.

  19. Immunomodulatory molecules of Fasciola hepatica: candidates for both vaccine and immunotherapeutic development.

    PubMed

    Dalton, John P; Robinson, Mark W; Mulcahy, Grace; O'Neill, Sandra M; Donnelly, Sheila

    2013-08-01

    The liver fluke, Fasciola hepatica, causes fascioliasis in domestic animals (sheep, cattle), a global disease that is also an important infection of humans. As soon as the parasite invades the gut wall its interaction with various host immune cells (e.g. dendritic cells, macrophages and mast cells) is complex. The parasite secretes a myriad of molecules that direct the immune response towards a favourable non-protective Th2-mediate/regulatory environment. These immunomodulatory molecules, such as cathepsin L peptidase (FhCL1), are under development as the first generation of fluke vaccines. However, this peptidase and other molecules, such as peroxiredoxin (FhPrx) and helminth defence molecule (FhHDM-1), exhibit various immunomodulatory properties that could be harnessed to help treat immune-related conditions in humans and animals.

  20. Proteomic analysis identifies differentially expressed proteins in AML1/ETO acute myeloid leukemia cells treated with DNMT inhibitors azacitidine and decitabine.

    PubMed

    Buchi, Francesca; Spinelli, Elena; Masala, Erico; Gozzini, Antonella; Sanna, Alessandro; Bosi, Alberto; Ferrari, Germano; Santini, Valeria

    2012-05-01

    Azacitidine and decitabine are DNA methyltransferase inhibitors used to treat myelodysplastic syndromes and acute myeloid leukemias. To further characterize different mechanisms between these two agents, cellular extracts from leukemic cells untreated or treated with either drug were analyzed using 2D electrophoresis. Numerous differentially expressed proteins were identified with MALDI-TOF/TOF-MS. Cyclophilin A, Catalase, Nucleophosmin and PCNA were decreased exclusively by azacitidine, TCP1 and hnRNP A2/B1 by both drugs; alpha-Enolase and Peroxiredoxin-1 by decitabine. Interestingly, the expression of the proinflammatory protein Cyclophilin A, also suggested as marker of cell necrosis, was stimulated by decitabine. Finally, a comprehensive pathway analysis of data highlighted a relationship between the identified proteins and potential effectors.

  1. The NADH oxidase-Prx system in Amphibacillus xylanus.

    PubMed

    Niimura, Youichi

    2007-01-01

    Amphibacillus NADH oxidase belongs to a growing new family of peroxiredoxin-linked oxidoreductases including alkyl hydroperoxide reductase F (AhpF). Like AhpF it displays extremely high hydroperoxide reductase activity in the presence of a Prx, thus making up the NADH oxidase-Prx system. The NADH oxidase primarily catalyzes the reduction of oxygen by NADH to form H2O2, while the Prx immediately reduces H2O2 (or ROOH) to water (or ROH). Consequently, the NADH oxidase-Prx system catalyzes the reduction of both oxygen and hydrogen peroxide to water with NADH as the preferred electron donor. The NADH oxidase-Prx system is widely distributed in aerobically growing bacteria lacking a respiratory chain and catalase, and plays an important role not only in scavenging hydroperoxides but also in regenerating NAD in these bacteria.

  2. Differential expression of Prx I and II in mouse testis and their up-regulation by radiation.

    PubMed

    Lee, Keesook; Park, Ji-Sun; Kim, Yun-Jeong; Soo Lee, Yong Soo; Sook Hwang, Tae Sook; Kim, Dae-Joong; Park, Eun-Mi; Park, Young-Mee

    2002-08-16

    Testis is one of the most sensitive organs to ionizing radiation. The present study was designed to unravel the possible role of antioxidant proteins, peroxiredoxin I and II (Prx I and II) in the testis. Our results show that Prx I and II are constitutively expressed in the testis and their expression levels are decreased to some extent as the testis develops. Interestingly, immunohistochemical analysis revealed a preferential expression of Prx I and II in Leydig and Sertoli cells, respectively. Neither Prx I nor Prx II expression was obvious in the testicular germ cells including spermatogonia and spermatocytes. Ionizing radiation exerted oxidative stress on the testis and induced apoptosis primarily in the germ cells. When the irradiated testis was examined, the Prx system was found to be transiently up-regulated. Taken together, we suggest that the relative radiation-resistance of Leydig and Sertoli cells could be attributed in part to the antioxidant function of the Prx system in these cells.

  3. Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

    PubMed Central

    Huang, Beijing K.; Sikes, Hadley D.

    2014-01-01

    Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS) in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies. PMID:25460730

  4. Proteomic analysis of pancreas derived from adult cloned pig

    SciTech Connect

    Chae, Jung-Il; Cho, Young Keun; Cho, Seong-Keun; Kim, Jin-Hoi; Han, Yong-Mahn; Koo, Deog-Bon Lee, Kyung-Kwang

    2008-02-08

    The potential medical applications of animal cloning include xenotransplantation, but the complex molecular cascades that control porcine organ development are not fully understood. Still, it has become apparent that organs derived from cloned pigs may be suitable for transplantation into humans. In this study, we examined the pancreas of an adult cloned pig developed through somatic cell nuclear transfer (SCNT) using two-dimensional electrophoresis (2-DE) and Western blotting. Proteomic analysis revealed 69 differentially regulated proteins, including such apoptosis-related species as annexins, lamins, and heat shock proteins, which were unanimously upregulated in the SCNT sample. Among the downregulated proteins in SCNT pancreas were peroxiredoxins and catalase. Western blot results indicate that several antioxidant enzymes and the anti-apoptotic protein were downregulated in SCNT pancreas, whereas several caspases were upregulated. Together, these data suggest that the accumulation of reactive oxygen species (ROS) in the pancreas of an adult cloned pig leads to apoptosis.

  5. Oxidative Stress in Fungi: Its Function in Signal Transduction, Interaction with Plant Hosts, and Lignocellulose Degradation

    PubMed Central

    Breitenbach, Michael; Weber, Manuela; Rinnerthaler, Mark; Karl, Thomas; Breitenbach-Koller, Lore

    2015-01-01

    In this review article, we want to present an overview of oxidative stress in fungal cells in relation to signal transduction, interaction of fungi with plant hosts, and lignocellulose degradation. We will discuss external oxidative stress which may occur through the interaction with other microorganisms or plant hosts as well as internally generated oxidative stress, which can for instance originate from NADPH oxidases or “leaky” mitochondria and may be modulated by the peroxiredoxin system or by protein disulfide isomerases thus contributing to redox signaling. Analyzing redox signaling in fungi with the tools of molecular genetics is presently only in its beginning. However, it is already clear that redox signaling in fungal cells often is linked to cell differentiation (like the formation of perithecia), virulence (in plant pathogens), hyphal growth and the successful passage through the stationary phase. PMID:25854186

  6. Effect of different glucose concentrations on proteome of Saccharomyces cerevisiae.

    PubMed

    Guidi, Francesca; Francesca, Guidi; Magherini, Francesca; Francesca, Magherini; Gamberi, Tania; Tania, Gamberi; Borro, Marina; Marina, Borro; Simmaco, Maurizio; Maurizio, Simmaco; Modesti, Alessandra; Alessandra, Modesti

    2010-07-01

    We performed a proteomic study to understand how Saccharomyces cerevisiae adapts its metabolism during the exponential growth on three different concentrations of glucose; this information will be necessary to understand yeast carbon metabolism in different environments. We induced a natural diauxic shift by growing yeast cells in glucose restriction thus having a fast and complete glucose exhaustion. We noticed differential expressions of groups of proteins. Cells in high glucose have a decreased growth rate during the initial phase of fermentation; in glucose restriction and in high glucose we found an over-expression of a protein (Peroxiredoxin) involved in protection against oxidative stress insult. The information obtained in our study validates the application of a proteomic approach for the identification of the molecular bases of environmental variations such as fermentation in high glucose and during a naturally induced diauxic shift.

  7. iTRAQ-based proteomics reveals novel biomarkers of osteoarthritis

    PubMed Central

    Ikeda, Daiki; Ageta, Hiroshi; Tsuchida, Kunihiro

    2013-01-01

    Objective: We performed comprehensive proteomic analyses of articular cartilage by using the isobaric tags for relative and absolute quantitation (iTRAQ) method, and searched for candidate biomarkers for osteoarthritis (OA). Methods: Articular cartilage was collected from patients with OA or femoral neck fracture for the control group. Molecular variations were detected by the iTRAQ method, and quantitative analyses were performed by western blot. Results: Using the iTRAQ method, we identified 76 proteins with different expression levels in OA patients and the control group. Among these proteins, we selected LECT2 (leukocyte cell-derived chemotaxin-2), BAALC (brain and acute leukemia, cytoplasmic), and PRDX6 (peroxiredoxin-6), which had not been reported as biomarkers for OA. Conclusions: Use of these proteins in combination with conventional OA biomarkers may better reflect the grade and prognosis of OA. PMID:23937207

  8. Redox Homeostasis and Cellular Antioxidant Systems: Crucial Players in Cancer Growth and Therapy

    PubMed Central

    Ciucis, Chiara De

    2016-01-01

    Reactive oxygen species (ROS) and their products are components of cell signaling pathways and play important roles in cellular physiology and pathophysiology. Under physiological conditions, cells control ROS levels by the use of scavenging systems such as superoxide dismutases, peroxiredoxins, and glutathione that balance ROS generation and elimination. Under oxidative stress conditions, excessive ROS can damage cellular proteins, lipids, and DNA, leading to cell damage that may contribute to carcinogenesis. Several studies have shown that cancer cells display an adaptive response to oxidative stress by increasing expression of antioxidant enzymes and molecules. As a double-edged sword, ROS influence signaling pathways determining beneficial or detrimental outcomes in cancer therapy. In this review, we address the role of redox homeostasis in cancer growth and therapy and examine the current literature regarding the redox regulatory systems that become upregulated in cancer and their role in promoting tumor progression and resistance to chemotherapy. PMID:27418953

  9. Proteomic analysis of acute responses to copper sulfate stress in larvae of the brine shrimp, Artemia sinica

    NASA Astrophysics Data System (ADS)

    Zhou, Qian; Wu, Changgong; Dong, Bo; Li, Fuhua; Liu, Fengqi; Xiang, Jianhai

    2010-03-01

    Proteomics was used to reveal the differential protein expression profiles of acute responses to copper sulfate exposure in larvae of Artemia sinica. Fourteen differentially displayed protein spots were detected and seven of them were identified. Three spots were up-expressed and identified: actin, heat shock protein 70, and chaperone subunit 1; three down-regulated proteins were identified: arginine kinase, elongation factor-2, and glycine-rich protein; and a newly expressed protein was identified as peroxiredoxin. The study indicates the involvement of all the differentially expressed proteins in the early responses of protein expression, and in the survival of A. sinica in the presence of copper and other heavy metals; the findings improve understanding of the organism’s adaptive responses and resistance.

  10. Control of Mitochondrial Remodeling by the ATPase Inhibitory Factor 1 Unveils a Pro-survival Relay via OPA1.

    PubMed

    Faccenda, Danilo; Nakamura, Junji; Gorini, Giulia; Dhoot, Gurtej K; Piacentini, Mauro; Yoshida, Masusuke; Campanella, Michelangelo

    2017-02-21

    The ubiquitously expressed ATPase inhibitory factor 1 (IF1) is a mitochondrial protein that blocks the reversal of the F1Fo-ATPsynthase, preventing dissipation of cellular ATP and ischemic damage. IF1 suppresses programmed cell death, enhancing tumor invasion and chemoresistance, and is expressed in various types of human cancers. In this study, we examined its effect on mitochondrial redox balance and apoptotic cristae remodeling, finding that, by maintaining ATP levels, IF1 reduces glutathione (GSH) consumption and inactivation of peroxiredoxin 3 (Prx3) during apoptosis. This correlates with inhibition of metallopeptidase OMA1-mediated processing of the pro-fusion dynamin-related protein optic atrophy 1 (OPA1). Stabilization of OPA1 impedes cristae remodeling and completion of apoptosis. Taken together, these data suggest that IF1 acts on both mitochondrial bioenergetics and structure, is involved in mitochondrial signaling in tumor cells, and may underlie their proliferative capacity.

  11. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals.

    PubMed

    Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-Ichi; Kotani, Eiji; Hirano, Tomoko; Nakajima, Yumiko; Matsumoto, Goichi; Mori, Hajime

    2014-09-01

    Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase-Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6.

  12. dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao.

    PubMed

    Caribé dos Santos, A C; Sena, J A L; Santos, S C; Dias, C V; Pirovani, C P; Pungartnik, C; Valle, R R; Cascardo, J C M; Vincentz, M

    2009-11-01

    The genome sequence of the hemibiotrophic fungus Moniliophthora perniciosa revealed genes possibly participating in the RNAi machinery. Therefore, studies were performed in order to investigate the efficiency of gene silencing by dsRNA. We showed that the reporter gfp gene stably introduced into the fungus genome can be silenced by transfection of in vitro synthesized gfpdsRNA. In addition, successful dsRNA-induced silencing of endogenous genes coding for hydrophobins and a peroxiredoxin were also achieved. All genes showed a silencing efficiency ranging from 18% to 98% when compared to controls even 28d after dsRNA treatment, suggesting systemic silencing. Reduction of GFP fluorescence, peroxidase activity levels and survival responses to H(2)O(2) were consistent with the reduction of GFP and peroxidase mRNA levels, respectively. dsRNA transformation of M. perniciosa is shown here to efficiently promote genetic knockdown and can thus be used to assess gene function in this pathogen.

  13. Interplay between cellular redox oscillations and circadian clocks.

    PubMed

    Rey, G; Reddy, A B

    2015-09-01

    The circadian clock is a cellular timekeeping mechanism that helps organisms from bacteria to humans to organize their behaviour and physiology around the solar cycle. Current models for circadian timekeeping incorporate transcriptional/translational feedback loop mechanisms in the predominant model systems. However, recent evidence suggests that non-transcriptional oscillations such as metabolic and redox cycles may play a fundamental role in circadian timekeeping. Peroxiredoxins, an antioxidant protein family, undergo rhythmic oxidation on the circadian time scale in a variety of species, including bacteria, insects and mammals, but also in red blood cells, a naturally occurring, non-transcriptional system. The profound interconnectivity between circadian and redox pathways strongly suggests that a conserved timekeeping mechanism based on redox cycles could be integral to generating circadian rhythms.

  14. Cross-talk between circadian clocks, sleep-wake cycles, and metabolic networks: Dispelling the darkness.

    PubMed

    Ray, Sandipan; Reddy, Akhilesh B

    2016-04-01

    Integration of knowledge concerning circadian rhythms, metabolic networks, and sleep-wake cycles is imperative for unraveling the mysteries of biological cycles and their underlying mechanisms. During the last decade, enormous progress in circadian biology research has provided a plethora of new insights into the molecular architecture of circadian clocks. However, the recent identification of autonomous redox oscillations in cells has expanded our view of the clockwork beyond conventional transcription/translation feedback loop models, which have been dominant since the first circadian period mutants were identified in fruit fly. Consequently, non-transcriptional timekeeping mechanisms have been proposed, and the antioxidant peroxiredoxin proteins have been identified as conserved markers for 24-hour rhythms. Here, we review recent advances in our understanding of interdependencies amongst circadian rhythms, sleep homeostasis, redox cycles, and other cellular metabolic networks. We speculate that systems-level investigations implementing integrated multi-omics approaches could provide novel mechanistic insights into the connectivity between daily cycles and metabolic systems.

  15. Impaired gastric ulcer healing in diabetic mice: role of methylglyoxal.

    PubMed

    Naito, Y; Takagi, T; Oya-Ito, T; Okada, H; Suzuki, T; Hirata, I; Hirai, M; Uchiyama, K; Handa, O; Uchida, K; Yoshikawa, T

    2009-12-01

    Methylglyoxal is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine at arginine residue. The aim of the present study was to investigate the role of methylglyoxal in the delayed healing of gastric ulcer in diabetes, and to identify the methylglyoxal-modified proteins as a target molecule of this modification. Using male C57BL/6 mice, diabetes was induced by a single i.p. injection of streptozotocin and gastric ulcers were produced by the focal application of 40% of acetic acid to the serosal surface of the stomach. In order to evaluate the effect of OPB-9195, an inhibitor of methylglyoxal modification, on gastric ulcer healing, mice were given orally OPB-9195 (30 mg/kg) twice daily for 14 days, one week before and after the injection of streptozotocin. The area of gastric ulcer on day 7 was significantly increased in diabetic mice compared to non-diabetic mice, indicating delayed ulcer healing. This increase in ulcer area in diabetic mice was significantly reversed by the treatment with OPB-9195 without affecting blood glucose levels. Proteomics analysis showed the methylglyoxal-modification of peroxiredoxin 6 proteins in the diabetic gastric mucosa around gastric ulcer, and this modification was markedly inhibited by the treatment with OPB-9195. In conclusion, the present study suggests a link of increased methylglyoxal modification of proteins including peroxiredoxin 6 to the delayed gastric ulcer healing in diabetes, and also shows the therapeutic potential of the inhibitor of methylglyoxal modification for the treatment of diabetic gastric ulcers.

  16. Formaldehyde induces apoptosis through decreased Prx 2 via p38 MAPK in lung epithelial cells.

    PubMed

    Lim, Seul Ki; Kim, Jong Chun; Moon, Chang Jong; Kim, Gye Yeop; Han, Ho Jae; Park, Soo Hyun

    2010-05-27

    Formaldehyde (FA) is an important substance that induces sick house syndrome and diseases, such as asthma and allergies. Oxidative stress is involved in the development of respiratory disease, and diverse antioxidants may protect respiratory tract cells from apoptosis. Peroxiredoxin is a pivotal endogenous antioxidant. In the present study, FA induced death in A549 cells, a lung epithelial cell line, in a dose-dependent manner. FA also increased lipid peroxide formation (LPO) in A549 cells, suggesting a role for oxidative stress. Additionally, FA decreased peroxiredoxin 2 (Prx 2) protein levels after a 24 or 48h exposure to FA. We also examined whether the FA-induced decrease in Prx 2 was associated with apoptosis. Prx 2 overexpression protected against FA-induced cell apoptosis but not necrosis. Prx 2 overexpression blocked FA-induced increase in Bax, a pro-apoptotic molecule, and a decrease in Bcl-2, an anti-apoptotic molecule. Prx 2 overexpression also protected against FA-induced activation of some special apoptosis-associated proteins [caspase-3, caspase-9, and polypeptide poly (ADP-ribose) polymerase (PARP)]. Furthermore, we examined the signaling molecules involved in the FA-induced decrease in Prx 2 expression. The FA-induced decrease in Prx 2 and increase in cell apoptosis was restored by treatment with SB203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by SP600125 [a c-jun-N-terminal kinase (JNK) inhibitor]. Also, FA-induced events were blocked by treatment with p38 siRNA, but not by scrambled siRNA. Indeed, FA increased p38 MAPK activation, suggesting a role for p38 MAPK in FA action. In conclusion, FA mediated apoptosis in lung epithelial cells by decreasing Prx 2 via p38 MAPK.

  17. Production and radioimmunoimaging of novel fully human phage display recombinant antibodies and growth inhibition of lung adenocarcinoma cell line overexpressing Prx I.

    PubMed

    Luo, Yi; Pang, Hua; Li, Shujie; Cao, Hui; Peng, Zhiping; Fan, Chunbo; Li, Shaolin

    2009-07-01

    The Peroxiredoxin I (Prx I) is a member of the Peroxiredoxin family, which is overexpressed in many diverse tumor types and is an anti-apoptosis protein for tumor cell proliferation and survival. Therapeutic strategies targeting the Prx I may therefore be effective broad-spectrum anticancer agents. We constructed a phage display single-chain variable fragment (scFv) antibody library and sieve out the fully human, lung adenocarcinoma-sepcific monoclonal antibodies. The selection on Prx I was performed using above-mentioned lung adenocarcinoma-sepcific monoclonal antibodies with high affinity to Prx I overexpressing lung adenocarcinoma cells. The candidate scFv sequences, based on enzyme-linked immunosorbent assay (ELISA) screening data, were chosen for soluble expression, and a 30 kDa band was observed on polyacrylamide gel electrophoresis as predicted. The purified antibodies were characterized by immunoblotting and showed high specificity to Prx I-overexpressing lung adenocarcinoma cells A549. Radioimmunoimaging was taken to evaluate specificity and distribution of antibodies in vivo. The radiolocalization index (RI) of tumor/serum and tumor/muscle gradually increased, reaching its peak (4.06 +/- 0.13 and 5.17 +/- 0.97, respectively) at 48 h postadministration. Single photon emission computed tomography (SPECT) imaging showed the radioactivity was aggregated in tumor locations and tumor imaging was clearly observed. The internalized scFv resulted in antibody-mediated cell apoptosis and downregulation of Prx I expression. These results demonstrate that the scFv possesses strong antitumor activity on lung adenocarcinoma and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on Prx I for growth and survival.

  18. Medroxyprogesterone Acetate Antagonizes Estrogen Up-Regulation of Brain Mitochondrial Function

    PubMed Central

    Irwin, Ronald W.; Yao, Jia; Ahmed, Syeda S.; Hamilton, Ryan T.; Cadenas, Enrique

    2011-01-01

    The impact of clinical progestins used in contraception and hormone therapies on the metabolic capacity of the brain has long-term implications for neurological health in pre- and postmenopausal women. Previous analyses indicated that progesterone and 17β-estradiol (E2) sustain and enhance brain mitochondrial energy-transducing capacity. Herein we determined the impact of the clinical progestin, medroxyprogesterone acetate (MPA), on glycolysis, oxidative stress, and mitochondrial function in brain. Ovariectomized female rats were treated with MPA, E2, E2+MPA, or vehicle with ovary-intact rats serving as a positive control. MPA alone and MPA plus E2 resulted in diminished mitochondrial protein levels for pyruvate dehydrogenase, cytochrome oxidase, ATP synthase, manganese-superoxide dismutase, and peroxiredoxin V. MPA alone did not rescue the ovariectomy-induced decrease in mitochondrial bioenergetic function, whereas the coadministration of E2 and MPA exhibited moderate efficacy. However, the coadministration of MPA was detrimental to antioxidant defense, including manganese-superoxide dismutase activity/expression and peroxiredoxin V expression. Accumulated lipid peroxides were cleared by E2 treatment alone but not in combination with MPA. Furthermore, MPA abolished E2-induced enhancement of mitochondrial respiration in primary cultures of the hippocampal neurons and glia. Collectively these findings indicate that the effects of MPA differ significantly from the bioenergetic profile induced by progesterone and that, overall, MPA induced a decline in glycolytic and oxidative phosphorylation protein and activity. These preclinical findings on the basis of acute exposure to MPA raise concerns regarding neurological health after chronic use of MPA in contraceptive and hormone therapy. PMID:21159850

  19. Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

    SciTech Connect

    Guterres, Fernanda Augusta de Lima Barbosa; Martinez, Glaucia Regina; Rocha, Maria Eliane Merlin; Winnischofer, Sheila Maria Brochado

    2013-11-15

    Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated β-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 and p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process. - Highlights: • Lower concentrations of simvastatin can induce senescent phenotype in melanoma cells. • Simvastatin induces senescence in human melanoma cells via p53/p21 pathway. • Senescent phenotype is related with increased intracellular ROS. • Partial detoxification of ROS by catalase/peroxiredoxin-1 could lead cells to senescence rather than apoptosis.

  20. Extracellular matrix-associated proteome changes during non-host resistance in citrus-Xanthomonas interactions.

    PubMed

    Swaroopa Rani, Tirupaati; Podile, Appa Rao

    2014-04-01

    Non-host resistance (NHR) is a most durable broad-spectrum resistance employed by the plants to restrict majority of pathogens. Plant extracellular matrix (ECM) is a critical defense barrier. Understanding ECM responses during interaction with non-host pathogen will provide insights into molecular events of NHR. In this study, the ECM-associated proteome was compared during interaction of citrus with pathogen Xanthomonas axonopodis pv. citri (Xac) and non-host pathogen Xanthomonas oryzae pv. oryzae (Xoo) at 8, 16, 24 and 48 h post inoculation. Comprehensive analysis of ECM-associated proteins was performed by extracting wall-bound and soluble ECM components using both destructive and non-destructive procedures. A total of 53 proteins was differentially expressed in citrus-Xanthomonas host and non-host interaction, out of which 44 were identified by mass spectrometry. The differentially expressed proteins were related to (1) defense-response (5 pathogenesis-related proteins, 3 miraculin-like proteins (MIR, MIR1 and MIR2) and 2 proteases); (2) enzymes of reactive oxygen species (ROS) metabolism [Cu/Zn superoxide dismutase (SOD), Fe-SOD, ascorbate peroxidase and 2-cysteine-peroxiredoxin]; (3) signaling (lectin, curculin-like lectin and concanavalin A-like lectin kinase); and (4) cell-wall modification (α-xylosidase, glucan 1, 3 β-glucosidase, xyloglucan endotransglucosylase/hydrolase). The decrease in ascorbate peroxidase and cysteine-peroxiredoxin could be involved in maintenance of ROS levels. Increase in defense, cell-wall remodeling and signaling proteins in citrus-Xoo interaction suggests an active involvement of ECM in execution of NHR. Partially compromised NHR in citrus against Xoo, upon Brefeldin A pre-treatment supported the role of non-classical secretory proteins in this phenomenon.

  1. Protein Oxidation in the Lungs of C57BL/6J Mice Following X-Irradiation

    PubMed Central

    Barshishat-Kupper, Michal; McCart, Elizabeth A.; Freedy, James G.; Tipton, Ashlee J.; Nagy, Vitaly; Kim, Sung-Yop; Landauer, Michael R.; Mueller, Gregory P.; Day, Regina M.

    2015-01-01

    Damage to normal lung tissue is a limiting factor when ionizing radiation is used in clinical applications. In addition, radiation pneumonitis and fibrosis are a major cause of mortality following accidental radiation exposure in humans. Although clinical symptoms may not develop for months after radiation exposure, immediate events induced by radiation are believed to generate molecular and cellular cascades that proceed during a clinical latent period. Oxidative damage to DNA is considered a primary cause of radiation injury to cells. DNA can be repaired by highly efficient mechanisms while repair of oxidized proteins is limited. Oxidized proteins are often destined for degradation. We examined protein oxidation following 17 Gy (0.6 Gy/min) thoracic X-irradiation in C57BL/6J mice. Seventeen Gy thoracic irradiation resulted in 100% mortality of mice within 127–189 days postirradiation. Necropsy findings indicated that pneumonitis and pulmonary fibrosis were the leading cause of mortality. We investigated the oxidation of lung proteins at 24 h postirradiation following 17 Gy thoracic irradiation using 2-D gel electrophoresis and OxyBlot for the detection of protein carbonylation. Seven carbonylated proteins were identified using mass spectrometry: serum albumin, selenium binding protein-1, alpha antitrypsin, cytoplasmic actin-1, carbonic anhydrase-2, peroxiredoxin-6, and apolipoprotein A1. The carbonylation status of carbonic anhydrase-2, selenium binding protein, and peroxiredoxin-6 was higher in control lung tissue. Apolipoprotein A1 and serum albumin carbonylation were increased following X-irradiation, as confirmed by OxyBlot immunoprecipitation and Western blotting. Our findings indicate that the profile of specific protein oxidation in the lung is altered following radiation exposure. PMID:28248270

  2. The signature of seeds in resurrection plants: a molecular and physiological comparison of desiccation tolerance in seeds and vegetative tissues.

    PubMed

    Illing, Nicola; Denby, Katherine J; Collett, Helen; Shen, Arthur; Farrant, Jill M

    2005-11-01

    Desiccation-tolerance in vegetative tissues of angiosperms has a polyphyletic origin and could be due to 1) appropriation of the seed-specific program of gene expression that protects orthodox seeds against desiccation, and/or 2) a sustainable version of the abiotic stress response. We tested these hypotheses by comparing molecular and physiological data from the development of orthodox seeds, the response of desiccation-sensitive plants to abiotic stress, and the response of desiccation-tolerant plants to extreme water loss. Analysis of publicly-available gene expression data of 35 LEA proteins and 68 anti-oxidant enzymes in the desiccation-sensitive Arabidopsis thaliana identified 13 LEAs and 4 anti-oxidants exclusively expressed in seeds. Two (a LEA6 and 1-cys-peroxiredoxin) are not expressed in vegetative tissues in A. thaliana, but have orthologues that are specifically activated in desiccating leaves of Xerophyta humilis. A comparison of antioxidant enzyme activity in two desiccation-sensitive species of Eragrostis with the desiccation-tolerant E. nindensis showed equivalent responses upon initial dehydration, but activity was retained at low water content in E. nindensis only. We propose that these antioxidants are housekeeping enzymes and that they are protected from damage in the desiccation-tolerant species. Sucrose is considered an important protectant against desiccation in orthodox seeds, and we show that sucrose accumulates in drying leaves of E. nindensis, but not in the desiccation-sensitive Eragrostis species. The activation of "seed-specific" desiccation protection mechanisms (sucrose accumulation and expression of LEA6 and 1-cys-peroxiredoxin genes) in the vegetative tissues of desiccation-tolerant plants points towards acquisition of desiccation tolerance from seeds.

  3. Thiol-based redox signaling in the nitrogen-fixing symbiosis

    PubMed Central

    Frendo, Pierre; Matamoros, Manuel A.; Alloing, Geneviève; Becana, Manuel

    2013-01-01

    In nitrogen poor soils legumes establish a symbiotic interaction with rhizobia that results in the formation of root nodules. These are unique plant organs where bacteria differentiate into bacteroids, which express the nitrogenase enzyme complex that reduces atmospheric N 2 to ammonia. Nodule metabolism requires a tight control of the concentrations of reactive oxygen and nitrogen species (RONS) so that they can perform useful signaling roles while avoiding nitro-oxidative damage. In nodules a thiol-dependent regulatory network that senses, transmits and responds to redox changes is starting to be elucidated. A combination of enzymatic, immunological, pharmacological and molecular analyses has allowed us to conclude that glutathione and its legume-specific homolog, homoglutathione, are abundant in meristematic and infected cells, that their spatio-temporally distribution is correlated with the corresponding (homo)glutathione synthetase activities, and that they are crucial for nodule development and function. Glutathione is at high concentrations in the bacteroids and at moderate amounts in the mitochondria, cytosol and nuclei. Less information is available on other components of the network. The expression of multiple isoforms of glutathione peroxidases, peroxiredoxins, thioredoxins, glutaredoxins and NADPH-thioredoxin reductases has been detected in nodule cells using antibodies and proteomics. Peroxiredoxins and thioredoxins are essential to regulate and in some cases to detoxify RONS in nodules. Further research is necessary to clarify the regulation of the expression and activity of thiol redox-active proteins in response to abiotic, biotic and developmental cues, their interactions with downstream targets by disulfide-exchange reactions, and their participation in signaling cascades. The availability of mutants and transgenic lines will be crucial to facilitate systematic investigations into the function of the various proteins in the legume

  4. Toxicity of neurons treated with herbicides and neuroprotection by mitochondria-targeted antioxidant SS31.

    PubMed

    Reddy, Tejaswini P; Manczak, Maria; Calkins, Marcus J; Mao, Peizhong; Reddy, Arubala P; Shirendeb, Ulziibat; Park, Byung; Reddy, P Hemachandra

    2011-01-01

    The purpose of this study was to determine the neurotoxicity of two commonly used herbicides: picloram and triclopyr and the neuroprotective effects of the mitochondria-targeted antioxidant, SS31. Using mouse neuroblastoma (N2a) cells and primary neurons from C57BL/6 mice, we investigated the toxicity of these herbicides, and protective effects of SS1 peptide against picloram and triclopyr toxicity. We measured total RNA content, cell viability and mRNA expression of peroxiredoxins, neuroprotective genes, mitochondrial-encoded electron transport chain (ETC) genes in N2a cells treated with herbicides and SS31. Using primary neurons from C57BL/6 mice, neuronal survival was studied in neurons treated with herbicides, in neurons pretreated with SS31 plus treated with herbicides, neurons treated with SS31 alone, and untreated neurons. Significantly decreased total RNA content, and cell viability in N2a cells treated with picloram and triclopyr were found compared to untreated N2a cells. Decreased mRNA expression of neuroprotective genes, and ETC genes in cells treated with herbicides was found compared to untreated cells. Decreased mRNA expression of peroxiredoxins 1-6 in N2a cells treated with picloram was found, suggesting that picloram affects the antioxidant enzymes in N2a cells. Immunofluorescence analysis of primary neurons revealed that decreased neuronal branching and degenerating neurons in neurons treated with picloram and triclopyr. However, neurons pretreated with SS31 prevented degenerative process caused by herbicides. Based on these results, we propose that herbicides--picloram and triclopyr appear to damage neurons, and the SS31 peptide appears to protect neurons from herbicide toxicity.

  5. Toxicity of Neurons Treated with Herbicides and Neuroprotection by Mitochondria-Targeted Antioxidant SS31

    PubMed Central

    Reddy, Tejaswini P.; Manczak, Maria; Calkins, Marcus J.; Mao, Peizhong; Reddy, Arubala P.; Shirendeb, Ulziibat; Park, Byung; Reddy, P. Hemachandra

    2011-01-01

    The purpose of this study was to determine the neurotoxicity of two commonly used herbicides: picloram and triclopyr and the neuroprotective effects of the mitochondria-targeted antioxidant, SS31. Using mouse neuroblastoma (N2a) cells and primary neurons from C57BL/6 mice, we investigated the toxicity of these herbicides, and protective effects of SS1 peptide against picloram and triclopyr toxicity. We measured total RNA content, cell viability and mRNA expression of peroxiredoxins, neuroprotective genes, mitochondrial-encoded electron transport chain (ETC) genes in N2a cells treated with herbicides and SS31. Using primary neurons from C57BL/6 mice, neuronal survival was studied in neurons treated with herbicides, in neurons pretreated with SS31 plus treated with herbicides, neurons treated with SS31 alone, and untreated neurons. Significantly decreased total RNA content, and cell viability in N2a cells treated with picloram and triclopyr were found compared to untreated N2a cells. Decreased mRNA expression of neuroprotective genes, and ETC genes in cells treated with herbicides was found compared to untreated cells. Decreased mRNA expression of peroxiredoxins 1–6 in N2a cells treated with picloram was found, suggesting that picloram affects the antioxidant enzymes in N2a cells. Immunofluorescence analysis of primary neurons revealed that decreased neuronal branching and degenerating neurons in neurons treated with picloram and triclopyr. However, neurons pretreated with SS31 prevented degenerative process caused by herbicides. Based on these results, we propose that herbicides—picloram and triclopyr appear to damage neurons, and the SS31 peptide appears to protect neurons from herbicide toxicity. PMID:21318024

  6. Redoxins in peripheral neurons after sciatic nerve injury.

    PubMed

    Valek, Lucie; Kanngießer, Maike; Häussler, Annett; Agarwal, Nitin; Lillig, Christopher Horst; Tegeder, Irmgard

    2015-12-01

    Peripheral nerve injury causes redox stress in injured neurons by upregulations of pro-oxidative enzymes, but most neurons survive suggesting an activation of endogenous defense against the imbalance. As potential candidates we assessed thioredoxin-fold proteins, called redoxins, which maintain redox homeostasis by reduction of hydrogen peroxide or protein dithiol-disulfide exchange. Using a histologic approach, we show that the peroxiredoxins (Prdx1-6), the glutaredoxins (Glrx1, 2, 3 and 5), thioredoxin (Txn1 and 2) and their reductases (Txnrd1 and 2) are expressed in neurons, glial and/or vascular cells of the dorsal root ganglia (DRGs) and in the spinal cord. They show distinct cellular and subcellular locations in agreement with the GO terms for "cellular component". The expression and localization of Glrx, Txn and Txnrd proteins was not affected by sciatic nerve injury but peroxiredoxins were upregulated in the DRGs, Prdx1 and Prdx6 mainly in non-neuronal cells and Prdx4 and Prdx5 in DRG neurons, the latter associated with an increase of respective mRNAs and protein accumulation in peripheral and/or central fibers. The upregulation of Prdx4 and Prdx5 in DRG neurons was reduced in mice with a cre-loxP mediated deficiency of hypoxia inducible factor 1 alpha (HIF1α) in these neurons. The results identify Prdx4 and Prdx5 as endogenous HIF1α-dependent, transcriptionally regulated defenders of nerve injury evoked redox stress that may be important for neuronal survival and regeneration.

  7. Simulated Microgravity Regulates Gene Transcript Profiles of 2T3 Preosteoblasts: Comparison of the Random Positioning Machine and the Rotating Wall Vessel Bioreactor

    NASA Technical Reports Server (NTRS)

    Patel, Mamta J.; Liu, Wenbin; Sykes, Michelle C.; Ward, Nancy E.; Risin, Semyon A.; Risin, Diana; Hanjoong, Jo

    2007-01-01

    Microgravity of spaceflight induces bone loss due in part to decreased bone formation by osteoblasts. We have previously examined the microgravity-induced changes in gene expression profiles in 2T3 preosteoblasts using the Random Positioning Machine (RPM) to simulate microgravity conditions. Here, we hypothesized that exposure of preosteoblasts to an independent microgravity simulator, the Rotating Wall Vessel (RWV), induces similar changes in differentiation and gene transcript profiles, resulting in a more confined list of gravi-sensitive genes that may play a role in bone formation. In comparison to static 1g controls, exposure of 2T3 cells to RWV for 3 days inhibited alkaline phosphatase activity, a marker of differentiation, and downregulated 61 genes and upregulated 45 genes by more than two-fold as shown by microarray analysis. The microarray results were confirmed with real time PCR for downregulated genes osteomodulin, bone morphogenic protein 4 (BMP4), runx2, and parathyroid hormone receptor 1. Western blot analysis validated the expression of three downregulated genes, BMP4, peroxiredoxin IV, and osteoglycin, and one upregulated gene peroxiredoxin I. Comparison of the microarrays from the RPM and the RWV studies identified 14 gravi-sensitive genes that changed in the same direction in both systems. Further comparison of our results to a published database showing gene transcript profiles of mechanically loaded mouse tibiae revealed 16 genes upregulated by the loading that were shown to be downregulated by RWV and RPM. These mechanosensitive genes identified by the comparative studies may provide novel insights into understanding the mechanisms regulating bone formation and potential targets of countermeasure against decreased bone formation both in astronauts and in general patients with musculoskeletal disorders.

  8. Sequestering ability of some chelating agents towards methylmercury(II).

    PubMed

    Falcone, Gabriella; Foti, Claudia; Gianguzza, Antonio; Giuffrè, Ottavia; Napoli, Anna; Pettignano, Alberto; Piazzese, Daniela

    2013-01-01

    A study on the interactions between CH(3)Hg(+) and some S, N and O donor ligands (2-mercaptopropanoic acid (thiolactic acid (H(2)TLA)), 3-mercaptopropanoic acid (H(2)MPA), 2-mercaptosuccinic acid (thiomalic acid (H(3)TMA)), D,L-penicillamine (H(2)PSH), L-cysteine (H(2)CYS), glutathione (H(3)GSH), N,N'-bis(3-aminopropyl)-1-4-diaminobutane (spermine (SPER)), 1,2,3,4,5,6-benzenehexacarboxylic acid (mellitic acid (H(6)MLT)) and ethylenediaminetetraacetic acid (H(4)EDTA)) is reported. The speciation models in aqueous solution and the possible structures of the complexes formed are discussed on the basis of potentiometric, calorimetric, UV spectrophotometric and electrospray mass spectrometric results. For the CH(3)Hg(+)-S donor ligand systems, the formation of ML(1-z) and MLH(2-z) complex species is observed, together with a diprotonated MLH(2)(3-z) species for CYS(2-), PSH(2-) and GSH(3-) and the mixed hydrolytic one ML(OH)(-z) for TLA(2-) and MPA(2-). The dependence of the stability on ionic strength and on temperature is also analysed. In the other CH(3)Hg(+)-L systems (L = MLT(6-), SPER and EDTA(4-)), ML(1-z), MLH(2-z) and MLH(2)(3-z) complex species are formed, together with the MLH(3)(4-z) species for SPER, the mixed hydrolytic ML(OH)(-z) one for SPER and EDTA, and the M(2)L(2-z) for EDTA only. On the basis of the speciation models proposed, the sequestering ability of the ligands towards methylmercury(II) cation is evaluated. All S donor ligands show a good sequestering power (at 10(-11) mol L(-1) level, in the pH range 4 to 8) following the trend MPA(2-) < PSH(2-) < GSH(3-) < TLA(2-) < CYS(2-) < TMA(3-), while significantly lower is the sequestering ability of MLT, SPER and EDTA (at 10(-3)-10(-5) mol L(-1) level, in the pH range 4 to 8).

  9. Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8+T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii

    PubMed Central

    2010-01-01

    Background Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8+ T cells from seropositive but not seronegative persons. Results Herein, when these peptides were administered with the universal CD4+T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam2Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam2Cys is an effective way to elicit IFN-γ producing CD8+ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1224-232); AMLTAFFLR (GRA6164-172); RSFKDLLKK (GRA7134-142); STFWPCLLR (SAG2C13-21); SSAYVFSVK(SPA250-258); and AVVSLLRLLK(SPA89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite

  10. Spontaneous mobility of GABAA receptor M2 extracellular half relative to noncompetitive antagonist action.

    PubMed

    Chen, Ligong; Durkin, Kathleen A; Casida, John E

    2006-12-15

    The gamma-aminobutyric acid type A receptor beta(3) homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs) and Zn(2+). Our earlier study of the M2 cytoplasmic half (-1' to 10') established a model in which NCAs bind at pore-lining residues Ala(2)', Thr(6)', and Leu(9)'. To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extra cellular half of the beta(3) homopentamer (11' to 20'). Spontaneous disulfides formed with T13'C, L18'C, and E20'C from M2/M2 cross-linking and with I14'C (weak), H17'C, and R19'Con bridging M2/M3 intersubunits, based on single (M2 Cys only) and dual (M2 Cys plus M3 C289S) mutations. Induced disulfides also formed with T16'C, but there were few or none with M11'C, T12'C, and N15'C. These findings show conformational flexibility/mobility in the M2 extracellular half 17' to 20' region interpreted as a deformed beta-like conformation in the open channel. The NCA radioligands used were [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ([(3)H]EBOB) and [(3)H]3,3-bis-trifluoromethylbicyclo[2.2.1]heptane-2,2-dicarbonitrile with essentially the same results. NCA binding was disrupted by individual Cys substitutions at 13',14',16',17', and 19'. The inactivity of T13'C/T13'S may have been due to disturbance of the channel gate; I14'S and T16'S showed much better binding activity than their Cys counterparts, and the low activities of H17'C and R19'C were reversed by dithiothreitol. Zn(2+) potency for inhibition of [(3)H]EBOB binding was lowered 346-fold by the mutation H17'A. We propose that NCAs enter their binding site both directly, through the channel pore, and indirectly, through the water cavity of adjacent subunits.

  11. Formation of Hg(II) tetrathiolate complexes with cysteine at neutral pH

    SciTech Connect

    Warner, Thomas; Jalilehvand, Farideh

    2016-01-04

    Mercury(II) ions precipitate from aqueous cysteine (H2Cys) solutions containing H2Cys/Hg(II) mole ratio ≥ 2.0 as Hg(S-HCys)2. In absence of additional cysteine, the precipitate dissolves at pH ~12 with the [Hg(S,N-Cys)2]2- complex dominating. With excess cysteine (H2Cys/Hg(II) mole ratio ≥ 4.0), higher complexes form and the precipitate dissolves at lower pH values. Previously, we found that tetrathiolate [Hg(S-Cys)4]6- complexes form at pH = 11.0; in this work we extend the investigation to pH values of physiological interest. We examined two series of Hg(II)-cysteine solutions in which CHg(II) varied between 8 – 9 mM and 80 – 100 mM, respectively, with H2Cys/Hg(II) mole ratios from 4 to ~20. The solutions were prepared in the pH range 7.1 – 8.8, at the pH at which the initial Hg(S-HCys)2 precipitate dissolved. The variations in the Hg(II) speciation were followed by 199Hg NMR, X-ray absorption and Raman spectroscopic techniques. Our results show that in the dilute solutions (CHg(II) = 8 – 9 mM), mixtures of di-, tri- (major) and tetrathiolate complexes exist at moderate cysteine excess (CH2Cys ~ 0.16 M) at pH 7.1. In the more concentrated solutions (CHg(II) = 80 – 100 mM) with high cysteine excess (CH2Cys > 0.9 M), tetrathiolate [Hg(S-cysteinate)4]m-6 (m = 0 – 4) complexes dominate in the pH range 7.3 – 7.8, with lower charge than for the [Hg(S-Cys)4]6- complex due to protonation of some (m) of the amino groups of the coordinated cysteine ligands. In conclusion, the results of this investigation could provide a key to the mechanism of biosorption and accumulation of Hg(II) ions in biological / environmental systems.

  12. Role of hydroquinone-thiol conjugates in benzene-mediated toxicity.

    PubMed

    Lau, Serrine S; Kuhlman, Christopher L; Bratton, Shawn B; Monks, Terrence J

    2010-03-19

    Hydroquinone (HQ) is a metabolite of benzene, and in combination with phenol (PHE), reproduces benzene myelotoxicity. HQ readily oxidizes to 1,4-benzoquinone (1,4-BQ) followed by the reductive addition of glutathione (GSH). Subsequent cycles of oxidation and GSH addition give rise to a variety of mono-, and multi-GSH substituted conjugates. Following administration of PHE/HQ (1.1 mmol/kg/0.9 mmol/kg, ip) to male Sprague-Dawley (SD) rats, 2-(glutathion-S-yl)HQ [GS-HQ], 2,5-bis-(glutathion-S-yl)HQ [2,5-GS-HQ], 2,6-bis-(glutathion-S-yl)HQ [2,6-GS-HQ], and 2,3,5-tris-(glutathion-S-yl)HQ [2,3,5-GS-HQ] were all identified in bone marrow. 2-(Cystein-S-ylglycine)HQ [2-(CysGly)HQ], 2-(cystein-S-yl)HQ [2-(Cys)HQ], and 2-(N-acetylcystein-S-yl)HQ [2-(NACys)HQ] were also found in the bone marrow of PHE/HQ and benzene treated rats and mice, indicating the presence of an active mercapturic acid pathway within bone marrow. Moreover, 2,6-GS-HQ and 2,3,5-GS-HQ were hematotoxic when administered to rats. All of the HQ-GSH conjugates retain the ability to redox cycle and generate reactive oxygen species (ROS), and to arylate target proteins. Recent in vitro and in vivo studies in our laboratory revealed lysine and arginine residues as primary targets of 1,4-BQ, GS-HQ and 2-(NACys)HQ adduction. In contrast 1,4-BQ-adduction of cysteine residues may be a transient interaction, where physiological conditions dictate adduct stability. The generation of ROS and alkylation of proteins may both contribute to benzene-mediated myelotoxicity, and the two processes may be inter-dependent. However, the precise molecular mechanism by which benzene and HQ-GSH conjugates induce hematotoxicity remains to be determined. Within 18h of administration of PHE/HQ to SD rats a significant decrease in blood lymphocyte count was observed. At this early time point, erythrocyte counts and hemoglobin concentrations remained within the normal range. Concomitant with the decrease in lymphocyte count, western blot

  13. Role of Cys³⁶⁰² in the function and regulation of the cardiac ryanodine receptor.

    PubMed

    Mi, Tao; Xiao, Zhichao; Guo, Wenting; Tang, Yijun; Hiess, Florian; Xiao, Jianmin; Wang, Yundi; Zhang, Joe Z; Zhang, Lin; Wang, Ruiwu; Jones, Peter P; Chen, S R Wayne

    2015-04-01

    The cardiac Ca²⁺ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys³⁶³⁵ in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys³⁶⁰²) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys³⁶⁰² (C³⁶⁰²A) on store overload-induced Ca²⁺ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C³⁶⁰²A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca²⁺ release by reducing the termination threshold. As a result, C³⁶⁰²A markedly increased the fractional Ca²⁺ release. Furthermore, the C³⁶⁰²A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca²⁺ release, but it had no effect on the stimulatory action of 4,4'-dithiodipyridine (DTDP) on Ca²⁺ release. In addition, Cys³⁶⁰² mutations (C³⁶⁰²A or C³⁶⁰²R) did not abolish the effect of CaM on Ca²⁺-release termination. Therefore, RyR2-Cys³⁶⁰² is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys³⁶⁰² plays an important role in the activation and termination of Ca²⁺ release, but it is not essential for CaM regulation of RyR2.

  14. Al2O3 Nanoparticle Addition to Commercial Magnesium Alloys: Multiple Beneficial Effects

    PubMed Central

    Paramsothy, Muralidharan; Chan, Jimmy; Kwok, Richard; Gupta, Manoj

    2012-01-01

    The multiple beneficial effects of Al2O3 nanoparticle addition to cast magnesium based systems (followed by extrusion) were investigated, constituting either: (a) enhanced strength; or (b) simultaneously enhanced strength and ductility of the corresponding magnesium alloys. AZ31 and ZK60A nanocomposites containing Al2O3 nanoparticle reinforcement were each fabricated using solidification processing followed by hot extrusion. Compared to monolithic AZ31 (tension levels), the corresponding nanocomposite exhibited higher yield strength (0.2% tensile yield strength (TYS)), ultimate strength (UTS), failure strain and work of fracture (WOF) (+19%, +21%, +113% and +162%, respectively). Compared to monolithic AZ31 (compression levels), the corresponding nanocomposite exhibited higher yield strength (0.2% compressive yield strength (CYS)) and ultimate strength (UCS), lower failure strain and higher WOF (+5%, +5%, −4% and +11%, respectively). Compared to monolithic ZK60A (tension levels), the corresponding nanocomposite exhibited lower 0.2% TYS and higher UTS, failure strain and WOF (−4%, +13%, +170% and +200%, respectively). Compared to monolithic ZK60A (compression levels), the corresponding nanocomposite exhibited lower 0.2% CYS and higher UCS, failure strain and WOF (−10%, +7%, +15% and +26%, respectively). The capability of Al2O3 nanoparticles to enhance the properties of cast magnesium alloys in a way never seen before with micron length scale reinforcements is clearly demonstrated.

  15. Structural basis of redox-dependent modulation of galectin-1 dynamics and function

    PubMed Central

    Guardia, Carlos M; Caramelo, Julio J; Trujillo, Madia; Méndez-Huergo, Santiago P; Radi, Rafael; Estrin, Darío A; Rabinovich, Gabriel A

    2014-01-01

    Galectin-1 (Gal-1), a member of a family of multifunctional lectins, plays key roles in diverse biological processes including cell signaling, immunomodulation, neuroprotection and angiogenesis. The presence of an unusual number of six cysteine residues within Gal-1 sequence prompted a detailed analysis of the impact of the redox environment on the functional activity of this lectin. We examined the role of each cysteine residue in the structure and function of Gal-1 using both experimental and computational approaches. Our results show that: (i) only three cysteine residues present in each carbohydrate recognition domain (CRD) (Cys2, Cys16 and Cys88) were important in protein oxidation, (ii) oxidation promoted the formation of the Cys16–Cys88 disulfide bond, as well as multimers through Cys2, (iii) the oxidized protein did not bind to lactose, probably due to poor interactions with Arg48 and Glu71, (iv) in vitro oxidation by air was completely reversible and (v) oxidation by hydrogen peroxide was relatively slow (1.7 ± 0.2 M−1 s−1 at pH 7.4 and 25°C). Finally, an analysis of key cysteines in other human galectins is also provided in order to predict their behaviour in response to redox variations. Collectively, our data provide new insights into the structural basis of Gal-1 redox regulation with critical implications in physiology and pathology. PMID:24451991

  16. Organotin(IV) Derivatives of L-Cysteine and their in vitro Anti-Tumor Properties

    PubMed Central

    Chasapis, Christos T.; Garoufis, Achilles; Bakas, Thomas; Kubicki, Maciej; Ming, Yang

    2004-01-01

    The synthesis and characterization of the organotin compounds [(n-C4H9)2Sn(cys)] (1), [(C6H5)2Sn(cys)] (2), [(C6H5)3Sn(Hcys).(H2o)] (3), {[(CH3)2Sn(Kcys)2].2(H20)} (4), {[(n-C4H9)2Sn(Kcys)2].2(H20)} (5) and {[(C6H5)2Sn(Kcys)2].2(H20)} (6) (where H2cys = L-cysteine) are reported. The compounds have been characterized by elemental analysis and 1H-NMR, Uv-Vis, FT-IR and MOssbauer spectroscopic techniques. Attempted recrystallization of (2) in DMSO/methanol 2:1 solution yielded after several days unexpectedly the dimeric compound bis(tri-phenyltin)sulphide {[(C6H5)3Sn]2S} (7) which has been characterized by x-ray analysis. The structure of the parent complex (2) as well as the mechanism of the decomposition of cysteine are being further investigated. The in vitro anticancer activity of complexes (I)- (6), against human leukemia (HL60), human liver (Bel7402), human stomach (BGC823) and human cervix epithelial human carcinoma (Hela), nasopharyngeal carcinoma (KB) and lung cancer (PG) tumor cells, were evaluated. PMID:18365068

  17. Mn-catalase (Alr0998) protects the photosynthetic, nitrogen-fixing cyanobacterium Anabaena PCC7120 from oxidative stress.

    PubMed

    Banerjee, Manisha; Ballal, Anand; Apte, Shree Kumar

    2012-11-01

    Role of the non-haem, manganese catalase (Mn-catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn-catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat(+). The Alr0998 protein could be immunodetected in AnKat(+) cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat(+) cells but not in the wild-type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn-catalase. In response to oxidative stress, the AnKat(+) showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress-inducible 2-Cys-Prx protein. Treatment of wild-type Anabaena PCC7120 with H(2)O(2) caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO(2) fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat(+) strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn-catalase.

  18. Structural specializations of A2, a force-sensing domain in the ultralarge vascular protein von Willebrand factor

    SciTech Connect

    Zhang, Qing; Zhou, Yan-Feng; Zhang, Cheng-Zhong; Zhang, Xiaohui; Lu, Chafen; Springer, Timothy A.; Harvard-Med

    2009-06-30

    The lengths of von Willebrand factor (VWF) concatamers correlate with hemostatic potency. After secretion in plasma, length is regulated by hydrodynamic shear force-dependent unfolding of the A2 domain, which is then cleaved by a specific protease. The 1.9-{angstrom} crystal structure of the A2 domain demonstrates evolutionary adaptations to this shear sensor function. Unique among VWF A (VWA) domains, A2 contains a loop in place of the {alpha}4 helix, and a cis-proline. The central {beta}4-strand is poorly packed, with multiple side-chain rotamers. The Tyr-Met cleavage site is buried in the {beta}4-strand in the central hydrophobic core, and the Tyr structurally links to the C-terminal {alpha}6-helix. The {alpha}6-helix ends in 2 Cys residues that are linked by an unusual vicinal disulfide bond that is buried in a hydrophobic pocket. These features may narrow the force range over which unfolding occurs and may also slow refolding. Von Willebrand disease mutations, which presumably lower the force at which A2 unfolds, are illuminated by the structure.

  19. Specific N-terminal CGRP fragments mitigate chronic hypoxic pulmonary hypertension in rats.

    PubMed

    Qing, Xin; Wimalawansa, Sunil J; Keith, Ingegerd M

    2003-01-31

    Chronic hypoxic pulmonary hypertension (HPH) is characterized by elevated pulmonary arterial pressure (P(PA)), right ventricular hypertrophy (RVH), pulmonary vascular remodeling, pulmonary edema and polycythemia. Currently, there is no safe and effective treatment for HPH. Calcitonin gene-related peptide (CGRP) is the most potent peptide vasodilator discovered thus far. We previously demonstrated that exogenous CGRP reversed HPH in rats. However, the CGRP1 receptor antagonist CGRP(8-37) and smaller inhibitory C-terminal CGRP fragments that can be formed by enzymatic cleavage in vivo may compromise the beneficial effects of endogenous or exogenous CGRP. We here examine the agonistic efficacy of N-terminal rat alpha-CGRP peptides containing the disulfide bridge (Cys(2)-Cys(7)) with amidated C-terminal in prevention of HPH. Chronic infusion of CGRP(1-8), CGRP(1-13), or CGRP(1-14) at 7 nmol/h/rat via the right jugular vein during 14 days of hypobaric hypoxia (10% inspired O(2)) significantly decreased the P(PA), RVH and pulmonary arterial medial thickness in comparison with controls, suggesting that these CGRP sequences can mitigate chronic HPH in rats. Systemic pressure was unchanged by infused peptides indicating no carry-over effect. In conclusion, N-terminal CGRP fragments (CGRP(1-8), CGRP(1-13) and CGRP(1-14)) may have a protective role in hypoxic pulmonary hypertension.

  20. Different Arms of the Adaptive Immune System Induced by a Combination Vaccine Work in Concert to Provide Enhanced Clearance of Influenza

    PubMed Central

    Cobbin, Joanna C. A.; Zeng, Weiguang; Jackson, David C.; Brown, Lorena E.

    2014-01-01

    Current split influenza virus vaccines that induce strain-specific neutralising antibodies provide some degree of protection against influenza infection but there is a clear need to improve their effectiveness. The constant antigenic drift of influenza viruses means that vaccines are often not an exact match to the circulating strain and so levels of relevant antibodies may not be sufficiently high to afford protection. In the situation where the emergent influenza virus is completely novel, as is the case with pandemic strains, existing vaccines may provide no benefit. In this study we tested the concept of a combination vaccine consisting of sub-optimal doses of split influenza virus vaccine mixed with a cross-protective T-cell inducing lipopeptide containing the TLR2 ligand Pam2Cys. Mice immunised with combination vaccines showed superior levels of lung viral clearance after challenge compared to either split virus or lipopeptide alone, mediated through activation of enhanced humoral and/or additional cellular responses. The mechanism of action of these vaccines was dependent on the route of administration, with intranasal administration being superior to subcutaneous and intramuscular routes, potentially through the induction of memory CD8+ T cells in the lungs. This immunisation strategy not only provides a mechanism for minimising the dose of split virus antigen but also, through the induction of cross-protective CD8+ T cells, proves a breadth of immunity to provide potential benefit upon encounter with serologically diverse influenza isolates. PMID:25522323

  1. A novel transcription factor gene FHS1 is involved in the DNA damage response in Fusarium graminearum

    PubMed Central

    Son, Hokyoung; Fu, Minmin; Lee, Yoonji; Lim, Jae Yun; Min, Kyunghun; Kim, Jin-Cheol; Choi, Gyung Ja; Lee, Yin-Won

    2016-01-01

    Cell cycle regulation and the maintenance of genome integrity are crucial for the development and virulence of the pathogenic plant fungus Fusarium graminearum. To identify transcription factors (TFs) related to these processes, four DNA-damaging agents were applied to screen a F. graminearum TF mutant library. Sixteen TFs were identified to be likely involved in DNA damage responses. Fhs1 is a fungal specific Zn(II)2Cys6 TF that localises exclusively to nuclei. fhs1 deletion mutants were hypersensitive to hydroxyurea and defective in mitotic cell division. Moreover, deletion of FHS1 resulted in defects in perithecia production and virulence and led to the accumulation of DNA damage. Our genetic evidence demonstrated that the FHS1-associated signalling pathway for DNA damage response is independent of the ATM or ATR pathways. This study identified sixteen genes involved in the DNA damage response and is the first to characterise the novel transcription factor gene FHS1, which is involved in the DNA damage response. The results provide new insights into mechanisms underlying DNA damage responses in fungi, including F. graminearum. PMID:26888604

  2. DNA Macroarray Profiling of Lactococcus lactis subsp. lactis IL1403 Gene Expression during Environmental Stresses†

    PubMed Central

    Xie, Yi; Chou, Lan-szu; Cutler, Adele; Weimer, Bart

    2004-01-01

    This report describes the use of an oligonucleotide macroarray to profile the expression of 375 genes in Lactococcus lactis subsp. lactis IL1403 during heat, acid, and osmotic stress. A set of known stress-associated genes in IL1403 was used as the internal control on the array. Every stress response was accurately detected using the macroarray, compared to data from previous reports. As a group, the expression patterns of the investigated metabolic genes were significantly altered by heat, acid, and osmotic stresses. Specifically, 13 to 18% of the investigated genes were differentially expressed in each of the environmental stress treatments. Interestingly, the methionine biosynthesis pathway genes (metA-metB1 and metB2-cysK) were induced during heat shock, but methionine utilization genes, such as metK, were induced during acid stress. These data provide a possible explanation for the differences between acid tolerance mechanisms of L. lactis strains IL1403 and MG1363 reported previously. Several groups of transcriptional responses were common among the stress treatments, such as repression of peptide transporter genes, including the opt operon (also known as dpp) and dtpT. Reduction of peptide transport due to environmental stress will have important implications in the cheese ripening process. Although stress responses in lactococci were extensively studied during the last decade, additional information about this bacterium was gained from the use of this metabolic array. PMID:15528540

  3. Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris*

    PubMed Central

    Wang, Xiaolong; Wang, Qi; Wang, Jinjia; Bai, Peng; Shi, Lei; Shen, Wei; Zhou, Mian; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

    2016-01-01

    The alcohol oxidase 1 (AOX1) promoter (PAOX1) of Pichia pastoris is the most powerful and commonly used promoter for driving protein expression. However, mechanisms regulating its transcriptional activity are unclear. Here, we identified a Zn(II)2Cys6-type methanol-induced transcription factor 1 (Mit1) and elucidated its roles in regulating PAOX1 activity in response to glycerol and methanol. Mit1 regulated the expression of many genes involved in methanol utilization pathway, including AOX1, but did not participate in peroxisome proliferation and transportation of peroxisomal proteins during methanol metabolism. Structural analysis of Mit1 by performing domain deletions confirmed its specific and critical role in the strict repression of PAOX1 in glycerol medium. Importantly, Mit1, Mxr1, and Prm1, which positively regulated PAOX1 in response to methanol, were bound to PAOX1 at different sites and did not interact with each other. However, these factors cooperatively activated PAOX1 through a cascade. Mxr1 mainly functioned during carbon derepression, whereas Mit1 and Prm1 functioned during methanol induction, with Prm1 transmitting methanol signal to Mit1 by binding to the MIT1 promoter (PMIT1), thus increasingly expressing Mit1 and subsequently activating PAOX1. PMID:26828066

  4. Echistatin disulfide bridges: selective reduction and linkage assignment.

    PubMed Central

    Gray, W. R.

    1993-01-01

    Echistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with tris-(2-carboxyethyl)-phosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 degrees C a single bridge linking Cys 2-Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 degrees C the bridges Cys 7-Cys 32 and Cys 8-Cys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 degrees C to a peptide that retains a single bridge linking Cys 20-Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 degrees C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2-11; 7-32; 8-37; 20-39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis. PMID:8251946

  5. Leukotriene E4 is a full functional agonist for human cysteinyl leukotriene type 1 receptor-dependent gene expression

    PubMed Central

    Foster, Holly R.; Fuerst, Elisabeth; Branchett, William; Lee, Tak H.; Cousins, David J.; Woszczek, Grzegorz

    2016-01-01

    Leukotriene E4 (LTE4) the most stable of the cysteinyl leukotrienes (cysLTs) binds poorly to classical type 1 (CysLT1) and 2 (CysLT2) receptors although it induces potent responses in human airways in vivo, such as bronchoconstriction, airway hyperresponsiveness and inflammatory cell influx suggesting the presence of a novel receptor that preferentially responds to LTE4. To identify such a receptor two human mast cell lines, LAD2 and LUVA, were selected that differentially responded to LTE4 when analysed by intracellular signalling and gene expression. Comparative transcriptome analysis and recombinant gene overexpression experiments revealed CysLT1 as a receptor responsible for potent LTE4-induced response in LAD2 but not in LUVA cells, an observation confirmed further by gene knockdown and selective inhibitors. Lentiviral overexpression of CysLT1 in LUVA cells augmented intracellular calcium signalling induced by LTE4 but did not restore full agonist responses at the gene expression level. Our data support a model where both an increased expression of Gαq-coupled CysLT1, and sustained intracellular calcium mobilisation and extracellular signal-regulated kinase (Erk) activation, are required for LTE4-mediated regulation of gene expression in human cells. Our study shows for the first time that CysLT1 expression is critically important for responsiveness to LTE4 within a human cell system. PMID:26830450

  6. Interaction between Kazal serine proteinase inhibitor SPIPm2 and viral protein WSV477 reduces the replication of white spot syndrome virus.

    PubMed

    Ponprateep, Sirikwan; Phiwsaiya, Kornsunee; Tassanakajon, Anchalee; Rimphanitchayakit, Vichien

    2013-09-01

    White spot syndrome (WSS) is a viral disease caused by white spot syndrome virus (WSSV) which leads to severe mortality in cultured penaeid shrimp. In response to WSSV infection in Penaeus monodon, a Kazal serine proteinase inhibitor SPIPm2, normally stored in the granules of granular and semi-granular hemocytes is up-regulated and found to deter the viral replication. By using yeast two-hybrid screening, we have identified a viral target protein, namely WSV477. Instead of being a proteinase, the WSV477 was reported to be a Cys2/Cys2-type zinc finger regulatory protein having ATP/GTP-binding activity. In vitro pull down assay confirmed the protein-protein interaction between rSPIPm2 and rWSV477. Confocal laser scanning microscopy demonstrated that the SPIPm2 and WSV477 were co-localized in the cytoplasm of shrimp hemocytes. Using RNA interference, the silencing of WSV477 resulted in down-regulated of viral late gene VP28, the same result obtained with SPIPm2. In this instance, the SPIPm2 does not function as proteinase inhibitor but inhibit the regulatory function of WSV477.

  7. Identification and quantification of adducts between oxidized rosmarinic acid and thiol compounds by UHPLC-LTQ-Orbitrap and MALDI-TOF/TOF tandem mass spectrometry.

    PubMed

    Tang, Chang-bo; Zhang, Wan-gang; Dai, Chen; Li, Hui-xia; Xu, Xing-lian; Zhou, Guang-hong

    2015-01-28

    LTQ Orbitrap MS/MS was used to identify the adducts between quinones derived from rosmarinic acid (RosA) and thiol compounds, including cysteine (Cys), glutathione (GSH), and peptides digested from myosin. Two adducts of quinone-RosA/Cys and quinone-RosA/2Cys, one quinone-RosA/GSH adduct, and three quinone-RosA/peptide adducts were identified by extracted ion and MS(2) fragment ion chromatograms. By using MALDI-TOF/TOF MS, the adduction reaction between RosA and myosin in myofibrillar protein isolates was determined, demonstrating that the accurate reaction site was at Cys949 of myosin. The effect of reaction conditions, including stirring time, temperature, and oxidative stress, on the formation of adducts was further investigated. The formation of quinone-RosA/Cys and quinone-RosA/GSH increased with stirring time. Both adducts increased with temperature, whereas the reactivity of the addition reaction of GSH was higher than that of Cys. With increasing oxidation stress, the formation of quinone-RosA/GSH adduct increased and that of quinone-RosA/Cys adduct decreased.

  8. Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain

    PubMed Central

    Liang, Zicai; Sottrup-Jensen, Lars; Aspán, Anna; Hall, Martin; Söderhäll, Kenneth

    1997-01-01

    A 155-kDa proteinase inhibitor, pacifastin, from plasma of the freshwater crayfish, Pacifastacus leniusculus, was found to be composed of two covalently linked subunits. The two subunits are encoded by two different mRNAs, which were cloned and sequenced. The heavy chain of pacifastin (105 kDa) is related to transferrins, containing three transferrin lobes, two of which seem to be active for iron binding. The light chain of pacifastin (44 kDa) is the inhibitory subunit, and has nine cysteine-rich inhibitory domains that are homologous to each other and to low molecular weight proteinase inhibitors isolated from the grasshopper, Locusta migratoria. The nine light chain domains and the Locusta inhibitors share a characteristic cysteine array (Cys-Xaa9–12-Cys-Xaa2-Cys-Xaa-Cys-Xaa6–8-Cys-Xaa4-Cys) distinct from any described proteinase inhibitor family, suggesting that they constitute a new family of proteinase inhibitors. Pacifastin is the first known protein that has combined properties of a transferrin-like molecule and a proteinase inhibitor. PMID:9192625

  9. Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum.

    PubMed

    O'Kane, D J; Lee, J

    1985-03-12

    The properties of lumazine proteins purified from the marine bioluminescent bacteria Photobacterium phosphoreum, a psychrophile, and Photobacterium leiognathi, a relatively thermophilic species, are compared. An accurate 1:1 stoichiometry of binding of the ligand 6,7-dimethyl-8-ribityllumazine to each lumazine protein is established by back-titration of the apoprotein with the authentic ligand, using both fluorescence and absorption measurements. Neither protein contains metal cofactors, organic phosphorus, or carbohydrate. Both proteins are anionic and hydrophilic. They each contain a single Trp residue and have blocked amino terminals but otherwise differ in amino acid composition and other properties (P. phosphoreum and P. leiognathi, respectively): Met (internal), 1, 2; Cys, 2, 1; Arg, 4, 7; pI, 4.78 and 4.83, 4.38 and 4.45; Mr, 19 750, 21 300. In the P. phosphoreum protein both Cys residues are accessible, but in the P. leiognathi protein the single Cys is "buried". Modification of this buried Cys and at least one Cys in the P. phosphoreum protein prevents binding of the ligand. The UV and visible absorption spectra of both lumazine proteins denatured in 6 M guanidine hydrochloride can be accurately modeled by using the number of equivalents of the lumazine derivative and blocked aromatic amino acid model compounds determined by chemical and spectrophotometric analyses for Trp, Tyr, and Phe.

  10. Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris.

    PubMed

    Wang, Xiaolong; Wang, Qi; Wang, Jinjia; Bai, Peng; Shi, Lei; Shen, Wei; Zhou, Mian; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

    2016-03-18

    The alcohol oxidase 1 (AOX1) promoter (P AOX1) of Pichia pastoris is the most powerful and commonly used promoter for driving protein expression. However, mechanisms regulating its transcriptional activity are unclear. Here, we identified a Zn(II)2Cys6-type methanol-induced transcription factor 1 (Mit1) and elucidated its roles in regulating PAOX1 activity in response to glycerol and methanol. Mit1 regulated the expression of many genes involved in methanol utilization pathway, including AOX1, but did not participate in peroxisome proliferation and transportation of peroxisomal proteins during methanol metabolism. Structural analysis of Mit1 by performing domain deletions confirmed its specific and critical role in the strict repression of P AOX1 in glycerol medium. Importantly, Mit1, Mxr1, and Prm1, which positively regulated P AOX1 in response to methanol, were bound to P AOX1 at different sites and did not interact with each other. However, these factors cooperatively activated P AOX1 through a cascade. Mxr1 mainly functioned during carbon derepression, whereas Mit1 and Prm1 functioned during methanol induction, with Prm1 transmitting methanol signal to Mit1 by binding to the MIT1 promoter (P MIT1), thus increasingly expressing Mit1 and subsequently activating P AOX1.

  11. Association with AflR in Endosomes Reveals New Functions for AflJ in Aflatoxin Biosynthesis

    PubMed Central

    Ehrlich, Kenneth C.; Mack, Brian M.; Wei, Qijian; Li, Ping; Roze, Ludmila V.; Dazzo, Frank; Cary, Jeffrey W.; Bhatnagar, Deepak; Linz, John E.

    2012-01-01

    Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus ΔaflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export. PMID:23342682

  12. A C-terminal Hydrophobic, Solvent-protected Core and a Flexible N-terminus are Potentially Required for Human Papillomavirus 18 E7 Protein Functionality

    SciTech Connect

    Liu, S.; Tian, Y; Greenaway, F; Sun, M

    2010-01-01

    The oncogenic potential of the high-risk human papillomavirus (HPV) relies on the expression of genes specifying the E7 and E6 proteins. To investigate further the variation in oligomeric structure that has been reported for different E7 proteins, an HPV-18 E7 cloned from a Hispanic woman with cervical intraepithelial neoplasia was purified to homogeneity most probably as a stable monomeric protein in aqueous solution. We determined that one zinc ion is present per HPV-18 E7 monomer by amino acid and inductively coupled plasma-atomic emission spectroscopy analysis. Intrinsic fluorescence and circular dichroism spectroscopic results indicate that the zinc ion is important for the correct folding and thermal stability of HPV-18 E7. Hydroxyl radical mediated protein footprinting coupled to mass spectrometry and other biochemical and biophysical data indicate that near the C-terminus, the four cysteines of the two Cys-X{sub 2}-Cys motifs that are coordinated to the zinc ion form a solvent inaccessible core. The N-terminal LXCXE pRb binding motif region is hydroxyl radical accessible and conformationally flexible. Both factors, the relative flexibility of the pRb binding motif at the N-terminus and the C-terminal metal-binding hydrophobic solvent-protected core, combine together and facilitate the biological functions of HPV-18 E7.

  13. A water-soluble and retrievable ruthenium-based probe for colorimetric recognition of Hg(II) and Cys

    NASA Astrophysics Data System (ADS)

    Cui, Yali; Hao, Yuanqiang; Zhang, Yintang; Liu, Baoxia; Zhu, Xu; Qu, Peng; Li, Deliang; Xu, Maotian

    2016-08-01

    A new ruthenium-based complex 1 [(bis(4,4‧-dimethylphosphonic-2,2‧-bipyridine) dithiocyanato ruthenium (II))] was developed as a colorimetric probe for the detection of Hg(II) and Cys (Cysteine). The obtained compound 1 can give interconversional color changes upon the alternating addition of Hg(II) and Cys in 100% aqueous solution. The specific coordination between NCS groups with Hg(II) can lead to the formation of 1-Hg2 + complex, which can induce a remarkable spectral changes of probe 1. Afterwards the formed 1-Hg2 + complex can act as effective colorimetric sensor for Cys. Owing to the stronger binding affinity of sulfhydryl group to Hg2 +, Cys can extract Hg2 + from 1-Hg2 + complex resulting in the release of 1 and the revival of absorption profile of the probe 1. By introducing the hydrophilic phosphonic acid groups, the proposed probe exhibited excellent water solubility. The limits of detection (LODs) of the assay for Hg2 + and Cys are calculated to be 15 nM and 200 nM, respectively.

  14. Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain.

    PubMed Central

    Minehart, P L; Magasanik, B

    1991-01-01

    The GLN3 gene of Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen. We cloned the GLN3 gene and constructed null alleles by gene disruption. GLN3 is not essential for growth, but increased copies of GLN3 lead to a drastic decrease in growth rate. The complete nucleotide sequence of the GLN3 gene was determined, revealing one open reading frame encoding a polypeptide of 730 amino acids, with a molecular weight of approximately 80,000. The GLN3 protein contains a single putative Cys2/Cys2 zinc finger which has homology to the Neurospora crassa NIT2 protein, the Aspergillus nidulans AREA protein, and the erythroid-specific transcription factor GATA-1. Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLN1, the gene encoding glutamine synthetase. Neither control of transcription nor control of initiation of translation of GLN3 is important for regulation in response to glutamine availability. Images PMID:1682800

  15. Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional chinese medicine

    PubMed Central

    2011-01-01

    Background Species in the ascomycete fungal genus Cordyceps have been proposed to be the teleomorphs of Metarhizium species. The latter have been widely used as insect biocontrol agents. Cordyceps species are highly prized for use in traditional Chinese medicines, but the genes responsible for biosynthesis of bioactive components, insect pathogenicity and the control of sexuality and fruiting have not been determined. Results Here, we report the genome sequence of the type species Cordyceps militaris. Phylogenomic analysis suggests that different species in the Cordyceps/Metarhizium genera have evolved into insect pathogens independently of each other, and that their similar large secretomes and gene family expansions are due to convergent evolution. However, relative to other fungi, including Metarhizium spp., many protein families are reduced in C. militaris, which suggests a more restricted ecology. Consistent with its long track record of safe usage as a medicine, the Cordyceps genome does not contain genes for known human mycotoxins. We establish that C. militaris is sexually heterothallic but, very unusually, fruiting can occur without an opposite mating-type partner. Transcriptional profiling indicates that fruiting involves induction of the Zn2Cys6-type transcription factors and MAPK pathway; unlike other fungi, however, the PKA pathway is not activated. Conclusions The data offer a better understanding of Cordyceps biology and will facilitate the exploitation of medicinal compounds produced by the fungus. PMID:22112802

  16. Two sulfhydryl groups near the active site of soybean beta-amylase.

    PubMed

    Mikami, B; Nomura, K; Morita, Y

    1994-01-01

    The less reactive SH groups of soybean beta-amylase, SH4, SH5, and SH6, were modified with p-chloromercuribenzoic acid or N-ethylmaleimide, after the reactive SH groups, SH1, SH2, and SH3, were blocked with 5,5'-dithiobis-(2-nitrobenzoic acid) and cyanide. The enzyme activity decreased, accompanied by the modification of SH4. alpha-Cyclodextrin protected SH4 from the modification more effectively than maltose. The SH4-modified enzyme still bound to glucose, maltose, and alpha-cyclodextrin. SH4 was concerned with neither the catalysis nor substrate binding but its large substituent affected the substrate binding site. The sequencing of the 5-(iodoacetoamidoethyl)-aminoaphthalene-1-sulfonate-labeled peptides showed that SH4, SH5, and SH6 are Cys343, Cys82, and Cys208, respectively. Comparison of the primary structure of beta-amylases also showed that the sequence around SH4 (Cys343), as well as SH2 (Cys95), is strongly conserved between higher plant and bacterial beta-amylases. These results agree with the structure model deduced from X-ray crystallography of soybean beta-amylase.

  17. A novel therapeutic strategy of lipidated promiscuous peptide against Mycobacterium tuberculosis by eliciting Th1 and Th17 immunity of host

    PubMed Central

    Rai, Pradeep K; Chodisetti, Sathi Babu; Nadeem, Sajid; Maurya, Sudeep K; Gowthaman, Uthaman; Zeng, Weiguang; Janmeja, Ashok K; Jackson, David C; Agrewala, Javed N

    2016-01-01

    Regardless of the fact that potent drug-regimen is currently available, tuberculosis continues to kill 1.5 million people annually. Tuberculosis patients are not only inflicted by the trauma of disease but they also suffer from the harmful side-effects, immune suppression and drug resistance instigated by prolonged therapy. It is an exigency to introduce radical changes in the existing drug-regime and discover safer and better therapeutic measures. Hence, we designed a novel therapeutic strategy by reinforcing the efficacy of drugs to kill Mtb by concurrently boosting host immunity by L91. L91 is chimera of promiscuous epitope of Acr1 antigen of Mtb and TLR-2 agonist Pam2Cys. The adjunct therapy using drugs and L91 (D-L91) significantly declined the bacterial load in Mtb infected animals. The mechanism involved was through enhancement of IFN-γ+TNF-α+ polyfunctional Th1 cells and IL-17A+IFN-γ+ Th17 cells, enduring memory CD4 T cells and downregulation of PD-1. The down-regulation of PD-1 prevents CD4 T cells from undergoing exhaustion and improves their function against Mtb. Importantly, the immune response observed in animals could be replicated using T cells of tuberculosis patients on drug therapy. In future, D-L91 therapy can invigorate drugs potency to treat tuberculosis patients and reduce the dose and duration of drug-regime. PMID:27052185

  18. Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function.

    PubMed

    Shim, Joong-Youn; Rudd, James; Ding, Tomas T

    2011-02-01

    The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.

  19. Centipede venom peptide SsmTX-I with two intramolecular disulfide bonds shows analgesic activities in animal models.

    PubMed

    Wang, Ying; Li, Xiaojie; Yang, Meifeng; Wu, Chunyun; Zou, Zhirong; Tang, Jing; Yang, Xinwang

    2017-03-01

    Pain is a major symptom of many diseases and results in enormous pressures on human body or society. Currently, clinically used analgesic drugs, including opioids and nonsteroidal anti-inflammatory drugs, have adverse reactions, and thus, the development of new types of analgesic drug candidates is urgently needed. Animal venom peptides have proven to have potential as new types of analgesic medicine. In this research, we describe the isolation and characterization of an analgesic peptide from the crude venom of centipede, Scolopendra subspinipes mutilans. The amino acid sequence of this peptide was identical with SsmTX-I that was previously reported as a specific Kv2.1 ion channel blocker. Our results revealed that SsmTX-I was produced by posttranslational processing of a 73-residue prepropeptide. The intramolecular disulfide bridge motifs of SsmTX-I was Cys1-Cys3 and Cys2-Cys4. Functional assay revealed that SsmTX-I showed potential analgesic activities in formalin-induced paw licking, thermal pain, and acetic acid-induced abdominal writhing mice models. Our research provides the first report of cDNA sequences, disulfide motif, successful synthesis, and analgesic potential of SsmTX-I for the development of pain-killing drugs. It indicates that centipede peptide toxins could be a treasure trove for the search of novel analgesic drug candidates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  20. Electrochemical detection of Cu2+ through Ag nanoparticle assembly regulated by copper-catalyzed oxidation of cysteamine.

    PubMed

    Cui, Lin; Wu, Jie; Li, Jie; Ge, Yanqiu; Ju, Huangxian

    2014-05-15

    A highly sensitive and selective electrochemical sensor was developed for the detection of Cu(2+) by the assembly of Ag nanoparticles (AgNPs) at dithiobis[succinimidylpropionate] encapsulated Au nanoparticles (DSP-AuNPs), which was regulated by copper-catalyzed oxidation of cysteamine (Cys). The electrochemical sensor was constructed by layer-by-layer modification of glassy carbon electrode with carbon nanotubes, poly(amidoamine) dendrimers and DSP-AuNPs. In the absence of Cu(2+), Cys could bind to the surface of citrate-stabilized AgNPs via Ag-S bond, thus AgNPs could be assembled on the sensor surface through the reaction between DSP and Cys. In contrast, the copper-catalyzed oxidation of Cys by dissolved oxygen in the presence of Cu(2+) inhibited the Cys-induced aggregation of AgNPs, leading to the decrease of the electrochemical stripping signal of AgNPs. Under the optimized conditions, this method could detect Cu(2+) in the range of 1.0-1000 nM with a detection limit of 0.48 nM. The proposed Cu(2+) sensor showed good reproducibility, stability and selectivity. It has been satisfactorily applied to determine Cu(2+) in water samples.

  1. In Azotobacter vinelandii hydrogenase, substitution of serine for the cysteine residues at positions 62, 65, 294, and 297 in the small (HoxK) subunit affects H2 oxidation [corrected

    PubMed Central

    Sayavedra-Soto, L A; Arp, D J

    1993-01-01

    The essential role of the small (HoxK) subunit of hydrogenase of Azotobacter vinelandii in H2 oxidation was established. This was achieved by modification of the two Cys-X2-Cys amino acid motifs at the N and C termini of the HoxK subunit (Cys-62, -65, -294, and -297). The Cys codons were individually mutated to Ser codons. Modifications in these two motifs resulted in loss of hydrogenase activity. At the N terminus, the mutations of the codons for the motif Cys-62-Thr-Cys-64-Cys-65 decreased the activity of hydrogenase to levels no higher than 30% of those of the parental strain. H2 oxidation with the alternate electron acceptors methylene blue and benzyl viologen was decreased. H2 evolution and exchange activities were also affected. Cys-64 possibly substitutes for either Cys-62 or Cys-65, allowing for partial activity. Mutation of the codons for Cys-294 and Cys-297 to Ser codons resulted in no hydrogenase activity. The results are consistent with alterations of the ligands of FeS clusters in the HoxK subunit of hydrogenase [corrected]. Images PMID:8501046

  2. Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis.

    PubMed

    Kowalska, Jolanta; Pszczoła, Katarzyna; Wilimowska-Pelc, Anna; Lorenc-Kubis, Irena; Zuziak, Ewa; Ługowski, Mateusz; Łegowska, Anna; Kwiatkowska, Anna; Sleszyńska, Małgorzata; Lesner, Adam; Walewska, Aleksandra; Zabłotna, Ewa; Rolka, Krzysztof; Wilusz, Tadeusz

    2007-06-01

    Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.

  3. Combined synthetic and recombinant techniques for the development of lipoprotein-based, self-adjuvanting vaccines targeting human papillomavirus type-16 associated tumors.

    PubMed

    Moyle, Peter M; Dai, Wei; Liu, Tzu-Yu; Hussein, Waleed M; Maruthayanar, Pirashanthini; Wells, James W; McMillan, Nigel A J; Skwarczynski, Mariusz; Toth, Istvan

    2015-12-01

    Human papillomaviruses (HPVs) are associated with various cancers, with HPV16 linked to more than half of cervical cancer cases. Vaccines to prevent HPV infection and cancer development have proven effective, but are not useful in individuals with prior HPV exposure. Treatment vaccines to eradicate or control HPV-associated lesions are therefore desirable for these patients. Herein we describe the development of a process to enable the production of semisynthetic vaccines based on the site-specific attachment of synthetic bacterial lipid analogs (e.g., Pam2Cys) to a non-oncogenic mutant HPV16 E7 protein to generate molecularly defined vaccines. Many cytotoxic lymphocyte (CTL) epitopes from E7 are delivered by this approach; potentially ensuring that large numbers of immunized individuals can generate CTLs to clear HPV infected cells. Delivery of this construct reduced the growth of HPV16-associated tumors in a TC1 mouse model, the effects of which were better than the potent CTL epitope HPV16 E7(44-57) administered with Montanide ISA51 adjuvant.

  4. A New Type of Metal-Binding Site in Cobalt- And Zinc-Containing Adenylate Kinases Isolated From Sulfate-Reducers D. Gigas And D. Desulfuricans ATCC 27774

    SciTech Connect

    Gavel, O.Y.; Bursakov, S.A.; Rocco, G.Di; Trincao, J.; Pickering, I.J.; George, G.N.; Calvete, J.J.; Brondino, C.; Pereira, A.S.; Lampreia, J.; Tavares, P.; Moura, J.J.G.; Moura, I.

    2009-05-18

    Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterized in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the 'LID' domain. The sequence {sup 129}Cys-X{sub 5}-His-X{sub 15}-Cys-X{sub 2}-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

  5. A novel therapeutic strategy of lipidated promiscuous peptide against Mycobacterium tuberculosis by eliciting Th1 and Th17 immunity of host.

    PubMed

    Rai, Pradeep K; Chodisetti, Sathi Babu; Nadeem, Sajid; Maurya, Sudeep K; Gowthaman, Uthaman; Zeng, Weiguang; Janmeja, Ashok K; Jackson, David C; Agrewala, Javed N

    2016-04-07

    Regardless of the fact that potent drug-regimen is currently available, tuberculosis continues to kill 1.5 million people annually. Tuberculosis patients are not only inflicted by the trauma of disease but they also suffer from the harmful side-effects, immune suppression and drug resistance instigated by prolonged therapy. It is an exigency to introduce radical changes in the existing drug-regime and discover safer and better therapeutic measures. Hence, we designed a novel therapeutic strategy by reinforcing the efficacy of drugs to kill Mtb by concurrently boosting host immunity by L91. L91 is chimera of promiscuous epitope of Acr1 antigen of Mtb and TLR-2 agonist Pam2Cys. The adjunct therapy using drugs and L91 (D-L91) significantly declined the bacterial load in Mtb infected animals. The mechanism involved was through enhancement of IFN-γ(+)TNF-α(+) polyfunctional Th1 cells and IL-17A(+)IFN-γ(+) Th17 cells, enduring memory CD4 T cells and downregulation of PD-1. The down-regulation of PD-1 prevents CD4 T cells from undergoing exhaustion and improves their function against Mtb. Importantly, the immune response observed in animals could be replicated using T cells of tuberculosis patients on drug therapy. In future, D-L91 therapy can invigorate drugs potency to treat tuberculosis patients and reduce the dose and duration of drug-regime.

  6. Hydrogen Peroxide Cycling in Acidic Geothermal Environments and Potential Implications for Oxidative Stress

    NASA Astrophysics Data System (ADS)

    Mesle, M.; Beam, J.; Jay, Z.; Bodle, B.; Bogenschutz, E.; Inskeep, W.

    2014-12-01

    Hydrogen peroxide (H2O2) may be produced in natural waters via photochemical reactions between dissolved oxygen, organic carbon and light. Other reactive oxygen species (ROS) such as superoxide and hydroxyl radicals are potentially formed in environments with high concentrations of ferrous iron (Fe(II), ~10-100 μM) by reaction between H2O2 and Fe(II) (i.e., Fenton chemistry). Thermophilic archaea and bacteria inhabiting acidic iron-oxide mats have defense mechanisms against both extracellular and intracellular peroxide, such as peroxiredoxins (which can degrade H2O2) and against other ROS, such as superoxide dismutases. Biological cycling of H2O2 is not well understood in geothermal ecosystems, and geochemical measurements combined with molecular investigations will contribute to our understanding of microbial response to oxidative stress. We measured H2O2 and other dissolved compounds (Fe(II), Fe(III), H2S, O2), as well as photon flux, pH and temperature, over time in surface geothermal waters of several acidic springs in Norris Geyser Basin, Yellowstone National Park, WY (Beowulf Spring and One Hundred Spring Plain). Iron-oxide mats were sampled in Beowulf Spring for on-going analysis of metatranscriptomes and RT-qPCR assays of specific stress-response gene transcription (e.g., superoxide dismutases, peroxiredoxins, thioredoxins, and peroxidases). In situ analyses show that H2O2 concentrations are lowest in the source waters of sulfidic systems (ca. 1 μM), and increase by two-fold in oxygenated waters corresponding to Fe(III)-oxide mat formation (ca. 2 - 3 μM). Channel transects confirm increases in H2O2 as a function of oxygenation (distance). The temporal dynamics of H2O2, O2, Fe(II), and H2S in Beowulf geothermal waters were also measured during a diel cycle, and increases in H2O2 were observed during peak photon flux. These results suggest that photochemical reactions may contribute to changes in H2O2. We hypothesize that increases in H2O2 and O2

  7. Supramolecular self-assembly of graphene oxide and metal nanoparticles into stacked multilayers by means of a multitasking protein ring

    NASA Astrophysics Data System (ADS)

    Ardini, Matteo; Golia, Giordana; Passaretti, Paolo; Cimini, Annamaria; Pitari, Giuseppina; Giansanti, Francesco; Leandro, Luana Di; Ottaviano, Luca; Perrozzi, Francesco; Santucci, Sandro; Morandi, Vittorio; Ortolani, Luca; Christian, Meganne; Treossi, Emanuele; Palermo, Vincenzo; Angelucci, Francesco; Ippoliti, Rodolfo

    2016-03-01

    Graphene oxide (GO) is rapidly emerging worldwide as a breakthrough precursor material for next-generation devices. However, this requires the transition of its two-dimensional layered structure into more accessible three-dimensional (3D) arrays. Peroxiredoxins (Prx) are a family of multitasking redox enzymes, self-assembling into ring-like architectures. Taking advantage of both their symmetric structure and function, 3D reduced GO-based composites are hereby built up. Results reveal that the ``double-faced'' Prx rings can adhere flat on single GO layers and partially reduce them by their sulfur-containing amino acids, driving their stacking into 3D multi-layer reduced GO-Prx composites. This process occurs in aqueous solution at a very low GO concentration, i.e. 0.2 mg ml-1. Further, protein engineering allows the Prx ring to be enriched with metal binding sites inside its lumen. This feature is exploited to both capture presynthesized gold nanoparticles and grow in situ palladium nanoparticles paving the way to straightforward and ``green'' routes to 3D reduced GO-metal composite materials.Graphene oxide (GO) is rapidly emerging worldwide as a breakthrough precursor material for next-generation devices. However, this requires the transition of its two-dimensional layered structure into more accessible three-dimensional (3D) arrays. Peroxiredoxins (Prx) are a family of multitasking redox enzymes, self-assembling into ring-like architectures. Taking advantage of both their symmetric structure and function, 3D reduced GO-based composites are hereby built up. Results reveal that the ``double-faced'' Prx rings can adhere flat on single GO layers and partially reduce them by their sulfur-containing amino acids, driving their stacking into 3D multi-layer reduced GO-Prx composites. This process occurs in aqueous solution at a very low GO concentration, i.e. 0.2 mg ml-1. Further, protein engineering allows the Prx ring to be enriched with metal binding sites inside its

  8. Radically different thioredoxin domain arrangement of ERp46, an efficient disulfide bond introducer of the mammalian PDI family.

    PubMed

    Kojima, Rieko; Okumura, Masaki; Masui, Shoji; Kanemura, Shingo; Inoue, Michio; Saiki, Masatoshi; Yamaguchi, Hiroshi; Hikima, Takaaki; Suzuki, Mamoru; Akiyama, Shuji; Inaba, Kenji

    2014-03-04

    The mammalian endoplasmic reticulum (ER) contains a diverse oxidative protein folding network in which ERp46, a member of the protein disulfide isomerase (PDI) family, serves as an efficient disulfide bond introducer together with Peroxiredoxin-4 (Prx4). We revealed a radically different molecular architecture of ERp46, in which the N-terminal two thioredoxin (Trx) domains with positively charged patches near their peptide-binding site and the C-terminal Trx are linked by unusually long loops and arranged extendedly, forming an opened V-shape. Whereas PDI catalyzes native disulfide bond formation by the cooperative action of two mutually facing redox-active sites on folding intermediates bound to the central cleft, ERp46 Trx domains are separated, act independently, and engage in rapid but promiscuous disulfide bond formation during early oxidative protein folding. Thus, multiple PDI family members likely contribute to different stages of oxidative folding and work cooperatively to ensure the efficient production of multi-disulfide proteins in the ER.

  9. Na2CO3-responsive mechanisms in halophyte Puccinellia tenuiflora roots revealed by physiological and proteomic analyses

    PubMed Central

    Zhao, Qi; Suo, Jinwei; Chen, Sixue; Jin, Yudan; Ma, Xiaolin; Yin, Zepeng; Zhang, Yuhong; Wang, Tai; Luo, Ji; Jin, Wenhai; Zhang, Xia; Zhou, Zhiqiang; Dai, Shaojun

    2016-01-01

    Soil alkalization severely affects crop growth and agricultural productivity. Alkali salts impose ionic, osmotic, and high pH stresses on plants. The alkali tolerance molecular mechanism in roots from halophyte Puccinellia tenuiflora is still unclear. Here, the changes associated with Na2CO3 tolerance in P. tenuiflora roots were assessed using physiological and iTRAQ-based quantitative proteomic analyses. We set up the first protein dataset in P. tenuiflora roots containing 2,671 non-redundant proteins. Our results showed that Na2CO3 slightly inhibited root growth, caused ROS accumulation, cell membrane damage, and ion imbalance, as well as reduction of transport and protein synthesis/turnover. The Na2CO3-responsive patterns of 72 proteins highlighted specific signaling and metabolic pathways in roots. Ca2+ signaling was activated to transmit alkali stress signals as inferred by the accumulation of calcium-binding proteins. Additionally, the activities of peroxidase and glutathione peroxidase, and the peroxiredoxin abundance were increased for ROS scavenging. Furthermore, ion toxicity was relieved through Na+ influx restriction and compartmentalization, and osmotic homeostasis reestablishment due to glycine betaine accumulation. Importantly, two transcription factors were increased for regulating specific alkali-responsive gene expression. Carbohydrate metabolism-related enzymes were increased for providing energy and carbon skeletons for cellular metabolism. All these provide new insights into alkali-tolerant mechanisms in roots. PMID:27596441

  10. Spirulina phycocyanin induces differential protein expression and apoptosis in SKOV-3 cells.

    PubMed

    Pan, Ruowang; Lu, Rongmao; Zhang, Ying; Zhu, Mei; Zhu, Wen; Yang, Rongrong; Zhang, Enyong; Ying, Jun; Xu, Teng; Yi, Huiguang; Li, Jinsong; Shi, Mengru; Zhou, Li; Xu, Zuyuan; Li, Peizhen; Bao, Qiyu

    2015-11-01

    The present study was designed to determine the effects of phycocyanin (PC) on Human ovarian cancer SKOV-3 cells and the underlying molecular mechanisms of action. The inhibitory effects of PC on the cell proliferation were detected by MTT assay. The IC50 values of PC were 182.0μM and 133.6μM for 24h and 48h exposure, respectively. PC induced apoptosis in SKOV-3 cells was observed by electron microscopy and flow cytometry. The apoptosis rate was increased from 1.6% to 19.8% after PC exposure. The fluorescence intensity of ROS and the activities of Caspase-3, Caspase-8, and Caspase-9 were increased. Differentiated expression protein spots were selected and identified using proteomic techniques. There were 698±73 and 683±79 protein spots resolved in untreated and PC-treated cells, respectively. Forty five differential protein spots were analyzed by MALDI-TOF-MS, including mtSSB, PSME3, and nucleolin. The mRNA expression profiles determined by RT-PCR were consistent with that of the two-dimensional electrophoresis. The decreased proteins such as HSP60, nucleolin, PPase, peroxiredoxin-4 and the increased protein (mtSSB) were identified in SKOV-3 cells after PC treatment, indicating that the effects of PC on tumor cell apoptosis may be relate to multiple target proteins. And the mitochondrial pathway may be the main pathway for PC-induced apoptosis.

  11. Energy and antioxidant responses of pacific oyster exposed to trace levels of pesticides.

    PubMed

    Epelboin, Yanouk; Quéré, Claudie; Pernet, Fabrice; Pichereau, Vianney; Corporeau, Charlotte

    2015-09-21

    Here, we assess the physiological effects induced by environmental concentrations of pesticides in Pacific oyster Crassostrea gigas. Oysters were exposed for 14 d to trace levels of metconazole (0.2 and 2 μg/L), isoproturon (0.1 and 1 μg/L), or both in a mixture (0.2 and 0.1 μg/L, respectively). Exposure to trace levels of pesticides had no effect on the filtration rate, growth, and energy reserves of oysters. However, oysters exposed to metconazole and isoproturon showed an overactivation of the sensing-kinase AMP-activated protein kinase α (AMPKα), a key enzyme involved in energy metabolism and more particularly glycolysis. In the meantime, these exposed oysters showed a decrease in hexokinase and pyruvate kinase activities, whereas 2-DE proteomic revealed that fructose-1,6-bisphosphatase (F-1,6-BP), a key enzyme of gluconeogenesis, was up-regulated. Activities of antioxidant enzymes were higher in oysters exposed to the highest pesticide concentrations. Both pesticides enhanced the superoxide dismutase activity of oysters. Isoproturon enhanced catalase activity, and metconazole enhanced peroxiredoxin activity. Overall, our results show that environmental concentrations of metconazole or isoproturon induced subtle changes in the energy and antioxidant metabolisms of oysters.

  12. Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pKa values by MALDI-TOF mass spectrometry

    PubMed Central

    Nelson, Kimberly J.; Day, Amanda E.; Zeng, Bubing B.; King, S. Bruce; Poole, Leslie B.

    2008-01-01

    Cysteine reactivity in enzymes is imparted to a large extent by the stabilization of the deprotonated form of the reduced cysteine (i.e. the thiolate) within the active site. While this is likely to be an important chemical attribute of many thiol-based enzymes including cysteine-dependent peroxidases (peroxiredoxins) and proteases, only relatively few pKa values have been determined experimentally. Presented here is a new technique for determining the pKa value of cysteine residues through quantitative mass spectrometry following chemical modification with an iodoacetamide-based reagent over a range of pH buffers. This isotope-coded reagent, N-phenyl iodoacetamide (iodoacetanilide), is readily prepared in deuterated (d5) and protiated (d0) versions and is more reactive toward free cysteine than is iodoacetamide. Using this approach, the pKa values for the two cysteine residues in Escherichia coli thioredoxin were determined to be 6.5 and > 10, in good agreement with previous reports using chemical modification approaches. This technique allows the pKa of specific cysteine residues to be determined in a clear, fast, and simple manner and, because cysteine residues on separate tryptic peptides are measured separately, is not complicated by the presence of multiple cysteines within the protein of interest. PMID:18162165

  13. Molecular basis underlying the biological effects elicited by extremely low-frequency magnetic field (ELF-MF) on neuroblastoma cells.

    PubMed

    Sulpizio, Marilisa; Falone, Stefano; Amicarelli, Fernanda; Marchisio, Marco; Di Giuseppe, Fabrizio; Eleuterio, Enrica; Di Ilio, Carmine; Angelucci, Stefania

    2011-12-01

    Extremely low-frequency magnetic fields (ELF-MFs) may affect human health because of the possible associations with leukemia but also with cancer, cardiovascular, and neurological disorders. In the present work, human SH-SY5Y neuroblastoma cells were exposed to a 50 Hz, 1 mT sinusoidal ELF-MF at three different times, that is, 5 days (T5), 10 days (T10), and 15 days (T15) and then the effects of ELF-MF on proteome expression and biological behavior were investigated. Through comparative analysis between treated and control samples, we analyzed the proteome changes induced by ELF-MF exposure. Nine new proteins resolved in sample after a 15-day treatment were involved in a cellular defense mechanism and/or in cellular organization and proliferation such as peroxiredoxin isoenzymes (2, 3, and 6), 3-mercaptopyruvate sulfurtransferase, actin cytoplasmatic 2, t-complex protein subunit beta, ropporin-1A, and profilin-2 and spindlin-1. Our results indicated that ELF-MFs exposure altered the proliferative status and other important cell biology-related parameters, such as cell growth pattern, and cytoskeletal organization. These findings support our hypothesis that ELF radiation could trigger a shift toward a more invasive phenotype.

  14. Comparative Proteomic Analysis of Advanced Ovarian Cancer Tissue to Identify Potential Biomarkers of Responders and Nonresponders to First-Line Chemotherapy of Carboplatin and Paclitaxel

    PubMed Central

    Sehrawat, Urmila; Pokhriyal, Ruchika; Gupta, Ashish Kumar; Hariprasad, Roopa; Khan, Mohd Imran; Gupta, Divya; Naru, Jasmine; Singh, Sundararajan Baskar; Mohanty, Ashok Kumar; Vanamail, Perumal; Kumar, Lalit; Kumar, Sunesh; Hariprasad, Gururao

    2016-01-01

    Conventional treatment for advanced ovarian cancer is an initial debulking surgery followed by chemotherapy combination of carboplatin and paclitaxel. Despite initial high response, three-fourths of these women experience disease recurrence with a dismal prognosis. Patients with advanced-stage ovarian cancer who underwent cytoreductive surgery were enrolled and tissue samples were collected. Post surgery, these patients were started on chemotherapy and followed up till the end of the cycle. Fluorescence-based differential in-gel expression coupled with mass spectrometric analysis was used for discovery phase of experiments, and real-time polymerase chain reaction, Western blotting, and pathway analysis were performed for expression and functional validation of differentially expressed proteins. While aldehyde reductase, hnRNP, cyclophilin A, heat shock protein-27, and actin are upregulated in responders, prohibitin, enoyl-coA hydratase, peroxiredoxin, and fibrin-β are upregulated in the nonresponders. The expressions of some of these proteins correlated with increased apoptotic activity in responders and decreased apoptotic activity in nonresponders. Therefore, the proteins qualify as potential biomarkers to predict chemotherapy response. PMID:26997873

  15. Reactive oxygen species in response of plants to gravity stress

    NASA Astrophysics Data System (ADS)

    Jadko, Sergiy

    2016-07-01

    Reactive oxygen species (ROS) as second messengers can induce stress response of plants. Thioredoxins (Trx) and peroxiredoxins (Prx) can function as sensors and transmitters of the ROS in stress signaling and antioxidant response. 12-14 days old tissue culture of Arabidopsis thaliana have been investigated. Hypergravity stress was induced by centrifugation at 10 and 20 g during 30 and 90 min and than intensity of spontaneous chemiluminescence (SChL/ROS content), Trx and Prx activities were determined. All experiments were repeated from 3 to 5 times and the obtained data were statistically treated. In the tissue culture under development of the stress there were an increase in intensity of SChL and Trx and Prx activities. Thus, under hypergravity stress in the plant occurred early increase in the ROS level and the ROS induced the increase in the Trx and Prx activities. Prx and Trx can also participate in the formation of stress respons as acceptors and transducers of the redox signals. Increase in the activity of these enzymes primarily aimed at increasing of the total antioxidant activity in the cells to prevent of the plant to development of oxidative degradation by ROS.

  16. Intracerebral injection of oil cyst content of human craniopharyngioma (oil machinery fluid) as a toxic model in the rat brain.

    PubMed

    Tena-Suck, Martha Lilia; Hernández-Campos, Ma Elena; Ortiz-Plata, Alma; Salinas-Lara, Citlaltepetl; Colín-González, Ana Laura; Santamaría, Abel

    2014-04-01

    Craniopharyngiomas (CPs) are benign epithelial cystic tumors of the sellar and suprasellar region with a high survival rate and high recurrence in children. CPs contain dense oily fluid, but little is known yet about this content and its contribution to tissue damage and tumoral growth. In this study, we developed a simple experimental model produced by intracortical injection to rats of the cyst fluid content collected from human CPs to explore its possible contribution to brain tissue damage. The cyst fluid of the CPs ("oil machinery fluid") was collected during surgical removal, briefly preserved and further tested in rats through intracortical infusion. The group receiving "oil machinery fluid" presented increased reactive oxygen species formation, oxidative damage to lipids and reactive gliosis accompanied by augmented immunoreactivity to peroxiredoxin and thioredoxin reductase 1 at 15, 30 and 45 days post-injection. Other markers of inflammation and cell damage were stimulated at all post-lesion days tested. There was also a body weight gain. The persistence of tissue damage and oxidative stress suggests that "oil machinery fluid" exerts progressive alterations similar to those observed in patients with CPs, supporting the concept that some components of cyst fluid may contribute to brain tissue damage in these patients.

  17. Proteomic analyses of methamphetamine (METH)-induced differential protein expression by immature dendritic cells (IDC).

    PubMed

    Reynolds, Jessica L; Mahajan, Supriya D; Sykes, Donald E; Schwartz, Stanley A; Nair, Madhavan P N

    2007-04-01

    In the US, the increase in methamphetamine (METH) use has been associated with increased human immunodeficiency virus (HIV-1) infection. Dendritic cells (DC) are the first line of defense against HIV-1. DC play a critical role in harboring HIV-1 and facilitate the infection of neighboring T cells. However, the role of METH on HIV-1 infectivity and the expression of the proteome of immature dendritic cells (IDC) has not been elucidated. We hypothesize that METH modulates the expression of a number of proteins by IDC that foster the immunopathogenesis of HIV-1 infection. We utilized LTR amplification, p24 antigen assay and the proteomic method of difference gel electrophoresis (DIGE) combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to analyze the effects of METH on HIV-1 infectivity (HIV-1 IIIB; CXCR4-tropic, X4 strain) and the proteomic profile of IDC. Our results demonstrate that METH potentiates HIV-1 replication in IDC. Furthermore, METH significantly differentially regulates the expression of several proteins including CXCR3, protein disulfide isomerase, procathepsin B, peroxiredoxin and galectin-1. Identification of unique, METH-induced proteins may help to develop novel markers for diagnostic, preventive and therapeutic targeting in METH using subjects.

  18. Integrative Study of Physiological Changes Associated with Bacterial Infection in Pacific Oyster Larvae

    PubMed Central

    Genard, Bertrand; Miner, Philippe; Nicolas, Jean-Louis; Moraga, Dario; Boudry, Pierre; Pernet, Fabrice; Tremblay, Réjean

    2013-01-01

    Background Bacterial infections are common in bivalve larvae and can lead to significant mortality, notably in hatcheries. Numerous studies have identified the pathogenic bacteria involved in such mortalities, but physiological changes associated with pathogen exposure at larval stage are still poorly understood. In the present study, we used an integrative approach including physiological, enzymatic, biochemical, and molecular analyses to investigate changes in energy metabolism, lipid remodelling, cellular stress, and immune status of Crassostrea gigas larvae subjected to experimental infection with the pathogenic bacteria Vibrio coralliilyticus. Findings Our results showed that V. coralliilyticus exposure induced (1) limited but significant increase of larvae mortality compared with controls, (2) declined feeding activity, which resulted in energy status changes (i.e. reserve consumption, β-oxidation, decline of metabolic rate), (3) fatty acid remodeling of polar lipids (changes in phosphatidylinositol and lysophosphatidylcholine composition`, non-methylene–interrupted fatty acids accumulation, lower content of major C20 polyunsaturated fatty acids as well as activation of desaturases, phospholipase and lipoxygenase), (4) activation of antioxidant defenses (catalase, superoxide dismutase, peroxiredoxin) and cytoprotective processes (heat shock protein 70, pernin), and (5) activation of the immune response (non-self recognition, NF-κκ signaling pathway, haematopoiesis, eiconosoids and lysophosphatidyl acid synthesis, inhibitor of metalloproteinase and antimicrobial peptides). Conclusion Overall, our results allowed us to propose an integrative view of changes induced by a bacterial infection in Pacific oyster larvae, opening new perspectives on the response of marine bivalve larvae to infections. PMID:23704993

  19. Light and hydrogen peroxide inhibit C. elegans feeding through gustatory receptor orthologs and pharyngeal neurons

    PubMed Central

    Bhatla, Nikhil; Horvitz, H. Robert

    2015-01-01

    SUMMARY While gustatory sensing of the five primary flavors (sweet, salty, sour, bitter, and savory) has been extensively studied, pathways that detect non-canonical taste stimuli remain relatively unexplored. In particular, while reactive oxygen species cause generalized damage to biological systems, no gustatory mechanism to prevent ingestion of such material has been identified in any organism. We observed that light inhibits C. elegans feeding and used light as a tool to uncover molecular and neural mechanisms for gustation. Light can generate hydrogen peroxide, and we discovered that hydrogen peroxide similarly inhibits feeding. The gustatory receptor family members LITE-1 and GUR-3 are required for the inhibition of feeding by light and hydrogen peroxide. The I2 pharyngeal neurons increase calcium in response to light and hydrogen peroxide, and these responses require GUR-3 and a conserved antioxidant enzyme peroxiredoxin PRDX-2. Our results demonstrate a gustatory mechanism that mediates the detection and blocks ingestion of a non-canonical taste stimulus, hydrogen peroxide. PMID:25640076

  20. Thioredoxin in vascular biology: role in hypertension.

    PubMed

    Ebrahimian, Talin; Touyz, Rhian M

    2008-06-01

    The thioredoxin (TRX) system consists of TRX, TRX reductase, and NAD(P)H, and is able to reduce reactive oxygen species (ROS) through interactions with the redox-active center of TRX, which in turn can be reduced by TRX reductase in the presence of NAD(P)H. Among the TRX superfamily is peroxiredoxin (PRX), a family of non-heme peroxidases that catalyzes the reduction of hydroperoxides into water and alcohol. The TRX system is active in the vessel wall and functions either as an important endogenous antioxidant or interacts directly with signaling molecules to influence cell growth, apoptosis, and inflammation. Recent evidence implicates TRX in cardiovascular disease associated with oxidative stress, such as cardiac failure, arrhythmia, ischemia reperfusion injury, and hypertension. Thioredoxin activity is influenced by many mechanisms, including transcription, protein-protein interaction, and post-translational modification. Regulation of TRX in hypertensive models seems to be related to oxidative stress and is tissue- and cell-specific. Depending on the models of hypertension, TRX system could be upregulated or downregulated. The present review focuses on the role of TRX in vascular biology, describing its redox activities and biological properties in the media and endothelium of the vessel wall. In addition, the pathopysiological role of TRX in hypertension and other cardiovascular diseases is addressed.

  1. Redox regulation and pro-oxidant reactions in the physiology of circadian systems.

    PubMed

    Méndez, Isabel; Vázquez-Martínez, Olivia; Hernández-Muñoz, Rolando; Valente-Godínez, Héctor; Díaz-Muñoz, Mauricio

    2016-05-01

    Rhythms of approximately 24 h are pervasive in most organisms and are known as circadian. There is a molecular circadian clock in each cell sustained by a feedback system of interconnected "clock" genes and transcription factors. In mammals, the timing system is formed by a central pacemaker, the suprachiasmatic nucleus, in coordination with a collection of peripheral oscillators. Recently, an extensive interconnection has been recognized between the molecular circadian clock and the set of biochemical pathways that underlie the bioenergetics of the cell. A principle regulator of metabolic networks is the flow of electrons between electron donors and acceptors. The concomitant reduction and oxidation (redox) reactions directly influence the balance between anabolic and catabolic processes. This review summarizes and discusses recent findings concerning the mutual and dynamic interactions between the molecular circadian clock, redox reactions, and redox signaling. The scope includes the regulatory role played by redox coenzymes (NAD(P)+/NAD(P)H, GSH/GSSG), reactive oxygen species (superoxide anion, hydrogen peroxide), antioxidants (melatonin), and physiological events that modulate the redox state (feeding condition, circadian rhythms) in determining the timing capacity of the molecular circadian clock. In addition, we discuss a purely metabolic circadian clock, which is based on the redox enzymes known as peroxiredoxins and is present in mammalian red blood cells and in other biological systems. Both the timing system and the metabolic network are key to a better understanding of widespread pathological conditions such as the metabolic syndrome, obesity, and diabetes.

  2. Heterogeneous Porphyromonas gingivalis LPS modulates immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in human gingival fibroblasts

    PubMed Central

    Herath, Thanuja D. K.; Darveau, Richard P.; Seneviratne, Chaminda J.; Wang, Cun-Yu; Wang, Yu; Jin, Lijian

    2016-01-01

    Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis. PMID:27538450

  3. Modifications of plasma proteome in long-lived rats fed on a coenzyme Q10-supplemented diet.

    PubMed

    Santos-González, Mónica; Gómez Díaz, Consuelo; Navas, Plácido; Villalba, José Manuel

    2007-08-01

    Dietary coenzyme Q(10) prolongs life span of rats fed on a PUFAn-6-enriched diet. Our aim was to analyze changes in the levels of plasma proteins of rats fed on a PUFAn-6 plus coenzyme Q(10)-based diet. This approach could give novel insights into the mechanisms of life span extension by dietary coenzyme Q(10) in the rat. Serum albumin, which decreases with aging in the rat, was significantly increased by coenzyme Q(10) supplementation both at 6 and 24 months. After depletion of the most abundant proteins by affinity chromatography, levels of less abundant plasma proteins were also studied by using 2D-electrophoresis and MALDI-TOF mass fingerprinting analysis. Our results have shown that lifelong dietary supplementation with coenzyme Q(10) induced significant decreases of plasma hemopexin, apolipoprotein H and inter-alpha-inhibitor H4P heavy chain (at both 6 and 24 months), preprohaptoglobin, fibrinogen gamma-chain precursor, and fetuin-like protein (at 6 months), and alpha-1-antitrypsin precursor and type II peroxiredoxin (at 24 months). On the other hand, coenzyme Q(10) supplementation resulted in significant increases of serine protease inhibitor 3, vitamin D-binding protein (at 6 months), and Apo A-I (at 24 months). Our results support a beneficial role of dietary coenzyme Q(10) decreasing oxidative stress and cardiovascular risk, and modulating inflammation during aging.

  4. Phosphoproteomic analysis of the striatum from pleiotrophin knockout and midkine knockout mice treated with cocaine reveals regulation of oxidative stress-related proteins potentially underlying cocaine-induced neurotoxicity and neurodegeneration.

    PubMed

    Vicente-Rodríguez, Marta; Gramage, Esther; Herradón, Gonzalo; Pérez-García, Carmen

    2013-12-06

    The neurotrophic factors pleiotrophin (PTN) and midkine (MK) are highly upregulated in different brain areas relevant to drug addiction after administr