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ABSTRACT: Arenicola marina lives in marine environments where hydrogen peroxide concentrations reach micromolar levels. The annelid also forms reactive species through metabolic pathways. Its antioxidant systems include a cytosolic peroxiredoxin, peroxiredoxin 6 (AmPrx6 or AmPRDX6) that shows high homology to the mammalian 1-Cys peroxiredoxin. Previous work confirmed the peroxidase activity of AmPrx6 in the presence of dithiotreitol. Herein, we performed an in vitro kinetic characterization of the recombinant enzyme. AmPrx6 reduced hydrogen peroxide and peroxynitrite with rate constants of 1.1×10(7) and 2×10(6)M(-1)s(-1), respectively, at pH 7.4 and 25°C. Reduction of tert-butyl hydroperoxide was slower. The pK(a) of the peroxidatic thiol of AmPrx6 was determined as 5.1±0.2, indicating that it exists as thiolate, the reactive species, at physiological pH. The reductive part of the catalytic cycle was also explored. Hydrogen sulfide, present in millimolar concentrations in marine sediments where the annelid lives and that is able to reduce the mammalian 1-Cys peroxiredoxin, did not support AmPrx6 peroxidase activity. The enzyme was not reduced by other potential physiological reductants tested. Our data indicate that in this annelid, Prx6 could contribute to peroxide detoxification in the presence of a so far unidentified reducing counterpart.
Archives of Biochemistry and Biophysics 07/2011; 514(1-2):1-7. · 2.93 Impact Factor
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ABSTRACT: Peroxiredoxin 5 (PRDX5) is a thioredoxin peroxidase able to reduce hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. In human, PRDX5 was reported to be localized in the cytosol, the mitochondria, the peroxisomes and the nucleus. Mitochondrial localization results from the presence of an N-terminal mitochondrial targeting sequence (MTS). Here, we examined the conservation of mitochondrial localization of PRDX5 in animal species. We found that PRDX5 MTS is present and functional in the annelid lugworm Arenicola marina. Surprisingly, although mitochondrial targeting is well conserved among animals, PRDX5 is missing in mitochondria of domestic pig. Thus, it appears that mitochondrial targeting of PRDX5 may have been lost throughout evolution in animal species, including pig, with unknown functional consequences.
Mitochondrion 07/2011; 11(6):973-81. · 3.62 Impact Factor
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ABSTRACT: The search for protein ligands is a crucial step in the inhibitor design process. Fragment screening represents an interesting method to rapidly find lead molecules, as it enables the exploration of a larger portion of the chemical space with a smaller number of compounds as compared to screening based on drug-sized molecules. Moreover, fragment screening usually leads to hit molecules that form few but optimal interactions with the target, thus displaying high ligand efficiencies. Here we report the screening of a homemade library composed of 200 highly diverse fragments against the human Peroxiredoxin 5 protein. Peroxiredoxins compose a family of peroxidases that share the ability to reduce peroxides through a conserved cysteine. The three-dimensional structures of these enzymes ubiquitously found throughout evolution have been extensively studied, however, their biological functions are still not well understood and to date few inhibitors have been discovered against these enzymes. Six fragments from the library were shown to bind to the Peroxiredoxin 5 active site and ligand-induced chemical shift changes were used to drive the docking of these small molecules into the protein structure. The orientation of the fragments in the binding pocket was confirmed by the study of fragment homologues, highlighting the role of hydroxyl functions that hang the ligands to the Peroxiredoxin 5 protein. Among the hit fragments, the small catechol molecule was shown to significantly inhibit Peroxiredoxin 5 activity in a thioredoxin peroxidase assay. This study reports novel data about the ligand-Peroxiredoxin interactions that will help considerably the development of potential Peroxiredoxin inhibitors.
PLoS ONE 01/2010; 5(3):e9744. · 4.09 Impact Factor
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ABSTRACT: Islets from adult rat possess weak antioxidant defense leading to unbalance between superoxide dismutase (SOD) and hydrogen peroxide-inactivating enzymatic activities, catalase (CAT) and glutathione peroxidase (GPX) rending them susceptible to oxidative stress. We have shown that this vulnerability is influenced by maternal diet during gestation and lactation.
The present study investigated if low antioxidant activity in islets is already observed at birth and if maternal protein restriction influences the development of islet antioxidant defenses. Rats were fed a control diet (C group) or a low protein diet during gestation (LP) or until weaning (LPT), after which offspring received the control diet. We found that antioxidant enzymatic activities varied with age. At birth and after weaning, normal islets possessed an efficient GPX activity. However, the antioxidant capacity decreased thereafter increasing the potential vulnerability to oxidative stress. Maternal protein malnutrition changed the antioxidant enzymatic activities in islets of the progeny. At 3 months, SOD activity was increased in LP and LPT islets with no concomitant activation of CAT and GPX. This unbalance could lead to higher hydrogen peroxide production, which may concur to oxidative stress causing defective insulin gene expression due to modification of critical factors that modulate the insulin promoter. We found indeed that insulin mRNA level was reduced in both groups of malnourished offspring compared to controls. Analyzing the expression of such critical factors, we found that c-Myc expression was strongly increased in islets from both protein-restricted groups compared to controls.
Modification in antioxidant activity by maternal low protein diet could predispose to pancreatic islet dysfunction later in life and provide new insights to define a molecular mechanism responsible for intrauterine programming of endocrine pancreas.
PLoS ONE 02/2009; 4(7):e6110. · 4.09 Impact Factor
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ABSTRACT: Peroxiredoxins (PRDXs) are a superfamily of thiol-dependent peroxidases found in all phyla. PRDXs are mechanistically divided into three subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs. To reduce peroxides, the N-terminal peroxidatic Cys of PRDXs is first oxidized into sulfenic acid. This intermediate is reduced by forming a disulfide bond either with a resolving Cys of another monomeric entity (typical 2-Cys) or of the same molecule (atypical 2-Cys). In 1-Cys PRDXs, the resolving Cys is missing and the sulfenic acid of the peroxidatic Cys is reduced by a heterologous thiol-containing reductant. In search of a homolog of human 1-Cys PRDX6 in Arenicola marina, an annelid worm living in intertidal sediments, we have cloned and characterized a PRDX exhibiting high sequence homology with its mammalian counterpart. However, A. marina PRDX6 possesses five Cys among which two Cys function as peroxidatic and resolving Cys of typical 2-Cys PRDXs. Thus, A. marina PRDX6 belongs to a transient group exhibiting sequence homologies with mammalian 1-Cys PRDX6 but must be mechanistically classified into typical 2-Cys PRDXs. Moreover, PRDX6 is highly expressed in tissues directly exposed to the external environment, suggesting that this PRDX may be of particular importance for protection against exogenous oxidative attacks.
Free Radical Biology and Medicine 09/2008; 45(4):482-93. · 5.42 Impact Factor
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ABSTRACT: 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 A 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.
Protein Science 05/2008; 17(4):700-10. · 2.80 Impact Factor
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ABSTRACT: Human peroxiredoxin 5 (PRDX5) catalyzes different peroxides reduction by enzymatic substitution mechanisms. Enzyme oxidation caused an increase in Trp84 fluorescence, allowing performing pre-steady state kinetic measurements. The technique was validated by comparing with data available from the literature or obtained herein by alternative approaches. PRDX5 reacted with organic hydroperoxides with rate constants in the 10(6)-10(7)M(-1)s(-1) range, similar to peroxynitrite-mediated PRDX5 oxidation, whereas its reaction with hydrogen peroxide was slower (10(5)M(-1)s(-1)). The method allowed determining the kinetics of intramolecular disulfide formation as well as thioredoxin 2-mediated reduction. The reactivities of PRDXs with peroxides were surprisingly high considering thiol pK(a), indicating that other protein determinants are involved in PRDXs specialization. The order of reactivities between PRDX5 towards oxidizing substrates differ from other PRDXs studied, pointing to a selective action of PRDXs with respect to peroxide detoxification, helping to rationalize the multiple enzyme isoforms present even in the same cellular compartment.
Archives of Biochemistry and Biophysics 12/2007; 467(1):95-106. · 2.93 Impact Factor
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ABSTRACT: Rationale: Uteroglobin-related protein 1 (UGRP1) and Clara cell protein (CC16), members of the secretoglobin family, increasingly appear to play a role in airway inflammatory response.
To explore levels of UGRP1 and CC16 in induced sputum of patients with asthma and rhinitis.
Induced-sputum samples of patients with asthma or rhinitis (n = 32 each; atopic asthma, n = 24; atopic rhinitis, n = 20) and from 19 nonsmoking nonatopic control subjects were analyzed for cytology and levels of UGRP1, CC16, and albumin. Measurements and main results: Sputum UGRP1 increased in both asthma and rhinitis, most strikingly so in asthma, in which changes were most significant in atopic individuals. By contrast, sputum CC16 did not change significantly in either condition, although it was positively correlated with UGRP1 in patients and control subjects. Changes in sputum UGRP1 in atopic asthma were not linked to permeability changes reflected by increased albumin levels but correlated positively with sputum macrophages and negatively with eosinophils. The observed differences in UGRP1 and CC16 may be linked to different cell populations being responsible for their secretion; UGRP1 is mainly secreted in larger conducting airways, whereas CC16 is mainly secreted by the nasal and peripheral airways epithelium.
The increase in UGRP1 but not of CC16 in asthma and rhinitis suggests that UGRP1 may play a role in these inflammatory diseases.
Chest 02/2007; 131(1):172-9. · 5.25 Impact Factor
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ABSTRACT: Peroxiredoxin 5 is a thioredoxin peroxidase ubiquitously expressed in mammalian tissues. Peroxiredoxin 5 can be addressed intracellularly to mitochondria, peroxisomes, the cytosol and the nucleus. Here, we show that mitochondrial human peroxiredoxin 5 protects mitochondrial DNA (mtDNA) from oxidative attacks. In an acellular assay, recombinant peroxiredoxin 5 was shown to protect plasmid DNA from damages induced by metal-catalyzed generation of reactive oxygen species. In Chinese hamster ovary cells, overexpression of mitochondrial peroxiredoxin 5 significantly decreased mtDNA damages caused by exogenously added hydrogen peroxide. Altogether our results suggest that mitochondrial peroxiredoxin 5 may play an important role in mitochondrial genome stability.
FEBS Letters 05/2005; 579(11):2327-33. · 3.54 Impact Factor
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ABSTRACT: Peroxiredoxins are an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Peroxiredoxin 5, which is the last discovered mammalian member, was previously shown to reduce peroxides with the use of reducing equivalents derived from thioredoxin. We report here that human peroxiredoxin 5 is also a peroxynitrite reductase. Analysis of peroxiredoxin 5 mutants, in which each of the cysteine residues was mutated, suggests that the nucleophilic attack on the O-O bond of peroxynitrite is performed by the N-terminal peroxidatic Cys(47). Moreover, with the use of pulse radiolysis, we show that human peroxiredoxin 5 reduces peroxynitrite with an unequalled high rate constant of (7+/-3)x10(7) M(-1)s(-1).
FEBS Letters 08/2004; 571(1-3):161-5. · 3.54 Impact Factor
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ABSTRACT: Peroxiredoxin 5 is the last discovered mammalian member of an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Mammalian peroxiredoxin 5 has been recently classified as an atypical 2-Cys peroxiredoxin due to the presence of a conserved peroxidatic N-terminal cysteine (Cys47) and an unconserved resolving C-terminal cysteine residue (Cys151) forming an intramolecular disulfide intermediate in the oxidized enzyme. We have recently reported the crystal structure of human peroxiredoxin 5 in its reduced form. Here, a new crystal form of human peroxiredoxin 5 is described at 2.0 A resolution. The asymmetric unit contains three polypeptide chains. Surprisingly, beside two reduced chains, the third one is oxidized although the enzyme was crystallized under initial reducing conditions in the presence of 1 mM 1,4-dithio-dl-threitol. The oxidized polypeptide chain forms an homodimer with a symmetry-related one through intermolecular disulfide bonds between Cys47 and Cys151. The formation of these disulfide bonds is accompanied by the partial unwinding of the N-terminal parts of the alpha2 helix, which, in the reduced form, contains the peroxidatic Cys47 and the alpha6 helix, which is sequentially close to the resolving residue Cys151. In each monomer of the oxidized chain, the C-terminal part including the alpha6 helix is completely reorganized and is isolated from the rest of the protein on an extended arm. In the oxidized dimer, the arm belonging to the first monomer now appears at the surface of the second subunit and vice versa.
Journal of Molecular Biology 05/2004; 337(5):1079-90. · 4.00 Impact Factor
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ABSTRACT: Clara-cell protein (CC16), the predominant protein secreted by bronchiolar Clara cells, increasingly appears to protect the respiratory tract against oxidative stress and inflammation. The aim of this study was to test in inbred strains of mice whether the lung susceptibility to O3 correlates with the transepithelial leakage of CC16, with the mRNA and protein levels of CC16, and possibly with specific isoforms of the protein in the respiratory tract. Five strains of mouse with increasing sensitivity to O3 (C3H, AKR, SJL, CBA, and C57Bl) were exposed to 1.8 ppm O3 for 3 h and examined at 0 and 6 h postexposure. The most sensitive (C57Bl) and resistant (C3H) mice were also continuously exposed to 0.11 ppm O3 for up to 3 days. Lung injury was evaluated by measuring in bronchoalveolar lavage fluid (BALF) the levels of total protein, albumin, lactate dehydrogenase (LDH), and inflammatory cells. The patterns of proteins in BALF were also analyzed by two-dimensional electrophoresis (2-DE). Exposure to 1.8 or 0.11 ppm O3 caused a transient elevation of CC16 in serum that was maximal immediately after exposure and closely correlated with the extent of lung injury evaluated by BALF markers. The epithelial damage assessed on the basis of serum CC16 or BALF markers showed an inverse relation with the preexposure levels of CC16 in BALF. Since preexposure levels of CC16 mRNA were similar between the strains and since lung epithelium damage was also negatively correlated with preexposure levels of albumin in BALF, these findings identify basal lung epithelium permeability as a determinant of susceptibility to O3. The 2-DE mapping of proteins in BALF of these two strains revealed the existence of two distinct isoforms of CC16 with pI values of 4.9 and 5.2. The most acidic form was significantly less concentrated in the C57Bl strain, the most sensitive to O3, a difference that might be related to the higher permeability of the lung epithelium or to some post-transcriptional variations. In conclusion, these results suggest that the permeability of the lung epithelial barrier may be an important determinant of the lung susceptibility to O3, controlling the intrapulmonary levels of CC16 and possibly of other antioxidant/inflammatory proteins.
Inhalation Toxicology 11/2003; 15(12):1209-30. · 1.92 Impact Factor
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ABSTRACT: The pathophysiology of brain lesions associated with cerebral palsy is multifactorial and likely involves excess release of glutamate and excess production of free radicals, among other factors. Theoretically, antioxidants could limit the severity of these brain lesions. Peroxiredoxins are a family of peroxidases widely distributed in eukaryotes and prokaryotes. Peroxiredoxin 5 (PRDX5) is a recently discovered mammalian member of this family of antioxidant enzymes that is able to reduce hydrogen peroxide and alkyl hydroperoxides. The present study was designed to examine the neuroprotective effects of recombinant PRDX5 against neonatal excitotoxic challenge in both in vivo and in vitro experiments. For in vivo experiments, mice (postnatal day 5) were injected intraneopallially with ibotenate acting on NMDA and metabotropic receptors, or S-bromowillardiine acting on AMPA-kainate receptors to produce excitotoxic stress and brain lesions. Systemically administered recombinant PRDX5 provided protection against ibotenate-induced excitotoxic stress. Brain lesions of animals given ibotenate and PRDX5 were up to 63% smaller than that given ibotenate alone. However, PRDX5 provided no prevention from lesions induced with S-bromowillardiine. A mutated recombinant PRDX5 that is devoid of peroxidase activity was also tested and showed no protection against lesions induced by either ibotenate or S-bromowillardiine. Two classical antioxidants, N-acetylcysteine and catalase-PEG, provided the same neuroprotective effect as PRDX5. For in vitro experiments, neocortical neurons were exposed to 300 microM NMDA alone, NMDA plus recombinant PRDX5, or NMDA, recombinant PRDX5 and dithiothreitol, a classical electron donor for peroxiredoxins. Recombinant PRDX5 plus dithiothreitol displayed a synergistic neuroprotective effect on NMDA-induced neuronal death. These findings indicate that reactive oxygen species production participates in the formation of NMDA receptor-mediated brain lesions in newborn mice and that antioxidant compounds, such as PRDX5, provide some neuroprotection in these models.
Free Radical Biology and Medicine 05/2003; 34(7):862-72. · 5.42 Impact Factor
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ABSTRACT: Using two-dimensional electrophoresis, we have recently identified in human bronchoalveolar lavage fluid a novel protein,
termed B166, with a molecular mass of 17 kDa. Here, we report the cloning of human and rat cDNAs encoding B166, which has
been renamed AOEB166 for antioxidantenzyme B166. Indeed, the deduced amino acid sequence reveals that AOEB166 represents a new mammalian subfamily of AhpC/TSA peroxiredoxin
antioxidant enzymes. Human AOEB166 shares 63% similarity with Escherichia coli AhpC22 alkyl hydroperoxide reductase and 66% similarity with a recently identifiedSaccharomyces cerevisiae alkyl hydroperoxide reductase/thioredoxin peroxidase. Moreover, recombinant AOEB166 expressed in E. coli exhibits a peroxidase activity, and an antioxidant activity comparable with that of catalase was demonstrated with the glutamine
synthetase protection assay against dithiothreitol/Fe3+/O2 oxidation. The analysis of AOEB166 mRNA distribution in 30 different human tissues and in 10 cell lines shows that the gene
is widely expressed in the body. Of interest, the analysis of N- and C-terminal domains of both human and rat AOEB166 reveals
amino acid sequences presenting features of mitochondrial and peroxisomal targeting sequences. Furthermore, human AOEB166
expressed as a fusion protein with GFP in HepG2 cell line is sorted to these organelles. Finally, acute inflammation induced
in rat lung by lipopolysaccharide is associated with an increase of AOEB166 mRNA levels in lung, suggesting a protective role
for AOEB166 in oxidative and inflammatory processes.
Journal of Biological Chemistry 10/1999; 274(43):30451-30458. · 4.77 Impact Factor
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ABSTRACT: Peroxiredoxin 5 (PRDX5) is a mammalian thioredoxin peroxidase ubiquitously expressed in tissues. Its role as antioxidant enzyme has been previously supported in different pathological situations. In this study, we determined the complete human PRDX5 genomic organization and isolated the 5'-flanking region of the gene. Human PRDX5 gene is composed of six exons and five introns similarly to other chordate PRDX5 genes. Several single nucleotide polymorphisms were identified. Six out of them have amino acid substitutions in protein-coding region. Analysis of the 5'-flanking region of human PRDX5 revealed the presence of a TATA-less promoter containing a canonical CpG island and several putative response elements for transcription factors. To analyze the regulatory mechanisms controlling human PRDX5 expression, we characterized the 5'-flanking region by cloning various segments of this region in front of a luciferase reporter sequence. Transfection in HepG2 cells indicate that the 5'-flanking region contains regulatory elements for constitutive expression of human PRDX5. Multiple transcription start sites were also identified by 5'-RACE-PCR in human liver. Moreover, although no corresponding proteins were reported, we present new alternative splicing variants encoded specifically by human PRDX5 gene. The characterization of human PRDX5 gene revealed the complexity of its regulation and a high variability of sequences that might be associated with pathological situations.
Biochimica et Biophysica Acta 1769(7-8):472-83. · 4.66 Impact Factor
