André Clippe

Cloning and characterization of AOEB166, a novel mammalian antioxidant enzyme of the peroxiredoxin family.

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.

Human peroxiredoxin 5 is a peroxynitrite reductase.

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 Cys47. Moreover, with the use of pulse radiolysis, we show that human peroxiredoxin 5 reduces peroxynitrite with an unequalled high rate constant of (7 ± 3) × 107 M−1 s−1.

Human mitochondrial peroxiredoxin 5 protects from mitochondrial DNA damages induced by hydrogen peroxide

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.

Recombinant peroxiredoxin 5 protects against excitotoxic brain lesions in newborn mice

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.

Early low protein diet aggravates unbalance between antioxidant enzymes leading to islet dysfunction.

Background 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. Methodology/Principal Findings 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. Conclusion and Significance 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.

Combined transcriptomic and physiological approaches reveal strong differences between short‐ and long‐term response of rice (Oryza sativa) to iron toxicity

Ferrous iron toxicity is a mineral disorder frequently occurring under waterlogged soils where rice is cultivated. To decipher the main metabolic pathways involved in rice response to iron excess, seedlings have been exposed to 125 mg L(-1) FeSO(4) for 3 weeks. A combined transcriptomic, biochemical and physiological study has been performed after short-term (3 d) or long-term (3 weeks) exposure to iron in order to elucidate the strategy of stress adaptation with time. Our results showed that short- and long-term exposure involved a very different response in gene expression regarding both the number and function. A larger number of genes were up- or down-regulated after 3 d than after 3 weeks of iron treatment; these changes also occurred in shoot even though no significant difference in iron concentration was recorded. Those modifications in gene expression after 3 d affected not only genes involved in hormonal signalling but also genes involved in C-compound and carbohydrate metabolism, oxygen and electron transfer, oxidative stress, and iron homeostasis and transport. Modification in some gene expression can be followed by modification in corresponding metabolic products and physiological properties, or differed in time for some others, underlying the importance of an integrated study.

Deciphering priming-induced improvement of rapeseed (Brassica napus L.) germination through an integrated transcriptomic and proteomic approach

Rape seeds primed with -1.2 MPa polyethylene glycol 6000 showed improved germination performance. To better understand the beneficial effect of osmopriming on seed germination, a global expression profiling method was used to compare, for the first time, transcriptomic and proteomic data for osmoprimed seeds at the crucial phases of priming procedure (soaking, drying), whole priming process and subsequent germination. Brassica napus was used here as a model to dissect the process of osmopriming into its essential components. A total number of 952 genes and 75 proteins were affected during the main phases of priming and post-priming germination. Transcription was not coordinately associated with translation resulting in a limited correspondence between mRNAs level and protein abundance. Soaking, drying and final germination of primed seeds triggered distinct specific pathways since only a minority of genes and proteins were involved in all phases of osmopriming while a vast majority was involved in only one single phase. A particular attention was paid to genes and proteins involved in the transcription, translation, reserve mobilization, water uptake, cell cycle and oxidative stress processes.

Deconstructing the catalytic efficiency of peroxiredoxin-5 peroxidatic cysteine.

Human peroxiredoxin-5 (PRDX5) is a thiol peroxidase that reduces H2O2 10(5) times faster than free cysteine. To assess the influence of two conserved residues on the reactivity of the critical cysteine (C47), we determined the reaction rate constants of PRDX5, wild type (WT), T44V and R127Q with one substrate electrophile (H2O2) and a nonspecific electrophile (monobromobimane). We also studied the corresponding reactions of low molecular weight (LMW) thiolates in order to construct a framework against which we could compare our proteins. To obtain a detailed analysis of the structural and energetic changes involved in the reaction between WT PRDX5 and H2O2, we performed ONIOM quantum mechanics/molecular mechanics (QM/MM) calculations with a QM region including 60 atoms of substrate and active site described by the B3LYP density functional and the 6-31+G(d,p) basis set; the rest of the protein was included in the MM region. Brønsted correlations reveal that the absence of T44 can increase the general nucleophilicity of the C47 but decreases the specific reactivity toward H2O2 by a factor of 10(3). The R127Q mutation causes C47 to behave like a LMW thiolate in the two studied reactions. QM/MM results with WT PRDX5 showed that hydrogen bonds in the active site are the cornerstone of two effects that make catalysis possible: the enhancement of thiolate nucleophilicity upon substrate ingress and the stabilization of the transition state. In both effects, T44 has a central role. These effects occur in a precise temporal sequence that ensures that the selective nucleophilicity of C47 is available only for peroxide substrates.

Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus

In Streptococcus thermophilus, the ComRS regulatory system governs the transcriptional level of comX expression and, hence, controls the early stage of competence development. The present work focuses on the posttranslational control of the activity of the sigma factor ComX and, therefore, on the late stage of competence regulation. In silico analysis performed on the S. thermophilus genome revealed the presence of a homolog of mecA (mecA(St)), which codes for the adaptor protein that is involved in ComK degradation by ClpCP in Bacillus subtilis. Using reporter strains and microarray experiments, we showed that MecA(St) represses late competence genes without affecting the early competence stage under conditions that are not permissive for competence development. In addition, this repression mechanism was found not only to act downstream of comX expression but also to be fully dependent on the presence of a functional comX gene. This negative control was similarly released in strains deleted for clpC, mecA, and clpC-mecA. Under artificial conditions of comX expression, we next showed that the abundance of ComX is higher in the absence of MecA or ClpC. Finally, results of bacterial two-hybrid assays strongly suggested that MecA interacts with both ComX and ClpC. Based on these results, we proposed that ClpC and MecA act together in the same regulatory circuit to control the abundance of ComX in S. thermophilus.

SOS Response Activation and Competence Development Are Antagonistic Mechanisms in Streptococcus thermophilus

Streptococcus includes species that either contain or lack the LexA-like repressor (HdiR) of the classical SOS response. In Streptococcus pneumoniae, a species which belongs to the latter group, SOS response inducers (e.g., mitomycin C [Mc] and fluoroquinolones) were shown to induce natural transformation, leading to the hypothesis that DNA damage-induced competence could contribute to genomic plasticity and stress resistance. Using reporter strains and microarray experiments, we investigated the impact of the SOS response inducers mitomycin C and norfloxacin and the role of HdiR on competence development in Streptococcus thermophilus. We show that both the addition of SOS response inducers and HdiR inactivation have a dual effect, i.e., induction of the expression of SOS genes and reduction of transformability. Reduction of transformability results from two different mechanisms, since HdiR inactivation has no major effect on the expression of competence (com) genes, while mitomycin C downregulates the expression of early and late com genes in a dose-dependent manner. The downregulation of com genes by mitomycin C was shown to take place at the level of the activation of the ComRS signaling system by an unknown mechanism. Conversely, we show that a ComX-deficient strain is more resistant to mitomycin C and norfloxacin in a viability plate assay, which indicates that competence development negatively affects the resistance of S. thermophilus to DNA-damaging agents. Altogether, our results strongly suggest that SOS response activation and competence development are antagonistic processes in S. thermophilus.