RESUMEN

In addition to their role in the breakdown of H2O2, some peroxiredoxins (Prxs) have chaperone and H2O2 sensing functions. Acting as an H2O2 sensor, Prx Gpx3 transfers the oxidant signal to the transcription factor Yap1, involved in the antioxidant response in Saccharomyces cerevisiae. We have shown that Aspergillus nidulans Yap1 ortholog NapA is necessary for the antioxidant response, the utilization of arabinose, fructose and ethanol, and for proper development. To address the Prx roles in these processes, we generated and characterized mutants lacking peroxiredoxins PrxA, PrxB, PrxC, or TpxC. Our results show that the elimination of peroxiredoxins PrxC or TpxC does not produce any distinguishable phenotype. In contrast, the elimination of atypical 2-cysteine peroxiredoxins PrxA and PrxB produce different mutant phenotypes. ΔprxA, ΔnapA and ΔprxA ΔnapA mutants are equally sensitive to H2O2 and menadione, while PrxB is dispensable for this. However, the sensitivity of ΔprxA and ΔprxA ΔnapA mutants is increased by the lack of PrxB. Moreover, PrxB is required for arabinose and ethanol utilization and fruiting body cell wall pigmentation. PrxA expression is partially independent of NapA, and the replacement of peroxidatic cysteine 61 by serine (C61S) is enough to cause oxidative stress sensitivity and prevent NapA nuclear accumulation in response to H2O2, indicating its critical role in H2O2 sensing. Our results show that despite their high similarity, PrxA and PrxB play differential roles in Aspergillus nidulans antioxidant response, carbon utilization and development.

Asunto(s)

Antioxidantes , Aspergillus nidulans , Antioxidantes/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Peróxido de Hidrógeno/metabolismo , Cisteína/metabolismo , Arabinosa , Estrés Oxidativo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Etanol , Carbono , Oxidación-Reducción

RESUMEN

Peroxiredoxins (Prdxs) are thiol-dependent enzymes that scavenge peroxides. Previously, we found that Prdxs were hyperoxidized in a Parkinson's disease model induced by paraquat (PQ), which led to their inactivation, perpetuating reactive oxygen species (ROS) formation. Herein, we evaluated the redox state of the typical 2-Cys-Prx subgroup. We found that PQ induces ROS compartmentalization in different organelles, reflected by the 2-Cys-Prdx hyperoxidation pattern detected by redox eastern blotting. 2-Cys Prdxs are most vulnerable to hyperoxidation, while atypical 2-Cys Peroxiredoxin 5 (Prdx5) is resistant and is expressed in multiple organelles, such as mitochondria, peroxisomes, and cytoplasm. Therefore, we overexpressed human Prdx5 in the dopaminergic SHSY-5Y cell line using the adenoviral vector Ad-hPrdx5. Prdx5 overexpression was confirmed by western blotting and immunofluorescence (IF) and effectively decreased PQ-mediated mitochondrial and cytoplasmic ROS assessed with a mitochondrial superoxide indicator and DHE through IF or flow cytometry. Decreased ROS mediated by Prdx5 in the main subcellular compartments led to overall cell protection against PQ-induced cell death, which was demonstrated by flow cytometry using Annexin V labeling and 7-AAD. Therefore, Prdx5 is an attractive therapeutic target for PD, as its overexpression protects dopaminergic cells from ROS and death, which warrants further experimental animal studies for its subsequent application in clinical trials.

Asunto(s)

Estrés Oxidativo , Paraquat , Animales , Humanos , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Paraquat/farmacología , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Peroxirredoxinas/farmacología , Muerte Celular/genética

RESUMEN

Neospora caninum, an apicomplexan parasite, is the etiological agent of neosporosis, a disease that leads to neurological symptoms in dogs and abortion in cattle. Vaccine or drug treatments for neosporosis remain to be determined. Therefore, it is of undeniable relevance to investigate new molecules involved in the parasite's successful survival within the host cell. The aim of this study was to characterize the N. caninum peroxiredoxin (NcPrx), an enzyme involved in the redox system of the parasite. The NcPrx amino acid sequence showed high identity and similarity compared to homologues representatives of Apicomplexa phylum. The recombinant NcPrx (rNcPrx) was cloned and expressed in Escherichia coli (BL21) with the predicted molecular weight (22 kDa), and the identity of monomer and dimer forms of rNcPrx was confirmed by mass spectrometry. Native and recombinant NcPrx were detected by ELISA and western blot, using the polyclonal anti-rNcPrx serum. Multiphoton analysis showed that NcPrx is localized in tachyzoite cytosol. H2O2 treatment increased the rNcPrx dimerization in vitro, and associated with the in silico data, we suggest that NcPrx belongs to typical 2-Cys Prx group (AhpC/Prx1 family). rNcPrx also increased the H2O2 clearance and protected plasmidial DNA under oxidative conditions. Finally, H2O2 increased the NcPrx dimerization in intracellular and extracellular tachyzoites suggesting that it is enrolled in H2O2 clearance and sensing in N. caninum.

Asunto(s)

Coccidiosis , Neospora , Animales , Antioxidantes/metabolismo , Bovinos , Coccidiosis/parasitología , Coccidiosis/veterinaria , Perros , Femenino , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Peroxidasa/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Embarazo

RESUMEN

Trypanosoma cruzi is the causative agent of Chagas disease which is currently treated by nifurtimox (NFX) and benznidazole (BZ). Nevertheless, the mechanism of action of NFX is not completely established. Herein, we show the protective effects of T. cruzi mitochondrial peroxiredoxin (MPX) in macrophage infections and in response to NFX toxicity. After a 3-day treatment of epimastigotes with NFX, MPX content increased (2.5-fold) with respect to control, and interestingly, an MPX-overexpressing strain was more resistant to the drug. The generation of mitochondrial reactive species and the redox status of the low molecular weight thiols of the parasite were not affected by NFX treatment indicating the absence of oxidative stress in this condition. Since MPX was shown to be protective and overexpressed in drug-challenged parasites, non-classical peroxiredoxin activity was studied. We found that recombinant MPX exhibits holdase activity independently of its redox state and that its overexpression was also observed in temperature-challenged parasites. Moreover, increased holdase activity (2-fold) together with an augmented protease activity (proteasome-related) and an enhancement in ubiquitinylated proteins was found in NFX-treated parasites. These results suggest a protective role of MPX holdase activity toward NFX toxicity. Trypanosoma cruzi has a complex life cycle, part of which involves the invasion of mammalian cells, where parasite replication inside the host occurs. In the early stages of the infection, macrophages recognize and engulf T. cruzi with the generation of reactive oxygen and nitrogen species toward the internalized parasite. Parasites overexpressing MPX produced higher macrophage infection yield compared with wild-type parasites. The relevance of peroxidase vs. holdase activity of MPX during macrophage infections was assessed using conoidin A (CA), a covalent, cell-permeable inhibitor of peroxiredoxin peroxidase activity. Covalent adducts of MPX were detected in CA-treated parasites, which proves its action in vivo. The pretreatment of parasites with CA led to a reduced infection index in macrophages revealing that the peroxidase activity of peroxiredoxin is crucial during this infection process. Our results confirm the importance of peroxidase activity during macrophage infection and provide insights for the relevance of MPX holdase activity in NFX resistance.

Asunto(s)

Enfermedad de Chagas , Trypanosoma cruzi , Animales , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Macrófagos , Mamíferos , Nifurtimox/metabolismo , Nifurtimox/farmacología , Nifurtimox/uso terapéutico , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Trypanosoma cruzi/metabolismo

RESUMEN

Pseudomonas aeruginosa is an opportunistic bacterium in patients with cystic fibrosis and hospital acquired infections. It presents a plethora of virulence factors and antioxidant enzymes that help to subvert the immune system. In this study, we identified the 2-Cys peroxiredoxin, alkyl-hydroperoxide reductase C1 (AhpC1), as a relevant scavenger of oxidants generated during inflammatory oxidative burst and a mechanism of P. aeruginosa (PA14) escaping from killing. Deletion of AhpC1 led to a higher sensitivity to hypochlorous acid (HOCl, IC50 3.2 ± 0.3 versus 19.1 ± 0.2 µM), hydrogen peroxide (IC50 91.2 ± 0.3 versus 496.5 ± 6.4 µM) and the organic peroxide urate hydroperoxide. ΔahpC1 strain was more sensitive to the killing by isolated neutrophils and less virulent in a mice model of infection. All mice intranasally instilled with ΔahpC1 survived as long as they were monitored (15 days), whereas 100% wild-type and ΔahpC1 complemented with ahpC1 gene (ΔahpC1 attB:ahpC1) died within 3 days. A significantly lower number of colonies was detected in the lung and spleen of ΔahpC1-infected mice. Total leucocytes, neutrophils, myeloperoxidase activity, pro-inflammatory cytokines, nitrite production and lipid peroxidation were much lower in lungs or bronchoalveolar liquid of mice infected with ΔahpC1. Purified AhpC neutralized the inflammatory organic peroxide, urate hydroperoxide, at a rate constant of 2.3 ± 0.1 × 106 M-1s-1, and only the ΔahpC1 strain was sensitive to this oxidant. Incubation of neutrophils with uric acid, the urate hydroperoxide precursor, impaired neutrophil killing of wild-type but improved the killing of ΔahpC1. Hyperuricemic mice presented higher levels of serum cytokines and succumbed much faster to PA14 infection when compared to normouricemic mice. In summary, ΔahpC1 PA14 presented a lower virulence, which was attributed to a poorer ability to neutralize the oxidants generated by inflammatory oxidative burst, leading to a more efficient killing by the host. The enzyme is particularly relevant in detoxifying the newly reported inflammatory organic peroxide, urate hydroperoxide.

Asunto(s)

Pseudomonas aeruginosa , Estallido Respiratorio , Animales , Humanos , Ratones , Oxidantes , Peroxirredoxinas/genética , Virulencia

RESUMEN

The oxidative and nitrosative responses generated by animals and plants are important defenses against infection and establishment of pathogenic microorganisms such as bacteria, fungi, and protozoa. Among distinct oxidant species, hydroperoxides are a group of chemically diverse compounds that comprise small hydrophilic molecules, such as hydrogen peroxide and peroxynitrite, and bulky hydrophobic species, such as organic hydroperoxides. Peroxiredoxins (Prx) are ubiquitous enzymes that use a highly reactive cysteine residue to decompose hydroperoxides and can also perform other functions, like molecular chaperone and phospholipase activities, contributing to microbial protection against the host defenses. Prx are present in distinct cell compartments and, in some cases, they can be secreted to the extracellular environment. Despite their high abundance, Prx expression can be further increased in response to oxidative stress promoted by host defense systems, by treatment with hydroperoxides or by antibiotics. In consequence, some isoforms have been described as virulence factors, highlighting their importance in pathogenesis. Prx are very diverse and are classified into six different classes (Prx1-AhpC, BCP-PrxQ, Tpx, Prx5, Prx6, and AhpE) based on structural and biochemical features. Some groups are absent in hosts, while others present structural peculiarities that differentiate them from the host's isoforms. In this context, the intrinsic characteristics of these enzymes may aid the development of new drugs to combat pathogenic microorganisms. Additionally, since some isoforms are also found in the extracellular environment, Prx emerge as attractive targets for the production of diagnostic tests and vaccines. KEY POINTS: • Peroxiredoxins are front-line defenses against host oxidative and nitrosative stress. • Functional and structural peculiarities differ pathogen and host enzymes. • Peroxiredoxins are potential targets to microbicidal drugs.

Asunto(s)

Peróxido de Hidrógeno , Peroxirredoxinas , Animales , Oxidación-Reducción , Estrés Oxidativo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Plantas/metabolismo

RESUMEN

Glutathione peroxidases (GPx) are a family of enzymes with the ability to reduce organic and inorganic hydroperoxides to the corresponding alcohols using glutathione or thioredoxin as an electron donor. Here, we report the functional and structural characterization of a GPx identified in Trichoderma reesei (TrGPx). TrGPx was recombinantly expressed in a bacterial host and purified using affinity. Using a thioredoxin coupled assay, TrGPx exhibited activity of 28 U and 12.5 U in the presence of the substrates H2O2 and t-BOOH, respectively, and no activity was observed when glutathione was used. These results indicated that TrGPx is a thioredoxin peroxidase and hydrolyses H2O2 better than t-BOOH. TrGPx kinetic parameters using a pyrogallol assay resulted at Kmapp = 11.7 mM, Vmaxapp = 10.9 IU/µg TrGPx, kcat = 19 s-1 and a catalytic efficiency of 1.6 mM-1 s-1 to H2O2 as substrate. Besides that, TrGPx demonstrated an optimum pH ranging from 9.0-12.0 and a half-life of 36 min at 80 °C. TrGPx 3D-structure was obtained in a reduced state and non-catalytic conformation. The overall fold is similar to the other phospholipid-hydroperoxide glutathione peroxidases. These data contribute to understand the antioxidant mechanism in fungi and provide information for using antioxidant enzymes in biotechnological applications.

Asunto(s)

Hypocreales/enzimología , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Secuencia de Aminoácidos , Antioxidantes/química , Antioxidantes/farmacología , Fraccionamiento Químico , Clonación Molecular , Activación Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Glutatión Peroxidasa/química , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Concentración de Iones de Hidrógeno , Hypocreales/genética , Modelos Moleculares , Peroxirredoxinas/genética , Peroxirredoxinas/aislamiento & purificación , Conformación Proteica , Relación Estructura-Actividad , Temperatura

RESUMEN

Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus that causes systemic paracoccidioidomycosis, a granulomatous disease. The massive production of reactive oxygen species (ROS) by the host's cellular immune response is an essential strategy to restrain the fungal growth. Among the ROS, the hydroperoxides are very toxic antimicrobial compounds and fungal peroxidases are part of the pathogen neutralizing antioxidant arsenal against the host's defense. Among them, the peroxiredoxins are highlighted, since some estimates suggest that they are capable of decomposing most of the hydroperoxides generated in the host's mitochondria and cytosol. We presently characterized a unique P. brasiliensis 1-Cys peroxiredoxin (PbPrx1). Our results reveal that it can decompose hydrogen peroxide and organic hydroperoxides very efficiently. We showed that dithiolic, but not monothiolic compounds or heterologous thioredoxin reductant systems, were able to retain the enzyme activity. Structural analysis revealed that PbPrx1 has an α/ß structure that is similar to the 1-Cys secondary structures described to date and that the quaternary conformation is represented by a dimer, independently of the redox state. We investigated the PbPrx1 localization using confocal microscopy, fluorescence-activated cell sorter, and immunoblot, and the results suggested that it localizes both in the cytoplasm and at the cell wall of the yeast and mycelial forms of P. brasiliensis, as well as in the yeast mitochondria. Our present results point to a possible role of this unique P. brasiliensis 1-Cys Prx1 in the fungal antioxidant defense mechanisms.

Asunto(s)

Paracoccidioides , Paracoccidioidomicosis , Proteínas de Saccharomyces cerevisiae , Humanos , Oxidación-Reducción , Peroxidasas/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMEN

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of CP, which occurs when a second molecule of peroxide reacts with the CP in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the CP sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

Asunto(s)

Oxidación-Reducción , Peroxirredoxinas/genética , Procesamiento Proteico-Postraduccional/genética , Tirosina/genética , Dominio Catalítico/genética , Cisteína/genética , Disulfuros/química , Humanos , Mutación/genética , Nitratos/metabolismo , Peroxidasa/genética , Peróxidos/metabolismo , Peroxirredoxinas/efectos de los fármacos , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/farmacología , Transducción de Señal/efectos de los fármacos

RESUMEN

The oxidative stress response of the highly resistant actinomycete Dietzia cinnamea P4 after treatment with hydrogen peroxide (H2O2) was assessed in order to depict the possible mechanisms underlying its intrinsic high resistance to DNA damaging agents. We used transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA levels after exposure to different concentrations of H2O2 at 10 min and 1 h following the addition of the stressor. Catalase and superoxide dismutase genes were induced in different ways, according to the condition applied. Moreover, alkyl hydroperoxide reductase ahpCF, thiol peroxidase, thioredoxin and glutathione genes were upregulated in the presence of H2O2. Expression of peroxidase genes was not detected during the experiment. Overall results point to an actinomycete strain endowed with a set of enzymatic defenses against oxidative stress and with the main genes belonging to a functional SOS system (lexA, recA, uvrD), including suppression of lexA repressor, concomitantly to recA and uvrD gene upregulation upon H2O2 challenge.

Asunto(s)

Actinomycetales/efectos de los fármacos , Actinomycetales/metabolismo , Peróxido de Hidrógeno/efectos adversos , Estrés Oxidativo , Respuesta SOS en Genética/fisiología , Actinomycetales/enzimología , Actinomycetales/genética , Proteínas Bacterianas/genética , Catalasa/clasificación , Catalasa/genética , Daño del ADN/efectos de los fármacos , ADN Helicasas/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos , Glutatión/genética , Cinética , Peroxidasas/genética , Peroxirredoxinas/genética , Filogenia , ARN Mensajero/metabolismo , Rec A Recombinasas/genética , Respuesta SOS en Genética/genética , Análisis de Secuencia , Serina Endopeptidasas/genética , Superóxido Dismutasa/genética , Tiorredoxinas/genética , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Mitochondria are main sites of peroxynitrite formation. While at low concentrations mitochondrial peroxynitrite has been associated with redox signaling actions, increased levels can disrupt mitochondrial homeostasis and lead to pathology. Peroxiredoxin 3 is exclusively located in mitochondria, where it has been previously shown to play a major role in hydrogen peroxide reduction. In turn, reduction of peroxynitrite by peroxiredoxin 3 has been inferred from its protective actions against tyrosine nitration and neurotoxicity in animal models, but was not experimentally addressed so far. Herein, we demonstrate the human peroxiredoxin 3 reduces peroxynitrite with a rate constant of 1 × 107 M-1 s-1 at pH 7.8 and 25 °C. Reaction with hydroperoxides caused biphasic changes in the intrinsic fluorescence of peroxiredoxin 3: the first phase corresponded to the peroxidatic cysteine oxidation to sulfenic acid. Peroxynitrite in excess led to peroxiredoxin 3 hyperoxidation and tyrosine nitration, oxidative post-translational modifications that had been previously identified in vivo. A significant fraction of the oxidant is expected to react with CO2 and generate secondary radicals, which participate in further oxidation and nitration reactions, particularly under metabolic conditions of active oxidative decarboxylations or increased hydroperoxide formation. Our results indicate that both peroxiredoxin 3 and 5 should be regarded as main targets for peroxynitrite in mitochondria.

Asunto(s)

Mitocondrias/metabolismo , Oxidantes/metabolismo , Peroxiredoxina III/genética , Peroxirredoxinas/genética , Dióxido de Carbono/metabolismo , Cisteína/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Cinética , Oxidación-Reducción , Peroxiredoxina III/metabolismo , Ácido Peroxinitroso/metabolismo , Procesamiento Proteico-Postraduccional/genética , Transducción de Señal/genética

RESUMEN

ß-thalassemia is a worldwide distributed monogenic red cell disorder, characterized by an absent or reduced beta globin chain synthesis. The unbalance of alpha-gamma chain and the presence of pathological free iron promote severe oxidative damage, playing crucial a role in erythrocyte hemolysis, exacerbating ineffective erythropoiesis and decreasing the lifespan of red blood cells (RBC). Catalase, glutathione peroxidase and peroxiredoxins act together to protect RBCs from hydrogen peroxide insult. Among them, peroxiredoxins stand out for their overall abundance and reactivity. In RBCs, Prdx2 is the third most abundant protein, although Prdxs 1 and 6 isoforms are also found in lower amounts. Despite the importance of these enzymes, Prdx1 and Prdx2 may have their peroxidase activity inactivated by hyperoxidation at high hydroperoxide concentrations, which also promotes the molecular chaperone activity of these proteins. Some studies have demonstrated the importance of Prdx1 and Prdx2 for the development and maintenance of erythrocytes in hemolytic anemia. Now, we performed a global analysis comparatively evaluating the expression profile of several antioxidant enzymes and their physiological reducing agents in patients with beta thalassemia intermedia (BTI) and healthy individuals. Furthermore, increased levels of ROS were observed not only in RBC, but also in neutrophils and mononuclear cells of BTI patients. The level of transcripts and the protein content of Prx1 were increased in reticulocyte and RBCs of BTI patients and the protein content was also found to be higher when compared to beta thalassemia major (BTM), suggesting that this peroxidase could cooperate with Prx2 in the removal of H2O2. Furthermore, Prdx2 production is highly increased in RBCs of BTM patients that present high amounts of hyperoxidized species. A significant increase in the content of Trx1, Srx1 and Sod1 in RBCs of BTI patients suggested protective roles for these enzymes in BTI patients. Finally, the upregulation of Nrf2 and Keap1 transcription factors found in BTI patients may be involved in the regulation of the antioxidant enzymes analyzed in this work.

Asunto(s)

Células Eritroides/metabolismo , Peroxirredoxinas/metabolismo , Talasemia beta/metabolismo , Talasemia beta/patología , Adolescente , Adulto , Western Blotting , Niño , Preescolar , Eritrocitos/citología , Eritrocitos/metabolismo , Femenino , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Neutrófilos/citología , Neutrófilos/metabolismo , Oxidación-Reducción , Peroxirredoxinas/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto Joven

RESUMEN

Clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis are differentially susceptible to 2-Thiophen Hydrazide (TCH); however its mechanism of action or the reasons for that difference are unknown. We report herein that under our experimental conditions, TCH inhibits M. tuberculosis in solid but not in liquid medium, and that in spite of resembling Isoniazid and Ethionamide, it does not affect mycolic acid synthesis. To understand the mechanisms of action of TCH we isolated M. tuberculosis TCH resistant mutants which fell into two groups; one resistant to TCH and Isoniazid but not to Ethionamide or Triclosan, and the other resistant only to TCH with no, or marginal, cross resistance to Isoniazid. A S315T katG mutation conferred resistance to TCH while katG expression from a plasmid reduced M. tuberculosis MIC to this drug, suggesting a possible involvement of KatG in TCH activation. Whole genome sequencing of mutants from this second group revealed a single mutation in the alkylhydroperoxide reductase ahpC promoter locus in half of the mutants, while the remaining contained mutations in dispensable genes. This is the first report of the genetics underlying the action of TCH and of the involvement of ahpC as the sole basis for resistance to an anti-tubercular compound.

Asunto(s)

Antituberculosos/farmacología , Ácidos Carboxílicos/farmacología , Catalasa/genética , Farmacorresistencia Bacteriana/genética , Etionamida/farmacología , Isoniazida/farmacología , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Peroxirredoxinas/genética , Regiones Promotoras Genéticas , Proteínas Bacterianas , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo

RESUMEN

OBJECTIVES: The aim of this study was to evaluate peroxiredoxin I (Prx I) participation in the cellular antioxidant response to low-dose X-rays through the analysis of its expression in buccal mucosa cells from patients of different ages following panoramic dental radiography. MATERIALS AND METHODS: Of the 50 patients included in this study, oral mucosa cells from six adults were collected for the immunofluorescence cytological analysis. The other 44 patients, 11 patients aged below 20 years; 22 patients aged between 20 and 50 years; and 11 patients aged above 50 years, were submitted to panoramic dental radiography, and oral mucosa cells were collected for the gene expression analysis before and 1 hour after exposure. RESULTS: The results demonstrated Prx I expression in the cytoplasm of oral mucosa cells either before or after radiation exposure. The quantitative analysis showed that in oral mucosa cells from patients aged below 50 years the mRNA levels of PRDX1 were significantly increased after radiation exposure. On the other hand, the cells from patients aged above 50 years presented significantly lower PRDX1 transcript levels after radiation exposition. CONCLUSIONS: Panoramic radiography leads to increased Prx I expression in buccal mucosa cells, probably as an adaptive response to eliminate X-ray-induced ROS, except in cells from elderly people. CLINICAL RELEVANCE: Even low doses of radiation employed for dental purposes are capable to provoke stress to cells, which was demonstrated via the induction of the antioxidant gene PRDX1. In elderly patients, such mechanism was demonstrated to be impaired.

Asunto(s)

Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Mucosa Bucal/metabolismo , Mucosa Bucal/efectos de la radiación , Peroxirredoxinas/genética , Radiografía Panorámica , Adulto , Factores de Edad , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Multiple myeloma (MM) is characterised by intense protein folding and, consequently endoplasmic reticulum (ER) stress. The prostaglandin 15d-PGJ2 is able to raise oxidative stress levels within the cell and potentially trigger cell death. The aim of this study was to evaluate the antineoplastic effect of 15d-PGJ2 on MM in vitro and in vivo via ER and oxidative stress pathways. MM.1R and MM.1S cell lines were treated with 15d-PGJ2 at 1-10µM and evaluated with regard to proliferation, mRNA expression of PRDX1, PRDX4, GRP78, GRP94, CHOP, BCL-2 and BAX. Stress data was validated via oxidized glutathione assays. MM.1R cells were inoculated into NOD/SCID mice, which were subsequently treated daily with 15d-PGJ2 at 4mg/kg or vehicle (control), with tumour volume being monitored for 14days. 15d-PGJ2 reduced cell proliferation, induced cell death and apoptosis at 5µM and 10µM and Stress-related genes were upregulated at the same doses. Oxidized glutathione levels were also increased. 15d-PGJ2 at 4mg/kg in vivo halted tumour growth. In conclusion, 15d-PGJ2 induced myeloma cell death via ER stress in vitro. 15d-PGJ2 in vivo also inhibited tumour growth.

Asunto(s)

Antineoplásicos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Prostaglandina D2/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Estrés Oxidativo/efectos de los fármacos , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Prostaglandina D2/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Regulación hacia Arriba , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas/genética , Cisteína/genética , Mycoplasma hyopneumoniae/enzimología , Peroxirredoxinas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Clonación Molecular , Análisis Mutacional de ADN , Electrones , Evolución Molecular , Genoma Bacteriano , Metales/química , Mutagénesis Sitio-Dirigida , Mycoplasma hyopneumoniae/genética , Oxígeno/química , Peroxidasas/metabolismo , Peroxirredoxinas/metabolismo , Filogenia , Proteínas Recombinantes/genética , Compuestos de Sulfhidrilo/química

RESUMEN

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 (3×106vs 6×103M-1s-1, respectively), while the rate constant of peroxynitrite reduction was (0.6-1.4) ×106M-1s-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 0.5×106 and 1×106M-1s-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.

Asunto(s)

Aldehído Oxidorreductasas/química , Proteínas Bacterianas/química , Ácidos Grasos/química , Mycobacterium tuberculosis/química , Peroxirredoxinas/química , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Grasos/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Cinética , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/enzimología , Oxidación-Reducción , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tiorredoxinas/genética , Tiorredoxinas/metabolismo

RESUMEN

OxyR proteins are LysR-type transcriptional regulators, which play an important role in responding to oxidative stress in bacteria. Azospirillum brasilense Sp7 harbours two copies of OxyR. The inactivation of the oxyR1, the gene organized divergently to ahpC in A. brasilense Sp7, led to an increased tolerance to alkyl hydroperoxides, which was corroborated by an increase in alkyl hydroperoxide reductase (AhpC) activity, enhanced expression of ahpC :lacZ fusion and increased synthesis of AhpC protein in the oxyR1::km mutant. The upstream region of ahpC promoter harboured a putative OxyR binding site, T-N11-A. Mutation of T, A or both in the T-N11-Amotif caused derepression of ahpC in A. brasilense suggesting that T-N11-A might be the binding site for a negative regulator. Retardation of the electrophoretic mobility of the T-N11-A motif harbouring oxyR1-ahpC intergenic DNA by recombinant OxyR1, under reducing as well as oxidizing conditions, indicated that OxyR1 acts as a negative regulator of ahpC in A. brasilense. Sequence of the promoter of ahpC, predicted on the basis of transcriptional start site, and an enhanced expression of ahpC:lacZ fusion in chrR2::km mutant background suggested that ahpC promoter was RpoE2 dependent. Thus, this study shows that in A. brasilense Sp7, ahpC expression is regulated negatively by OxyR1 but is regulated positively by RpoE2, an oxidative-stress-responsive sigma factor. It also shows that OxyR1 regulates the expression RpoE1, which is known to play an important role during photooxidative stress in A. brasilense.

Asunto(s)

Azospirillum brasilense/enzimología , Proteínas Bacterianas/genética , Regulación Enzimológica de la Expresión Génica , Peroxirredoxinas/genética , Proteínas Represoras/metabolismo , Factor sigma/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Peroxirredoxinas/metabolismo , Proteínas Represoras/genética , Factor sigma/genética

Asunto(s)

Proteínas de la Membrana/química , Peroxirredoxinas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae , Dominio Catalítico , Proteínas de la Membrana/genética , Modelos Moleculares , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Peroxirredoxinas/genética , Estructura Secundaria de Proteína , Proteínas de Saccharomyces cerevisiae/genética

RESUMEN

The water cavity of yeast thioredoxin 1 (yTrx1) is an ancestral, conserved structural element that is poorly understood. We recently demonstrated that the water cavity is involved in the complex protein dynamics that are responsible for the catalytically relevant event of coupling hydration, proton exchange, and motion at the interacting loops. Its main feature is the presence of the conserved polar residue, Asp24, which is buried in a hydrophobic cavity. Here, we evaluated the role of the solvation of Asp24 as the main element that is responsible for the formation of the water cavity in thioredoxins. We showed that the substitution of Asp24 with a hydrophobic residue (D24A) was not sufficient to completely close the cavity. The dynamics of the D24A mutant of yTrx1 at multiple time scales revealed that the D24A mutant presents motions at different time scales near the active site, interaction loops, and water cavity, revealing the existence of a smaller dissected cavity. Molecular dynamics simulation, along with experimental molecular dynamics, allowed a detailed description of the water cavity in wild-type yTrx1 and D24A. The cavity connects the interacting loops, the central ß-sheet, and α-helices 2 and 4. It is formed by three contiguous lobes, which we call lobes A-C. Lobe A is hydrophilic and the most superficial. It is formed primarily by the conserved Lys54. Lobe B is the central lobe formed by the catalytically important residues Cys33 and Asp24, which are strategically positioned. Lobe C is the most hydrophobic and is formed by the conserved cis-Pro73. The central lobe B is closed upon introduction of the D24A mutation, revealing that independent forces other than solvation of Asp24 maintain lobes A and C in the open configuration. These data allow us to better understand the properties of this enzyme.

Asunto(s)

Proteínas de la Membrana/química , Simulación de Dinámica Molecular , Peroxirredoxinas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Sustitución de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/genética , Mutación Missense , Peroxirredoxinas/genética , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-Actividad