RESUMEN
YbbN/CnoX are proteins that display a Thioredoxin (Trx) domain linked to a tetratricopeptide domain. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl. Here, we compared EcYbbN with YbbN proteins from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). EcYbbN presents a redox active Cys residue at Trx domain (Cys63), 24 residues away from SQHC motif (SQHC[N24]C) that can form mixed disulfides with target proteins. In contrast, XfYbbN and PaYbbN present two Cys residues in the CXXC (CAPC) motif, while only PaYbbN shows the Cys residue equivalent to Cys63 of EcYbbN. Our phylogenetic analysis revealed that most of the YbbN proteins are in the bacteria domain of life and that their members can be divided into four groups according to the conserved Cys residues. EcYbbN (SQHC[N24]C), XfYbbN (CAPC[N24]V) and PaYbbN (CAPC[N24]C) are representatives of three sub-families. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (DTNB) and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km = 1.27 x 107 M-1 s-1), similar to the canonical XfTrx (XfTsnC). Furthermore, EcYbbN and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function, when the SQHC was replaced by CQHC. In contrast, the XfYbbN CAPA mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. In all cases, the induction of the holdase activity was accompanied by YbbN oligomerization. Finally, we showed that deletion of ybbN gene did not render in P. aeruginosa more sensitive stressful treatments. Therefore, YbbN/CnoX proteins display distinct properties, depending on the presence of the three conserved Cys residues.
Asunto(s)
Escherichia coli , Oxidorreductasas , Pseudomonas aeruginosa , Tiorredoxinas , Xylella , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/química , Filogenia , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/química , Xylella/enzimología , Xylella/genética , Xylella/metabolismoRESUMEN
Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.
Asunto(s)
Linfoma de Burkitt , Deshidrocolesteroles , Ferroptosis , Neuroblastoma , Animales , Humanos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Supervivencia Celular , Deshidrocolesteroles/metabolismo , Peroxidación de Lípido , Trasplante de Neoplasias , Neuroblastoma/metabolismo , Neuroblastoma/patología , Oxidación-Reducción , Fenotipo , Reproducibilidad de los ResultadosRESUMEN
The emergence and re-emergence of bacterial strains resistant to multiple drugs represent a global health threat, and the search for novel biological targets is a worldwide concern. AhpC are enzymes involved in bacterial redox homeostasis by metabolizing diverse kinds of hydroperoxides. In pathogenic bacteria, AhpC are related to several functions, as some isoforms are characterized as virulence factors. However, no inhibitor has been systematically evaluated to date. Here we show that the natural ent-kaurane Adenanthin (Adn) efficiently inhibits AhpC and molecular interactions were explored by computer assisted simulations. Additionally, Adn interferes with growth and potentializes the effect of antibiotics (kanamycin and PMBN), positioning Adn as a promising compound to treat infections caused by multiresistant bacterial strains.
Asunto(s)
Diterpenos de Tipo Kaurano , Peroxirredoxinas , Antibacterianos/farmacología , Diterpenos de Tipo Kaurano/farmacología , Kanamicina , BacteriasRESUMEN
Typical 2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous Cys-based peroxidases, which are stable as decamers in the reduced state, and may dissociate into dimers upon disulfide bond formation. A peroxidatic Cys (CP) takes part of a catalytic triad, together with a Thr/Ser and an Arg. Previously, we described that the presence of Ser (instead of Thr) in the active site stabilizes yeast 2-Cys Prx as decamers. Here, we compared the hyperoxidation susceptibilities of yeast 2-Cys Prx. Notably, 2-Cys Prx containing Ser (named here Ser-Prx) were more resistant to hyperoxidation than enzymes containing Thr (Thr-Prx). In silico analysis revealed that Thr-Prx are more frequent in all domains of life, while Ser-Prx are more abundant in bacteria. As yeast 2-Cys Prx, bacterial Ser-Prx are more stable as decamers than Thr-Prx. However, bacterial Ser-Prx were only slightly more resistant to hyperoxidation than Thr-Prx. Furthermore, in all cases, organic hydroperoxide inhibited more the peroxidase activities of 2-Cys Prx than hydrogen peroxide. Moreover, bacterial Ser-Prx displayed increased thermal resistance and chaperone activity, which may be related with its enhanced stability as decamers compared to Thr-Prx. Therefore, the single substitution of Thr by Ser in the catalytic triad results in profound biochemical and structural differences in 2-Cys Prx.
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/metabolismoRESUMEN
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 , VirulenciaRESUMEN
Sulfenic acids are the primary product of thiol oxidation by hydrogen peroxide and other oxidants. Several aspects of sulfenic acid formation through thiol oxidation were established recently. In contrast, the reduction of sulfenic acids is still scarcely investigated. Here, we characterized the kinetics of the reduction of sulfenic acids by ascorbate in several proteins. Initially, we described the crystal structure of our model protein (Tsa2-C170S). There are other Tsa2 structures in distinct redox states in public databases and all of them are decamers, with the peroxidatic cysteine very accessible to reductants, convenient features to investigate kinetics. We determined that the reaction between Tsa2-C170S-Cys-SOH and ascorbate proceeded with a rate constant of 1.40 ± 0.08 × 103 M-1 s-1 through a competition assay developed here, employing 2,6-dichlorophenol-indophenol (DCPIP). A series of peroxiredoxin enzymes (Prx6 sub family) were also analyzed by this competition assay and we observed that the reduction of sulfenic acids by ascorbate was in the 0.4-2.2 × 103 M-1 s-1 range. We also evaluated the same reaction on glyceraldehyde 3-phosphate dehydrogenase and papain, as the reduction of their sulfenic acids by ascorbate were reported previously. Once again, the rate constants are in the 0.4-2.2 × 103 M-1 s-1 range. We also analyzed the reduction of Tsa2-C170S-SOH by ascorbate by a second, independent method, following hydrogen peroxide reduction through a specific electrode (ISO-HPO-2, World Precision Instruments) and employing a bi-substrate, steady state approach. The kcat/KMAsc was 7.4 ± 0.07 × 103 M-1 s-1, which was in the same order of magnitude as the value obtained by the DCPIP competition assay. In conclusion, our data indicates that reduction of sulfenic acid in various proteins proceed at moderate rate and probably this reaction is more relevant in biological systems where ascorbate concentrations are high.
Asunto(s)
Ácidos Sulfénicos , Compuestos de Sulfhidrilo , Cisteína/metabolismo , Peróxido de Hidrógeno , Oxidación-Reducción , Peroxirredoxinas/metabolismoRESUMEN
Human HSP27 is a small heat shock protein that modulates the ability of cells to respond to heat shock and oxidative stress, and also functions as a chaperone independent of ATP, participating in the proteasomal degradation of proteins. The expression of HSP27 is associated with survival in mammalian cells. In cancer cells, it confers resistance to chemotherapy; in neurons, HSP27 has a positive effect on neuronal viability in models of Alzheimer's and Parkinson's diseases. To better understand the mechanism by which HSP27 expression contributes to cell survival, we expressed human HSP27 in the budding yeast Saccharomyces cerevisiae under control of different mutant TEF promoters, that conferred nine levels of graded basal expression, and showed that replicative lifespan and proteasomal activity increase as well as the resistance to oxidative and thermal stresses. The profile of these phenotypes display a dose-response effect characteristic of hormesis, an adaptive phenomenon that is observed when cells are exposed to increasing amounts of stress or toxic substances. The hormetic response correlates with changes in expression levels of HSP27 and also with its oligomeric states when correlated to survival assays. Our results indicate that fine tuning of HSP27 concentration could be used as a strategy for cancer therapy, and also for improving neuronal survival in neurodegenerative diseases.
Asunto(s)
Proteínas de Choque Térmico HSP27 , Hormesis , Saccharomyces cerevisiae , Animales , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Respuesta al Choque Térmico , Humanos , Chaperonas Moleculares , Estrés Oxidativo , Saccharomyces cerevisiae/metabolismoRESUMEN
This study examined particularly relevant redox pathways such as glycolysis, pentose phosphate pathway (PPP), metHb reductase and nucleotide metabolism, in order to better address how sickle cells deal with redox metabolism disruption. We also investigated the generation of specific oxidative lesions, and the levels of an unexplored antioxidant that could act as a candidate biomarker for oxidative status in sickle cell anemia (SCA). We adopted rigorous exclusion criteria to obtain the studied groups, which were composed by 10 subjects without hemoglobinopathies and 10 SCA patients. We confirmed that sickle cells overwhelm the antioxidant defense system, leading to an impaired antioxidant capacity that significantly contributed to the increase in cholesterol oxidation (ChAld) and hemolysis. Among the antioxidants evaluated, ergothioneine levels decreased in SCA (two-fold). We found strong correlations of ergothioneine levels with other erythrocyte metabolism markers, suggesting its use as an antioxidant therapy alternative for SCA treatment. Moreover, we found higher activities of MetHb reductase, AChE, G6PDH, HXK, and LDH, as well as levels of NADPH, ATP and hypoxanthine in sickle cells. On this basis, we conclude that impaired antioxidant capacity leaves to a loss of glycolysis and PPP shifting mechanism control and further homeostasis rupture, contributing to a decreased lifespan of sickle cells.
Asunto(s)
Anemia de Células Falciformes/sangre , Antioxidantes/metabolismo , Eritrocitos/metabolismo , Homeostasis , Adulto , Anemia de Células Falciformes/fisiopatología , Biomarcadores/metabolismo , Brasil , Estudios de Casos y Controles , Colesterol/metabolismo , Ergotioneína/análisis , Eritrocitos/patología , Femenino , Glucólisis , Hemoglobinopatías/metabolismo , Hemólisis , Humanos , Hipoxantina/análisis , Inflamación , Peroxidación de Lípido , Masculino , Osmorregulación , Oxidación-Reducción , Vía de Pentosa Fosfato , Adulto JovenRESUMEN
A major pathway for the detoxification of organic hydroperoxides, such as cumene hydroperoxide (CHP), involves the MarR family transcriptional regulator OhrR and the peroxidase OhrA. However, the effect of these peroxides on the global transcriptome and the contribution of the OhrA/OhrR system to bacterial virulence remain poorly explored. Here, we analyzed the transcriptome profiles of Chromobacterium violaceum exposed to CHP and after the deletion of ohrR, and we show that OhrR controls the virulence of this human opportunistic pathogen. DNA microarray and Northern blot analyses of CHP-treated cells revealed the upregulation of genes related to the detoxification of peroxides (antioxidant enzymes and thiol-reducing systems), the degradation of the aromatic moiety of CHP (oxygenases), and protection against other secondary stresses (DNA repair, heat shock, iron limitation, and nitrogen starvation responses). Furthermore, we identified two upregulated genes (ohrA and a putative diguanylate cyclase with a GGDEF domain for cyclic di-GMP [c-di-GMP] synthesis) and three downregulated genes (hemolysin, chitinase, and collagenase) in the ohrR mutant by transcriptome analysis. Importantly, we show that OhrR directly repressed the expression of the putative diguanylate cyclase. Using a mouse infection model, we demonstrate that the ohrR mutant was attenuated for virulence and showed a decreased bacterial burden in the liver. Moreover, an ohrR-diguanylate cyclase double mutant displayed the same virulence as the wild-type strain. In conclusion, we have defined the transcriptional response to CHP, identified potential virulence factors such as diguanylate cyclase as members of the OhrR regulon, and shown that C. violaceum uses the transcriptional regulator OhrR to modulate its virulence.
Asunto(s)
Proteínas Bacterianas/metabolismo , Derivados del Benceno/metabolismo , Derivados del Benceno/farmacología , Chromobacterium/genética , Chromobacterium/patogenicidad , Proteínas Represoras/metabolismo , Transcripción Genética , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Quitinasas/genética , Colagenasas/genética , Proteínas de Escherichia coli/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Infecciones por Bacterias Gramnegativas/microbiología , Proteínas Hemolisinas , Humanos , Peróxido de Hidrógeno , Hígado/microbiología , Ratones , Oxigenasas/metabolismo , Peroxidasas/metabolismo , Liasas de Fósforo-Oxígeno/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Virulencia , Factores de Virulencia/genéticaRESUMEN
Docosahexaenoic acid (C22:6, n-3, DHA) is a polyunsaturated fatty acid highly enriched in the brain. This fatty acid can be easily oxidized yielding hydroperoxides as primary products. Cu, Zn-Superoxide dismutase (SOD1) aggregation is a common hallmark of Amyotrophic Lateral Sclerosis (ALS) and the molecular mechanisms behind their formation are not completely understood. Here we investigated the effect of DHA and its hydroperoxides (DHAOOH) on human SOD1 oligomerization in vitro. DHA induced the formation of high-molecular-weight (HMW) SOD1 species (>700 kDa). Aggregation was dependent on free thiols and occurred primarily with the protein in its apo-form. SOD1 incubation with DHA was accompanied by changes in protein structure leading to exposure of protein hydrophobic patches and formation of non-amyloid aggregates. Site-directed mutagenesis studies demonstrated that Cys 6 and Cys 111 in wild-type and Cys 6 in ALS-linked G93A mutant are required for aggregation. In contrast, DHAOOH did not induce HMW species formation but promoted abnormal covalent dimerization of apo-SOD1 that was resistant to SDS and thiol reductants. Overall, our data demonstrate that DHA and DHAOOH induce distinct types of apo-SOD1 oligomerization leading to the formation of HMW and low-molecular-weight species, respectively.
Asunto(s)
Ácidos Docosahexaenoicos/metabolismo , Peróxido de Hidrógeno/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ácidos Grasos/metabolismo , Humanos , Multimerización de Proteína , Compuestos de Sulfhidrilo/metabolismoRESUMEN
The lack of efficient refolding methodologies must be overcome to take full advantage of the fact that bacteria express high levels of aggregated recombinant proteins. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, dissociating aggregates, which makes HHP a useful tool to solubilize proteins for subsequent refolding. A process of refolding was set up by using as a model TsnC, a thioredoxin that catalyzes the disulfide reduction to a dithiol, a useful indication of biological activity. The inclusion bodies (IB) were dissociated at 2.4 kbar. The effect of incubation of IB suspensions at 1-800 bar, the guanidine hydrochloride concentration, the oxidized/reduced glutathione (GSH/GSSG) ratios, and the additives in the refolding buffer were analyzed. To assess the yields of fully biologically active protein obtained for each tested condition, it was crucial to analyze both the TsnC solubilization yield and its enzymatic activity. Application of 2.4 kbar to the IB suspension in the presence of 9 mM GSH, 1mM GSSG, 0.75 M guanidine hydrochloride, and 0.5M arginine with subsequent incubation at 1 bar furnished high refolding yield (81%). The experience gained in this study shall help to establish efficient HHP-based protein refolding processes for other proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bioquímica/métodos , Presión Hidrostática , Replegamiento Proteico , Tiorredoxinas/metabolismo , Xylella/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Dicroismo Circular , Disulfuros/metabolismo , Escherichia coli/metabolismo , Disulfuro de Glutatión/metabolismo , Guanidina/farmacología , Replegamiento Proteico/efectos de los fármacos , Estructura Secundaria de Proteína , Solubilidad , Tiorredoxinas/química , Tiorredoxinas/ultraestructuraRESUMEN
The killing of bacterial pathogens by macrophages occurs via the oxidative burst and bacteria have evolved to overcome this challenge and survive, using several virulence and defense strategies, including antioxidant mechanisms. We show here that the 1-Cys peroxiredoxin LsfA from the opportunistic pathogen Pseudomonas aeruginosa is endowed with thiol-dependent peroxidase activity that protects the bacteria from H(2)O(2) and that this protein is implicated in pathogenicity. LsfA belongs to the poorly studied Prx6 subfamily of peroxiredoxins. The function of these peroxiredoxins has not been characterized in bacteria, and their contribution to host-pathogen interactions remains unknown. Infection of macrophages with the lsfA mutant strains resulted in higher levels of the cytokine TNF-α production due to the activation of the NF-kB and MAPK pathways, that are partially inhibited by the wild-type P. aeruginosa strain. A redox fluorescent probe was more oxidized in the lsfA mutant-infected macrophages than it was in the macrophages infected with the wild-type strain, suggesting that the oxidative burst was overstimulated in the absence of LsfA. Although no differences in the phagocytosis rates were observed when macrophages were infected with wild-type and mutant bacteria in a gentamicin exclusion assay, a higher number of wild-type bacterial cells was found in the supernatant. This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase. In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria. LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions.
Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Macrófagos/inmunología , Peroxirredoxinas/farmacología , Infecciones por Pseudomonas/metabolismo , Animales , Humanos , Peróxido de Hidrógeno/farmacología , Macrófagos/efectos de los fármacos , Ratones , NADPH Oxidasas/metabolismo , Fagocitosis/inmunología , Infecciones por Pseudomonas/virología , Pseudomonas aeruginosa/patogenicidad , Estallido Respiratorio/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Virulencia/efectos de los fármacosRESUMEN
Francisella genus comprises Gram-negative facultative intracellular bacteria that are among the most infectious human pathogens. A protein of 14.7 KDa named as FTN_1133 was previously described as a novel hydroperoxide resistance protein in F. tularensis subsp. novicida, implicated in organic peroxide detoxification and virulence. Here, we describe a structural and biochemical characterization of FTN_1133. Contrary to previous assumptions, multiple amino acid sequence alignment analyses revealed that FTN_1133 does not share significant similarity with proteins of the Ohr/OsmC family or any other Cys-based, thiol dependent peroxidase, including conserved motifs around reactive cysteine residues. Circular dichroism analyses were consistent with the in silico prediction of an all-α-helix secondary structure. The pKa of its single cysteine residue, determined by a monobromobimane alkylation method, was shown to be 8.0±0.1, value that is elevated when compared with other Cys-based peroxidases, such as peroxiredoxins and Ohr/OsmC proteins. Attempts to determine a thiol peroxidase activity for FTN_1133 failed, using both dithiols (DTT, thioredoxin and lipoamide) and monothiols (glutathione or 2-mercaptoethanol) as reducing agents. Heterologous expression of FTN_1133 gene in ahpC and oxyR mutants of E. coli showed no complementation. Furthermore, analysis of FTN_1133 protein by non-reducing SDS-PAGE showed that an inter-molecular disulfide bond (not detected in Ohr proteins) can be generated under hydroperoxide treatment, but the observed rates were not comparable to those observed for other thiol-dependent peroxidases. All the biochemical and structural data taken together indicated that FTN_1133 displayed distinct characteristics from other thiol dependent peroxidases and, therefore, suggested that FTN_1133 is not directly involved in hydroperoxide detoxification.
Asunto(s)
Francisella tularensis/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Peroxidasas/química , Peroxidasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Secuencia Conservada , Modelos Moleculares , Estrés Oxidativo , Estructura Secundaria de ProteínaRESUMEN
Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 107M⻹ s⻹) and peroxynitrite ((3.7 ± 0.4) × 105 M⻹ s⻹) at pH 7.4 and 25°C.
Asunto(s)
Cisteína/análisis , Peroxidasa de Rábano Silvestre/análisis , Peroxirredoxinas/análisis , Proteínas Recombinantes/análisis , Animales , Química Analítica , Clonación Molecular , Cisteína/química , Cisteína/metabolismo , Escherichia coli , Gammaproteobacteria , Expresión Génica , Histidina/genética , Histidina/metabolismo , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/genética , Peroxidasa de Rábano Silvestre/metabolismo , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Oligopéptidos/genética , Oligopéptidos/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , EspectrofotometríaRESUMEN
The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 10(7) and 10(6) M(-1) S(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie approximately 12.3 A apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.
Asunto(s)
Proteínas Bacterianas/química , Peróxido de Hidrógeno/química , Modelos Químicos , Modelos Moleculares , Peroxirredoxinas/química , Ácido Peroxinitroso/química , Xylella/enzimología , Proteínas Bacterianas/genética , Catálisis , Cristalografía por Rayos X , Disulfuros/química , Ditiotreitol/química , Cinética , Mutagénesis Sitio-Dirigida , Peroxirredoxinas/genética , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Xylella/genéticaRESUMEN
Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower K(M) for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK(a) of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK(a) values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 A, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity.
Asunto(s)
Glutarredoxinas/química , Isoenzimas/química , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Saccharomyces cerevisiae cytosolic thioredoxin peroxidase 1 (cTPxI or Tsa1) is a bifunctional enzyme with protective roles in cellular defence against oxidative and thermal stress that exhibits both peroxidase and chaperone activities. Protein overoxidation and/or high temperatures induce great changes in its quaternary structure and lead to its assembly into large complexes that possess chaperone activity. A recombinant mutant of Tsa1 from S. cerevisiae, with Cys47 substituted by serine, was overexpressed in Escherichia coli as a His(6)-tagged fusion protein and purified by nickel-affinity chromatography. Crystals were obtained from protein previously treated with 1,4-dithiothreitol by the hanging-drop vapour-diffusion method using PEG 3000 as precipitant and sodium fluoride as an additive. Diffraction data were collected to 2.8 A resolution using a synchrotron-radiation source. The crystal structure was solved by molecular-replacement methods and structure refinement is currently in progress.