RESUMEN
Transient/chronic microenvironmental hypoxia that exists within a majority of solid tumors has been suggested to have a profound influence on tumor growth and therapeutic outcome. Since the functions of novel antioxidant proteins, peroxiredoxin I (Prx I) and II, have been implicated in regulating cell proliferation, differentiation, and apoptosis, it was of our special interest to probe a possible role of Prx I and II in the context of hypoxic tumor microenvironment. Since both Prx I and II use thioredoxin (Trx) as an electron donor and Trx is a substrate for thioredoxin reductase (TrxR), we investigated the regulation of Trx and TrxR as well as Prx expression following hypoxia. Here we show a dynamic change of glutathione homeostasis in lung cancer A549 cells and an up-regulation of Prx I and Trx following hypoxia. Western blot analysis of 10 human lung cancer and paired normal lung tissues also revealed an elevated expression of Prx I and Trx proteins in lung cancer tissues. Immunohistochemical analysis of the lung cancer tissues confirmed an augmented Prx I and Trx expression in cancer cells with respect to the parenchymal cells in adjacent normal lung tissue. Based on these results, we suggest that the redox changes in lung tumor microenvironment could have acted as a trigger for the up-regulation of Prx I and Trx in lung cancer cells. Although the clinical significance of our finding awaits more rigorous future study, preferential augmentation of the Prx I and Trx in lung cancer cells may well represent an attempt of cancer cells to manipulate a dynamic redox change in tumor microenvironment in a manner that is beneficial for their proliferation and malignant progression.
Asunto(s)
Hipoxia , Neoplasias Pulmonares/patología , Peroxidasas/biosíntesis , Tiorredoxinas/biosíntesis , Antioxidantes/farmacología , Apoptosis , Western Blotting , Diferenciación Celular , División Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Electrones , Glutatión/metabolismo , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Peroxirredoxinas , ARN/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Reductasa de Tiorredoxina-Disulfuro/biosíntesis , Factores de Tiempo , Regulación hacia ArribaRESUMEN
The peroxiredoxins (Prx) are a family of 25 kDa peroxidases that can reduce H2O2 using an electron from thioredoxin (Trx) or other substances. The mammalian Prx family is divided into six groups (Prx I-VI) on the basis of homology of amino acid sequences. They are located in the cytosol and play a role in the cell signaling system. Previous reports have shown that Prx II has proliferative and anti-apoptotic properties and thus may induce carcinogenic changes. We conducted this study to reveal the change in expression of Prx in human breast cancer in comparison to normal tissues. Western immunoblotting using Prx type I, II and III antibodies was undertaken on 24 human breast cancer tissues and normal counterparts. We used antibodies against purified recombinant NKEF-A/PAG, NKEF-B and MER 5 which are the Prx isoforms. Type I Prx was overexpressed in the cancer tissues of 21 patients (87.5%), type II in 18 patients (75%) and type III in 19 patients (79.2%) in relation to normal tissue. However, no significant relationship was found between Prx overexpression and clinicopathological parameters of breast cancer such as tumor size, lymphatic invasiveness, hormone receptor status or nuclear and histologic grade. In conclusion, Prx is overexpressed in breast cancer tissues to a great extent suggesting that Prx has a proliferative effect and may be related to cancer development or progression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Peroxidasas/biosíntesis , Peroxidasas/química , Adulto , Anticuerpos/metabolismo , Western Blotting , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Peroxiredoxina VI , Peroxirredoxinas , Isoformas de Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
Six distinct peroxiredoxin (Prx) proteins (Prx I-VI) from distinct genes have been identified in mammalian tissues. Prxs are members of a group of peroxidases that have conserved reactive cysteine residue(s) in the active site(s). An immediate physiological electron donor for the peroxidase catalysis for five Prx proteins (Prx I-V) has been identified as thioredoxin (Trx), but that for Prx VI (1-Cys Prx) is still unclear. To identify an immediate electron donor and a binding protein for Prx VI, we performed a Prx VI protein overlay assay. A 20-kDa binding protein was identified by the Prx VI protein overlay assay with flow-through fractions from a High-Q column with rat lung crude extracts. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MS-Fit, we identified the 20-kDa Prx VI-binding protein as a cyclophilin A (CyP-A). The binding of recombinant human CyP-A (hCyP-A) to Prx VI was confirmed by using the hCyP-A protein overlay assay and Western immunoblot analysis with hCyP-A-specific antibodies. hCyP-A enhanced the antioxidant activity of Prx VI, as well as the other known mammalian Prx isotypes. hCyP-A supported antioxidant activity of Prx II and Prx VI both against thiol (dithiothreitol)-containing metal-catalyzed oxidation (MCO) systems and ascorbate-containing MCO systems. Prx II was reduced by hCyP-A without help from any other reductant, and the reduction was cyclosporin A-independent. These results strongly suggest that CyP-A not only binds to Prx proteins but also supports its peroxidase activity as an immediate electron donor. In addition, Cys(115) and Cys(161) of hCyP-A were found to be involved in the activation and the reduction of Prx.
Asunto(s)
Ciclofilina A/farmacología , Peroxidasas/metabolismo , Animales , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Sitios de Unión , Western Blotting , Catálisis , Ciclosporina/farmacología , Cisteína/química , Relación Dosis-Respuesta a Droga , Activación Enzimática , Escherichia coli/metabolismo , Humanos , Pulmón/metabolismo , Peroxiredoxina VI , Peroxirredoxinas , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Fracciones Subcelulares , Factores de TiempoRESUMEN
Macrophage migration inhibitory factor (MIF) is an important mediator that plays a central role in the control of the host immune and inflammatory response. To investigate the molecular mechanism of MIF action, we have used the yeast two-hybrid system and identified PAG, a thiol-specific antioxidant protein, as an interacting partner of MIF. Association of MIF with PAG was found in 293T cells transiently expressing MIF and PAG. The use of PAG mutants (C52S, C71S, and C173S) revealed that this association was significantly affected by C173S, but not C52S and C71S, indicating that a disulfide involving Cys(173) of PAG is responsible for the formation of MIF-PAG complex. In addition, the interaction was highly dependent on the reducing conditions such as dithiothreitol or beta-mercaptoethanol but not in the presence of H2O2. Analysis of the activities of the interacting proteins showed that the D-dopachrome tautomerase activity of MIF was decreased in a dose-dependent manner by coexpression of wild-type PAG, C52S, and C71S, whereas C173S was almost ineffective, suggesting that the direct interaction may be involved in the control of D-dopachrome tautomerase activity of MIF. Moreover, MIF has been shown to bind to PAG and it also inhibits the antioxidant activity of PAG.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/metabolismo , Proteínas de Neoplasias , Peroxidasas/metabolismo , Línea Celular , Humanos , Peroxiredoxina III , Peroxirredoxinas , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
Peroxiredoxins are a family of peroxidases that are ubiquitously and abundantly expressed in mammalian tissues; however, comparatively less is known about their expression in the skin. In this study, we examined the expression of three isotypes of peroxiredoxins (I-III) in rat skin. Western blot analyses showed strong expression of peroxiredoxins I-III in the epidermis and dermis of intact skin. Additionally, they were expressed in cultured rat keratinocytes and fibroblasts. Confocal image analyses revealed that peroxiredoxin II was present in the cytoplasm as a diffuse, reticulated pattern. In immunohistochemical staining of rat skin, peroxiredoxin expression was mainly localized to the epidermis, hair follicles, and sebaceous glands. In the epidermis, peroxiredoxins I and II were expressed in all layers with a gradient of increasing expression to the granular layer. In contrast, peroxiredoxin III was expressed in all layers with a gradient of expression decreasing to the granular layer. In the hair follicle, peroxiredoxins I-III were mainly expressed in the outer root sheath, except peroxiredoxin II, which was strongly expressed in the inner root sheath. In situ hybridization showed that mRNA expression was commensurate with the level of protein. Ultraviolet B radiation increased peroxiredoxin II expression in rat skin within 15 min after irradiation. From this study we conclude that peroxiredoxin isoforms are ubiquitously expressed in rat skin, and expression of at least peroxiredoxin II can be regulated by ultraviolet irradiation as a peroxidase in the skin. J Invest Dermatol 115:1108-1114 2000
Asunto(s)
Antioxidantes/metabolismo , Peroxidasas/biosíntesis , Piel/metabolismo , Animales , Especificidad de Anticuerpos , Fibroblastos/química , Folículo Piloso/metabolismo , Isotipos de Inmunoglobulinas/biosíntesis , Isotipos de Inmunoglobulinas/inmunología , Inmunohistoquímica , Queratinocitos/química , Masculino , Peroxidasas/genética , Peroxirredoxinas , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Piel/efectos de la radiación , Rayos UltravioletaRESUMEN
Human peroxiredoxin II (Prx II) has been known to function as an antioxidant enzyme in cells. Using head-and-neck cancer cell lines, we investigated whether Prx II expression is related to the resistance of cells to radiation therapy in vivo and in vitro, and whether a Prx II antisense serves as a radiosensitizer. Increased expression of Prx II was observed in tissues isolated from the patients who did not respond to radiation therapy, whereas Prx II expression was weak in tissues from the patients with regressed tumors. Enhanced expression of Prx II in UMSCC-11A (11A) cells was also observed after treatment with gamma radiation. This increased expression conferred radiation resistance to cancer cells because overexpression of Prx II protected 11A cells from radiation-induced cell death, suggesting that blocking Prx II expression could enhance radiation sensitivity. Treatment of 11A cells with a Prx II antisense decreased induction of Prx II, enhancing the radiation sensitivity. From these results, we suggest that stress-induced overexpression of Prx II increases radiation resistance via protection of cancer cells from radiation-induced oxidative cytolysis and that a Prx II antisense can be used as a radiosensitizer.
Asunto(s)
Antioxidantes/uso terapéutico , Oligonucleótidos Antisentido/uso terapéutico , Peroxidasas/uso terapéutico , Tolerancia a Radiación/efectos de los fármacos , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Northern Blotting , Muerte Celular/efectos de los fármacos , Terapia Combinada , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Immunoblotting , Peroxidasas/metabolismo , Peroxirredoxinas , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
A peroxidase from yeast that reduces H2O2 with the use of electrons provided by thioredoxin (Trx) together with homologs from a wide variety of species constitute the peroxiredoxin (Prx) family of proteins. Twelve mammalian Prx members have been previously identified in association with various cellular functions apparently unrelated to peroxidase activity. These mammalian proteins have now been divided into three distinct types, Prx I, II, and III, on the basis of their deduced amino acid sequences and immunological reactivity. With the use of recombinant proteins, Prx I, II, and III have now been shown to possess peroxidase activity and to rely on Trx as a source of reducing equivalents. None of the three proteins exhibited peroxidase activity in the presence of glutaredoxin. All three enzymes showed similar kinetic properties: the Vmax was 6-13 micromol/min per mg at 37 degrees C, the Km for Trx was 3-6 microM, and the Km for H2O2 was < 20 microM. Immunoblot analysis of various rat tissues and cultured cells indicated that most cell types contain the three Prx isoforms, the sum of which amounts to approximately 1-10 microg per milligram of soluble protein. Prx I and II are cytosolic proteins, whereas Prx IlI is localized in mitochondria. These results suggest that, together with glutathione peroxidase and catalase, Prx enzymes likely play an important role in eliminating peroxides generated during metabolism as well as during stimulation of cell surface receptors.
Asunto(s)
Hígado/metabolismo , Peroxidasas/química , Peroxidasas/metabolismo , Células 3T3 , Animales , Antioxidantes/metabolismo , Línea Celular , Clonación Molecular , Escherichia coli , Glutatión Peroxidasa/metabolismo , Células HeLa , Humanos , Cinética , Mamíferos , Ratones , Peroxidasas/aislamiento & purificación , Peroxirredoxinas , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoAsunto(s)
Mapeo Cromosómico , Peroxidasas/genética , Animales , Arteriosclerosis/genética , Cruzamientos Genéticos , Marcadores Genéticos , Peróxido de Hidrógeno/metabolismo , Isoenzimas/genética , Ratones , Estrés Oxidativo , Peroxirredoxinas , Especies Reactivas de Oxígeno/metabolismo , Recombinación GenéticaAsunto(s)
Antioxidantes/metabolismo , Peroxidasas/metabolismo , Peróxidos/metabolismo , Tiorredoxinas/metabolismo , Animales , Antioxidantes/química , Encéfalo/enzimología , Bovinos , Eritrocitos/enzimología , Humanos , Mitocondrias/enzimología , Peroxidasas/química , Peroxidasas/aislamiento & purificación , Peroxirredoxinas , Isoformas de Proteínas , RatasRESUMEN
Peroxiredoxins (Prxs) are a newly defined family of antioxidant proteins that have been implicated, via their antioxidant activity, in a number of cellular functions, including cell proliferation and differentiation, protection of other proteins from oxidative damage, and intracellular signaling. We isolated genomic DNA sequences of the type II Prx (Prx II) gene from the mouse and analyzed their molecular genetic characteristics. In the mouse, the Prx II is found to form a small multigene family with three members. One of them, the Prx II-1 gene, is actively transcribed in a variety of adult tissues as well as in the developing embryos to produce a 1.1-kb mRNA. The Prx II-1 gene consists of six exons and five introns, and the whole transcription unit occupies about 4.5 kb in the mouse genome. The other two genes, Prx II-2 and Prx II-3, are encoded by single exons, and show 97.5 and 87% of nucleotide sequence homology with the Prx II-1 gene, respectively. Structural features of these genes and the results of RT-PCR analysis on RNAs from various tissue sources indicate that the Prx II-2 and Prx II-3 genes could be pseudogenes derived from the Prx II-1 gene by a mechanism involving retrotransposition. These results strongly suggest that only the Prx II-1 gene might be relevant for studying the function of the Prx II gene in the murine system.
Asunto(s)
Genes/genética , Familia de Multigenes/genética , Peroxidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/química , ADN/genética , ADN Complementario/química , ADN Complementario/genética , Evolución Molecular , Exones/genética , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Intrones/genética , Ratones , Datos de Secuencia Molecular , Peroxirredoxinas , Fosfolipasas A/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Distribución TisularRESUMEN
Mammalian tissues express three immunologically distinct peroxiredoxin (Prx) proteins (Prx I, II, and III), which are the products of distinct genes. With the use of recombinant proteins Prx I, II, and III, all have now been shown to possess peroxidase activity and to rely on Trx as a source of reducing equivalents for the reduction of H2O2. Prx I and II are cytosolic proteins, whereas Prx III is localized in mitochondria. Transient overexpression of Prx I or II in cultured cells showed that they were able to eliminate the intracellular H2O2 generated in response to growth factors. Moreover, the activation of nuclear factor kappaB (NFkappaB) induced by extracellularly added H2O2 or tumor necrosis factor-alpha was blocked by overproduction of Prx II. These results suggest that, together with glutathione peroxidase and catalase, Prx enzymes likely play an important role in eliminating peroxides generated during metabolism. In addition, Prx I and II might participate in the signaling cascades of growth factors and tumor necrosis factor-alpha by regulating the intracellular concentration of H2O2.
Asunto(s)
Citocinas/farmacología , Peróxido de Hidrógeno/metabolismo , Isoenzimas/metabolismo , Proteínas de Neoplasias , Peroxidasas/metabolismo , Tiorredoxinas/metabolismo , Animales , Glutatión Reductasa/metabolismo , Sustancias de Crecimiento/farmacología , Células HeLa , Humanos , Isoenzimas/genética , Ratones , FN-kappa B/metabolismo , Oxidación-Reducción , Peroxidasas/genética , Peroxiredoxina III , Peroxirredoxinas , Proteínas , Ratas , Proteínas Recombinantes/metabolismo , Transducción de Señal , Especificidad de la Especie , Fracciones Subcelulares/enzimología , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The 29 kDa surface protein of Entamoeba histolytica is an abundant antigenic protein expressed by pathogenic strains of this organism. The protein is a member of a widely-dispersed group of homologues which includes at least two cysteinyl peroxidases, Salmonella typhimurium alkyl hydroperoxidase C-22 protein (AhpC) and Saccharomyces cerevisiae thiol-specific antioxidant protein (TSA). Here, for the first time in a pathogenic eukaryote, we have demonstrated that the amoebic protein also possesses peroxidatic and antioxidant activities in the presence of reductants such as dithiothreitol or thioredoxin reductase plus thioredoxin. Although the S. typhimurium AhpF flavoprotein was not an effective reductant of the amoebic TSA protein, one inhibitory monoclonal antibody directed toward amoebic TSA was also partially inhibitory toward reduced but not oxidized bacterial AhpC. These antioxidant proteins are likely to be important not only in general cell protection, but also in the promotion of infection and invasion by these pathogenic organisms through protection against oxidative attack by activated host phagocytic cells.
Asunto(s)
Antioxidantes/metabolismo , Entamoeba histolytica/enzimología , Peroxidasa/metabolismo , Peroxidasas , Proteínas/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Reacciones Cruzadas , Entamoeba histolytica/inmunología , Entamoeba histolytica/patogenicidad , Glutamato-Amoníaco Ligasa/efectos de los fármacos , Glutamato-Amoníaco Ligasa/metabolismo , Oxidación-Reducción/efectos de los fármacos , Oxidorreductasas/inmunología , Peroxirredoxinas , Proteínas/inmunología , Proteínas Protozoarias/inmunología , Salmonella typhimurium/enzimología , Salmonella typhimurium/inmunologíaRESUMEN
Reduction-oxidation (redox) plays a critical role in NF-kappaB activation. Diverse stimuli appear to utilize reactive oxygen species (e.g. hydrogen peroxide) as common effectors for activating NF-kappaB. Antioxidants govern intracellular redox status, and many such molecules can reduce H2O2. However, functionally, it does appear that different antioxidants are variously selective for redox regulation of certain transcription factors such as NF-kappaB. For NF-kappaB, thioredoxin has been described to be a more potent antioxidant than either glutathione or N-acetylcysteine. Thioredoxin peroxidase is the immediate enzyme that links reduction of H2O2 to thioredoxin. Several putative human thioredoxin peroxidases have been identified using recursive sequence searches/alignments with yeast or prokaryotic enzymes. None has been characterized in detail for intracellular function(s). Here, we describe a new human thioredoxin peroxidase, antioxidant enzyme AOE372, identified by virtue of its protein-protein interaction with the product of a proliferation association gene, pag, which is also a thiol-specific antioxidant. In human cells, AOE372 defines a redox pathway that specifically regulates NF-kappaB activity via a modulation of IkappaB-alpha phosphorylation in the cytoplasm. We show that AOE372 activity is regulated through either homo- or heterodimerization with other thiol peroxidases, implicating subunit assortment as a mechanism for regulating antioxidant specificities. AOE372 function suggests thioredoxin peroxidase as an immediate regulator of H2O2-mediated activation of NF-kappaB.
Asunto(s)
Proteínas I-kappa B , FN-kappa B/metabolismo , Peroxidasas , Proteínas/fisiología , Secuencia de Aminoácidos , Antioxidantes/metabolismo , Secuencia de Bases , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Dimerización , Infecciones por VIH/enzimología , VIH-1 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Datos de Secuencia Molecular , Peso Molecular , Inhibidor NF-kappaB alfa , Proteínas de Neoplasias/metabolismo , Oxidación-Reducción , Peroxiredoxina III , Peroxirredoxinas , Fosforilación , Unión Proteica , Proteínas/química , Proteínas/genética , ARN Mensajero/biosíntesis , Distribución TisularRESUMEN
The thiol-specific antioxidant protein (TSA) protects glutamine synthetase from inactivation by a metal-catalyzed oxidation (MCO) system comprised of dithiothreitol (DTT)/Fe3+/O2 but not by the ascorbate/Fe3+/O2 MCO system. The removal of sulfur-centered radicals or H2O2 has been proposed as the protective mechanism of TSA. Like catalase, TSA prevents the initiation of the rapid O2 uptake phase during MCO of DTT but causes only partial inhibition when added after the reaction is well into the propagation phase. Stoichiometric studies showed that the antioxidant property of TSA is, at least in part, due to its ability to catalyze the destruction of H2O2 by the overall reaction 2 RSH + H2O2 --> RSSR + H2O. Results of kinetic studies demonstrate that the removal of H2O2 by TSA correlates with its ability to protect glutamine synthetase from inactivation. In the presence of thioredoxin, TSA is more active, whereas C170S (an active mutant of TSA in which cysteine 170 was replaced by a serine) and open reading frame 6 (a human antioxidant protein homologous to TSA with only one conserved cysteine residue) are only slightly affected. The thiol specificity of the protective activity of TSA derives from the fact that the oxidized form of TSA can be converted back to its sulfhydryl form by treatment with thiols but not by ascorbate.
Asunto(s)
Antioxidantes/metabolismo , Peróxido de Hidrógeno/metabolismo , Peroxidasas/metabolismo , Proteínas/metabolismo , Catalasa/metabolismo , Sistema Libre de Células , Ditiotreitol/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Hierro/metabolismo , Oxidación-Reducción , Peroxirredoxinas , Saccharomyces cerevisiae/enzimología , Relación Estructura-Actividad , Tiorredoxinas/metabolismoRESUMEN
Reactive oxygen species and free radicals that are produced during normal metabolism can potentially damage cellular macromolecules. Defenses against such damage include a number of antioxidant enzymes that specifically target the removal or dismutation of the reactive agent. We report here the isolation and regional mapping of a human gene, TDPX1, that encodes an enzyme homologous to a yeast thioredoxin-dependent peroxide reductase (thioredoxin peroxidase, TPX). The human TDPX1 coding sequence was determined from the product of a polymerase chain reaction (PCR) amplification of human cDNA. Based on PCR analysis of DNA from a human/rodent somatic cell hybrid panel, the TDPX1 locus was assigned to chromosome 13. Further localization of the locus to 13q12 was accomplished by fluorescence in situ hybridization analysis, using as a probe DNA from a yeast artificial chromosome (YAC) that contains the TDPX1 gene. It was also determined by PCR analysis of various YACs that the TDPX1 locus is in the region of the dinucleotide repeat markers D13S289 and D13S290. This regional mapping localizes the TDPX1 gene to a genomic region recently shown to contain the breast cancer susceptibility gene BRCA2 and a gene associated with a form of muscular dystrophy. Oxygen radical metabolism has been hypothesized to be important for cancer, muscular dystrophy, and other disorders, so TDPX1 should be considered a candidate gene for these diseases.
Asunto(s)
Cromosomas Humanos Par 13 , Saccharomyces cerevisiae/genética , Reductasa de Tiorredoxina-Disulfuro/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Mapeo Cromosómico , Cricetinae , Cartilla de ADN , Humanos , Células Híbridas , Ratones , Datos de Secuencia Molecular , Saccharomyces cerevisiae/enzimologíaAsunto(s)
Antioxidantes/metabolismo , Peroxidasas , Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Clonación Molecular , Cisteína , Glutamato-Amoníaco Ligasa/metabolismo , Mamíferos , Peroxirredoxinas , Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
A 25-kDa antioxidant enzyme that provides protection against oxidation systems capable of generating reactive oxygen and sulfur species has previously been identified. The nature of the oxidant eliminated by, and the physiological source of reducing equivalents for, this enzyme, however, were not known. The 25-kDa enzyme is now shown to be a peroxidase that reduces H2O2 and alkyl hydroperoxides with the use of hydrogens provided by thioredoxin, thioredoxin reductase, and NADPH. This protein is the first peroxidase to be identified that uses thioredoxin as the immediate hydrogen donor and is thus named thioredoxin peroxidase (TPx). TPx exists as a dimer of identical 25-kDa subunits that contain 2 cysteine residues, Cys47 and Cys170. Cys47-SH appears to be the site of oxidation by peroxides, and the oxidized Cys47 probably reacts with Cys170-SH of the other subunit to form an intermolecular disulfide. Mutant TPx proteins lacking either Cys47 or Cys170, therefore, do not exhibit thioredoxin-coupled peroxidase activity. The TPx disulfide is specifically reduced by thioredoxin, but can also be reduced (less effectively) by a small molecular size thiol. The Saccharomyces cerevisiae thioredoxin reductase gene was also cloned and sequenced, and the deduced amino sequence was shown to be 51% identical with that of the Escherichia coli enzyme.
Asunto(s)
Proteínas Fúngicas/metabolismo , Genes Fúngicos , Proteínas de Neoplasias , Peroxidasas/metabolismo , Proteínas/metabolismo , Saccharomyces cerevisiae/enzimología , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Antioxidantes/química , Ácido Ascórbico/química , Secuencia de Bases , Clonación Molecular , Cisteína/química , Ditiotreitol/química , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , NADP/metabolismo , Oxidación-Reducción , Peroxirredoxinas , Conformación Proteica , Relación Estructura-ActividadRESUMEN
A cDNA corresponding to a thiol-specific antioxidant enzyme (TSA) was isolated from a rat brain cDNA library with the use of antibodies to bovine TSA. The cDNA clone encoded an open reading frame capable of encoding a 198-residue polypeptide. The rat and yeast TSA proteins show significant sequence homology to the 21-kDa component (AhpC) of Salmonella typhimurium alkyl hydroperoxide reductase, and we have found that AhpC exhibits TSA activity. AhpC and TSA define a family of > 25 different proteins present in organisms from all kingdoms. The similarity among the family members extends over the entire sequence and ranges between 23% and 98% identity. A majority of the members of the AhpC/TSA family contain two conserved cysteines. At least eight of the genes encoding AhpC/TSA-like polypeptides are found in proximity to genes encoding other oxidoreductase activities, and the expression of several of the homologs has been correlated with pathogenicity. We suggest that the AhpC/TSA family represents a widely distributed class of antioxidant enzymes. We also report that a second family of proteins, defined by the 57-kDa component (AhpF) of alkyl hydroperoxide reductase and by thioredoxin reductase, has expanded to include six additional members.
Asunto(s)
Antioxidantes/metabolismo , Encéfalo/enzimología , Oxidorreductasas/metabolismo , Peroxidasas , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Antioxidantes/química , Secuencia de Bases , Bovinos , Clonación Molecular , ADN , Humanos , Datos de Secuencia Molecular , Oxidorreductasas/química , Peroxirredoxinas , Proteínas/química , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Thiol-specific antioxidant (TSA) from yeast contains cysteine residues at amino acid positions 47 and 170 but is not associated with obvious redox cofactors. These two cysteines are highly conserved in a family of proteins that exhibit sequence identity of 23-98% with TSA. The roles of Cys-47 and Cys-170 in yeast TSA were investigated by replacing them individually with serine and expressing the mutant TSA proteins (RC47S and RC170S, respectively), as well as wild-type TSA (RWT), in Escherichia coli. Wild-type TSA purified from yeast (YWT) and RWT were both shown to exist predominantly as dimers, whereas RC47S and RC170S existed mainly as monomers under a denaturing condition. This observation suggests that the dimerization of YWT and RWT requires disulfide linkage of Cys-47 and Cys-170. The presence of the Cys-47-Cys-170 linkage in YWT was directly shown by isolation of dimeric tryptic peptides, one monomer of which contained Cys-47 and the other contained Cys-170. A small percentage of YWT, RWT, RC47S, and RC170S molecules formed dimers linked by Cys-47-Cys-47 or Cys-170-Cys-170 disulfide bonds. The antioxidant activity of the various TSA proteins was evaluated from their ability to protect glutamine synthetase against the dithiothreitol/Fe3+/O2 oxidation system. YWT, RWT, and RC170S were equally protective, whereas RC47S was completely ineffective. Thus, Cys-47, but not Cys-170, constitutes the site of oxidation by putative substrate.