RESUMEN
Sestrin2 (SESN2) is a highly evolutionary conserved protein and involved in different cellular responses to various stresses. However, the potential function of SESN2 in immune system remains unclear. The present study was designed to test whether dendritic cells (DCs) could express SESN2, and investigate the underlying molecular mechanism as well as its potential significance. Herein, we firstly reported that SESN2 was expressed in DCs after high mobility group box-1 protein (HMGB1) stimulation and the apoptosis of DCs was obviously increased when SESN2 gene silenced by siRNA. Cells undergone SESN2-knockdown promoted endoplasmic reticulum (ER) stress (ERS)-related cell death, markedly exacerbated ER disruption as well as the formation of dilated and aggregated structures, and they significantly aggravated the extent of ERS response. Conversely, overexpressing SESN2 DCs markedly decreased apoptotic rates and attenuated HMGB1-induced ER morphology fragment together with inhibition of ERS-related protein translation. Furthermore, sesn2-/--deficient mice manifested increased DC apoptosis and aggravated ERS extent in septic model. These results indicate that SESN2 appears to be a potential regulator to inhibit apoptotic ERS signaling that exerts a protective effect on apoptosis of DCs in the setting of septic challenge.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteína HMGB1/farmacología , Peroxidasas/metabolismo , Sepsis/metabolismo , Animales , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Peroxidasas/deficiencia , Peroxidasas/genética , Sepsis/genética , Sepsis/microbiología , Sepsis/patología , Transducción de Señal , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Peroxidasin (PXDN) is a unique peroxidase containing extracellular matrix motifs and stabilizes collagen IV networks by forming sulfilimine crosslinks. PXDN gene knockout in Caenorhabditis elegans (C. elegans) and Drosophila results in the demise at the embryonic and larval stages. PXDN mutations lead to severe eye disorders, including microphthalmia, cataract, glaucoma, and anterior segment dysgenesis in humans and mice. To investigate how PXDN loss of function affects organ development, we generated Pxdn knockout mice by deletion of exon 1 and its 5' upstream sequences of the Pxdn gene using the CRISPR/Cas9 system. Loss of both PXDN expression and collagen IV sulfilimine cross-links was detected only in the homozygous mice, which showed completely or almost closed eyelids with small eyes, having no apparent external morphological defects in other organs. In histological analysis of eye tissues, the homozygous mice had extreme defects in eye development, including no eyeballs or drastically disorganized eye structures, whereas the heterozygous mice showed normal eye structure. Visual function tests also revealed no obvious functional abnormalities in the eyes between heterozygous mice and wild-type mice. Thus, these results suggest that PXDN activity is essential in eye development, and also indicate that a single allele of Pxdn gene is sufficient for eye-structure formation and normal visual function.
Asunto(s)
Anoftalmos , Ojo/crecimiento & desarrollo , Eliminación de Gen , Peroxidasas/deficiencia , Animales , Anoftalmos/genética , Anoftalmos/metabolismo , Anoftalmos/patología , Sistemas CRISPR-Cas , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Ojo/patología , Ratones , Ratones Noqueados , Peroxidasas/metabolismo , Visión Ocular/genéticaRESUMEN
Previous studies have demonstrated that NADPH oxidase (NOX)/vascular peroxidase (VPO1) pathway - mediated oxidative stress plays an important role in the pathogenesis of multiple cardiovascular diseases. This study aims to evaluate the correlation between NOX/VPO1 pathway and endothelial progenitor cells (EPCs) dysfunctions in hypoxia-induced pulmonary hypertension (PH). The rats were exposed to 10% hypoxia for 3 weeks to establish a PH model, which showed increases in right ventricle systolic pressure, right ventricular and pulmonary vascular remodeling, acceleration in apoptosis and impairment in functions of the peripheral blood derived - EPCs (the reduced abilities in adhesion, migration and tube formation), accompanied by up-regulation of NOX (NOX2 and NOX4) and VPO1. Next, normal EPCs were cultured under hypoxia to induce apoptosis in vitro. Consistent with the in vivo findings, hypoxia enhanced the apoptosis and dysfunctions of EPCs concomitant with an increase in NOX and VPO1 expression, hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production; these phenomena were attenuated by NOX2 or NOX4 siRNA. Knockdown of VPO1 showed similar results to that of NOX siRNA except no effect on NOX expression and H2O2 production. Based on these observations, we conclude that NOX/VPO1 pathway-derived reactive oxygen species promote the oxidative injury and dysfunctions of EPCs in PH, which may contribute to endothelial dysfunctions in PH.
Asunto(s)
Células Progenitoras Endoteliales/patología , Hemoproteínas/metabolismo , Hipertensión Pulmonar/enzimología , Hipertensión Pulmonar/patología , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 4/metabolismo , Peroxidasas/metabolismo , Animales , Apoptosis , Hipoxia de la Célula , Técnicas de Silenciamiento del Gen , Hemoproteínas/deficiencia , Hemoproteínas/genética , Hipertensión Pulmonar/genética , Masculino , NADPH Oxidasa 2/deficiencia , NADPH Oxidasa 2/genética , NADPH Oxidasa 4/deficiencia , NADPH Oxidasa 4/genética , Peroxidasas/deficiencia , Peroxidasas/genética , Fenotipo , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Doxorubicin is a chemotherapy medication widely used to treat a variety of cancers. Even though it offers one of the most effective anti-cancer treatments, its clinical use is limited because of its strong cardiotoxicity that can lead to fatal conditions. Here, we show that sestrin 1 and sestrin 2, members of the sestrin family of proteins that are stress-inducible regulators of metabolism, are critical for suppressing doxorubicin cardiotoxicity and coordinating the AMPK-mammalian target of rapamycin complex 1 (mTORC1) autophagy signaling network for cardioprotection. Expression of both sestrin 1 and sestrin 2 was highly increased in the mouse heart after doxorubicin injection. Genetic ablation of sestrin 1 and sestrin 2 rendered mice more vulnerable to doxorubicin and exacerbated doxorubicin-induced cardiac pathologies including cardiomyocyte apoptosis and cardiac dysfunction. These pathologies were associated with strong dysregulation of the cardiac signaling network, including suppression of the AMPK pathway and activation of the mTORC1 pathway. Consistent with AMPK downregulation and mTORC1 upregulation, autophagic activity of heart tissue was diminished, leading to prominent accumulation of autophagy substrate, p62/SQSTM1. Taken together, our results indicate that sestrin 1 and sestrin 2 are important cardioprotective proteins that coordinate metabolic signaling pathways and autophagy to minimize cardiac damage in response to doxorubicin insult. Augmenting this protective mechanism could provide a novel therapeutic rationale for prevention and treatment of doxorubicin cardiotoxicity. NEW & NOTEWORTHY Doxorubicin is a highly efficient chemotherapeutic medicine; however, its use is limited because of its strong cardiotoxicity. Here, we show that sestrin 1 and sestrin 2 are critical protectors of cardiomyocytes from doxorubicin damage. By upregulating AMPK and autophagic activities and suppressing mammalian target of rapamycin complex 1 and oxidative stress, sestrins counteract detrimental effects of doxorubicin on cardiomyocytes. Correspondingly, loss of sestrin 1 and sestrin 2 produced remarkable dysregulation of these pathways, leading to prominent cardiac cell death and deterioration of heart function.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Doxorrubicina/toxicidad , Cardiopatías/prevención & control , Miocitos Cardíacos/metabolismo , Peroxidasas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Apoptosis , Autofagia , Cardiotoxicidad , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Modelos Animales de Enfermedad , Cardiopatías/metabolismo , Cardiopatías/patología , Cardiopatías/fisiopatología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/patología , Estrés Oxidativo , Peroxidasas/deficiencia , Peroxidasas/genética , Proteína Sequestosoma-1/metabolismo , Transducción de SeñalRESUMEN
Renal fibrosis is the pathological hallmark of chronic kidney disease (CKD) and manifests as glomerulosclerosis and tubulointerstitial fibrosis. Reactive oxygen species contribute significantly to renal inflammation and fibrosis, but most research has focused on superoxide and hydrogen peroxide (H2O2). The animal heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPX), and peroxidasin (PXDN) uniquely metabolize H2O2 into highly reactive and destructive hypohalous acids, such as hypobromous and hypochlorous acid. However, the role of these peroxidases and their downstream hypohalous acids in the pathogenesis of renal fibrosis is unclear. Our study defines the contribution of MPO, EPX, and PXDN to renal inflammation and tubulointerstitial fibrosis in the murine unilateral ureteral obstruction (UUO) model. Using a nonspecific inhibitor of animal heme peroxidases and peroxidase-specific knockout mice, we find that loss of EPX or PXDN, but not MPO, reduces renal fibrosis. Furthermore, we demonstrate that eosinophils, the source of EPX, accumulate in the renal interstitium after UUO. These findings point to EPX and PXDN as potential therapeutic targets for renal fibrosis and CKD and suggest that eosinophils modulate the response to renal injury.
Asunto(s)
Peroxidasa del Eosinófilo/metabolismo , Eosinófilos/enzimología , Proteínas de la Matriz Extracelular/metabolismo , Riñón/enzimología , Nefritis Intersticial/enzimología , Peroxidasa/metabolismo , Peroxidasas/metabolismo , Obstrucción Ureteral/enzimología , Animales , Movimiento Celular , Modelos Animales de Enfermedad , Peroxidasa del Eosinófilo/deficiencia , Peroxidasa del Eosinófilo/genética , Eosinófilos/patología , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Femenino , Fibrosis , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis Intersticial/etiología , Nefritis Intersticial/patología , Nefritis Intersticial/prevención & control , Peroxidasa/deficiencia , Peroxidasa/genética , Peroxidasas/deficiencia , Peroxidasas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/patología , PeroxidasinaRESUMEN
Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.
Asunto(s)
Adaptación Fisiológica/genética , Aneuploidia , Deleción Cromosómica , Cromosomas Fúngicos/genética , Transporte de Electrón/fisiología , Proteínas Mitocondriales/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Antimicina A/farmacología , Citocromo-c Peroxidasa/genética , Citocromo-c Peroxidasa/fisiología , Eliminación de Gen , Dosificación de Gen , Genes Fúngicos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiología , Peróxido de Hidrógeno/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas Mitocondriales/genética , Oligomicinas/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/fisiología , Peroxidasas/deficiencia , Peroxidasas/genética , Especies Reactivas de Oxígeno/metabolismo , Rotenona/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.
Asunto(s)
Aptitud Genética , Mutación/genética , Peroxidasas/deficiencia , Peroxidasas/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Daño del ADN/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Mutación INDEL/genética , Tasa de Mutación , Fenotipo , Mutación Puntual/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Transcriptoma/genéticaRESUMEN
Reactive oxygen species and auxin play important roles in the networks that regulate plant development and morphogenetic changes. However, the molecular mechanisms underlying the interactions between them are poorly understood. This study isolated a mas (More Axillary Shoots) mutant, which was identified as an allele of the mitochondrial AAA-protease AtFtSH4, and characterized the function of the FtSH4 gene in regulating plant development by mediating the peroxidase-dependent interplay between hydrogen peroxide (H2O2) and auxin homeostasis. The phenotypes of dwarfism and increased axillary branches observed in the mas (renamed as ftsh4-4) mutant result from a decrease in the IAA concentration. The expression levels of several auxin signaling genes, including IAA1, IAA2, and IAA3, as well as several auxin binding and transport genes, decreased significantly in ftsh4-4 plants. However, the H2O2 and peroxidases levels, which also have IAA oxidase activity, were significantly elevated in ftsh4-4 plants. The ftsh4-4 phenotypes could be reversed by expressing the iaaM gene or by knocking down the peroxidase genes PRX34 and PRX33. Both approaches can increase auxin levels in the ftsh4-4 mutant. Taken together, these results provided direct molecular and genetic evidence for the interaction between mitochondrial ATP-dependent protease, H2O2, and auxin homeostasis to regulate plant growth and development.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Homeostasis , Ácidos Indolacéticos/metabolismo , Metaloproteasas/genética , Mitocondrias/genética , Peroxidasas/metabolismo , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Técnicas de Silenciamiento del Gen , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Mutación , Peroxidasas/deficiencia , Peroxidasas/genética , FenotipoRESUMEN
Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H(2)O(2) response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H(2)O(2). Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H(2)O(2). The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H(2)O(2) and other cellular proteins plays a secondary role.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Peroxidasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transducción de Señal/efectos de los fármacos , Secuencia de Bases , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/genética , Peroxidasas/deficiencia , Fenotipo , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Transducción de Señal/fisiologíaRESUMEN
Resistance of Listeria monocytogenes to reactive oxygen radicals may facilitate its survival in phagocytic cells and against some oxidizing sanitizers. The aim of this study was to investigate the function of the 2-cys peroxiredoxin (Prx) homologue in L. monocytogenes, particularly its survival in a hydrogen peroxide-containing environment. An in-frame prx deletion mutant and a complementation strain were constructed and evaluated for their growth and survival either in media containing different concentrations (0, 15, 20, 25, 50, and 294 mmol x L(-1)) hydrogen peroxide or in macrophages. Bacterial survival in various mouse organs was also investigated after intraperitoneal administration. We found that prx-defective L. monocytogenes was sensitive to hydrogen peroxide in in vitro growth media but not in mouse organs or in macrophages, suggesting that Prx promotes survival in the presence of exogenous hydrogen peroxide but not in mammalian cells or organs.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Listeria monocytogenes/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Peroxidasas/genética , Animales , Prueba de Complementación Genética , Listeria monocytogenes/genética , Listeria monocytogenes/crecimiento & desarrollo , Listeriosis/microbiología , Hígado/microbiología , Macrófagos Peritoneales/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana/genética , Peroxidasas/deficiencia , Peroxirredoxinas , Eliminación de Secuencia , Bazo/microbiología , Factores de TiempoRESUMEN
Mammalian 2-Cys peroxiredoxin II (Prx II) is a cellular peroxidase that eliminates endogenous H(2)O(2). The involvement of Prx II in the regulation of lipopolysaccharide (LPS) signaling is poorly understood. In this report, we show that LPS induces substantially enhanced inflammatory events, which include the signaling molecules nuclear factor kappaB and mitogen-activated protein kinase (MAPK), in Prx II-deficient macrophages. This effect of LPS was mediated by the robust up-regulation of the reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and the phosphorylation of p47(phox). Furthermore, challenge with LPS induced greater sensitivity to LPS-induced lethal shock in Prx II-deficient mice than in wild-type mice. Intravenous injection of Prx II-deficient mice with the adenovirus-encoding Prx II gene significantly rescued mice from LPS-induced lethal shock as compared with the injection of a control virus. The administration of catalase mimicked the reversal effects of Prx II on LPS-induced inflammatory responses in Prx II-deficient cells, which suggests that intracellular H(2)O(2) is attributable, at least in part, to the enhanced sensitivity to LPS. These results indicate that Prx II is an essential negative regulator of LPS-induced inflammatory signaling through modulation of ROS synthesis via NADPH oxidase activities and, therefore, is crucial for the prevention of excessive host responses to microbial products.
Asunto(s)
Endotoxinas/inmunología , Lipopolisacáridos/toxicidad , Peroxidasas/fisiología , Choque/inmunología , Choque/prevención & control , Animales , Línea Celular , Células Cultivadas , Inflamación/genética , Inflamación/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peroxidasas/deficiencia , Peroxidasas/genética , Peroxirredoxinas , Choque/genética , Choque/mortalidad , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Mice with knock-out of peroxiredoxin 6 (Prdx6), a recently described antioxidant enzyme, were evaluated for susceptibility to lung injury with paraquat (PQ) administration. With high dose PQ (30 mg/kg i.p.), all Prdx6-/- mice died (LT50 54 +/- 2.05 h, mean +/- SE) by 4 days, whereas 86% of the wild-type (WT) mice (C57BL/6) survived (n = 14). At 2 days after PQ, lung wet/dry weight ratio increased significantly (p < 0.05) to 7.57 +/- 0.37 in Prdx6-/- mice vs. 5.42 +/- 0.25 in WT mice. Total protein and nucleated cells in bronchoalveolar lavage fluid and TBARS and protein carbonyls in lung homogenate also showed more marked increases in Prdx6-/- mice. At 2.5 days after PQ, light microscopy of WT lungs showed mild injury while Prdx6-/- lungs showed epithelial cell necrosis, perivascular edema, and inflammatory cells. With low dose PQ (12.5 mg/kg), mortality and lung injury were less marked but were significantly greater with Prdx6-/- compared to WT mice. These results show that Prdx6-/- mice have increased susceptibility to lung injury with PQ administration. Thus, Prdx6 protects lungs against PQ toxicity as shown previously for hyperoxia, indicating that it functions as an important lung antioxidant enzyme.
Asunto(s)
Lesión Pulmonar , Estrés Oxidativo/efectos de los fármacos , Paraquat/toxicidad , Peroxidasas/genética , Animales , Líquido del Lavado Bronquioalveolar/química , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis , Peroxidasas/deficiencia , Peroxiredoxina VI , Peroxirredoxinas , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
Peroxiredoxin I (Prx-I), a key member of the peroxiredoxin family, reduces peroxides and equivalents through the thioredoxin system. Our previous work has shown that expression of Prx-I in mammalian cells increases following ionizing radiation (IR), indicating that Prx-I actively responds to IR-induced reactive oxygen species (ROS) and suggesting that Prx-I plays an important role in protecting cells from IR-induced death. To test this hypothesis, we suppressed the expression of Prx-I in SW480 cells by RNA interference. Our results show that IR induces the expression of Prx-I in SW480 cells in a dose- and time-dependent manner. The recombinant siRNA vector targeting Prx-I dramatically reduced the expression of Prx-I in SW480 cells. When Prx-I was knocked down in SW480 cells, the cells exhibited a decreased growth rate, a reduced antioxidant capability following IR and became more sensitive to IR-induced apoptosis. Together, our results demonstrate that Prx-I plays an important role in protecting cells from IR-induced cell death, which might be through scavenging IR-induced ROS in the cells.
Asunto(s)
Apoptosis/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Peroxidasas/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta en la Radiación , Silenciador del Gen , Proteínas de Choque Térmico/deficiencia , Proteínas de Choque Térmico/genética , Humanos , Peroxidasas/deficiencia , Peroxidasas/genética , Peroxirredoxinas , Dosis de RadiaciónRESUMEN
Platelet-derived growth factor (PDGF) is a potent mitogenic and migratory factor that regulates the tyrosine phosphorylation of a variety of signalling proteins via intracellular production of H2O2 (refs 1, 2-3). Mammalian 2-Cys peroxiredoxin type II (Prx II; gene symbol Prdx2) is a cellular peroxidase that eliminates endogenous H2O2 produced in response to growth factors such as PDGF and epidermal growth factor; however, its involvement in growth factor signalling is largely unknown. Here we show that Prx II is a negative regulator of PDGF signalling. Prx II deficiency results in increased production of H2O2, enhanced activation of PDGF receptor (PDGFR) and phospholipase Cgamma1, and subsequently increased cell proliferation and migration in response to PDGF. These responses are suppressed by expression of wild-type Prx II, but not an inactive mutant. Notably, Prx II is recruited to PDGFR upon PDGF stimulation, and suppresses protein tyrosine phosphatase inactivation. Prx II also leads to the suppression of PDGFR activation in primary culture and a murine restenosis model, including PDGF-dependent neointimal thickening of vascular smooth muscle cells. These results demonstrate a localized role for endogenous H2O2 in PDGF signalling, and indicate a biological function of Prx II in cardiovascular disease.
Asunto(s)
Neovascularización Fisiológica/efectos de los fármacos , Peroxidasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Aorta/citología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Células Cultivadas , Reestenosis Coronaria/metabolismo , Reestenosis Coronaria/patología , Activación Enzimática , Humanos , Ratones , Miocitos del Músculo Liso/citología , Peroxidasas/deficiencia , Peroxidasas/genética , Peroxirredoxinas , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Unión Proteica , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
PRDX6, a member of the peroxiredoxins (PRDXs) family, is a key player in the removal of reactive oxygen species (ROS). Using targeted inactivation of the Prdx6 gene, we present evidence that the corresponding protein offsets the deleterious effects of ROS on lens epithelial cells (LECs) and regulates gene expression by limiting its levels. PRDX6-depleted LECs displayed phenotypic alterations and elevated alpha-smooth muscle actin and betaig-h3 expression (markers for cataractogenesis), indistinguishable from transforming growth factor beta (TGFbeta)-induced changes. Biochemical assays disclosed enhanced levels of ROS, as well as high expression and activation of TGFbeta1 in Prdx6-/- LECs. A CAT assay revealed transcriptional repression of lens epithelium-derived growth factor (LEDGF), HSP27, and alphaB-crystallin promoter activities in these cells. A gel mobility shift assay demonstrated the attenuation of LEDGF binding to heat shock or stress response elements present in these genes. A supply of PRDX6 toPrdx6-/- LECs reversed these changes. Based on the above data, we propose a rheostat role for PRDX6 in regulating gene expression by controlling the ROS level to maintain cellular homeostasis.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Homeostasis/efectos de los fármacos , Cristalino/anomalías , Cristalino/metabolismo , Peroxidasas/deficiencia , Especies Reactivas de Oxígeno/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Apoptosis , Movimiento Celular , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cristalino/efectos de los fármacos , Cristalino/enzimología , Ratones , Estrés Oxidativo/efectos de los fármacos , Peroxidasas/genética , Peroxidasas/metabolismo , Peroxiredoxina VI , Peroxirredoxinas , Fenotipo , Transcripción Genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/farmacología , Cadena B de alfa-Cristalina/genéticaRESUMEN
Peroxidases figure prominently in biology and contribute significantly to cell biology, host defense against infection, and pathogenesis of several inflammatory diseases. These varied and diverse aspects of peroxidase biochemistry and its clinical implications will be the subjects of in-depth analysis at the 4th International peroxidase meeting held in Kyoto. Specific topics range from the molecular basis of peroxidase structure and function to the clinical consequences of autoantibodies generated against myeloperoxidase (MPO), the peroxidase present in circulating neutrophils. Consideration of novel aspects of peroxidase biology, both unanticipated biochemical properties of MPO and the potential role of MPO in the pathogenesis of inflammatory diseases such as atherosclerosis, will also be included. In addition to peroxidases, the newly expanded family of NADPH oxidases will be discussed. We hope that this collection of scientists who share a common interest in peroxidase biology but each possess expertise in distinctly different aspects of the subject will provide a setting for spirited discussion and a lively exchange of views to yield advances in understanding and to create new applications of those insights to benefit clinical medicine, agriculture and industry.
Asunto(s)
Peroxidasas/inmunología , Animales , Humanos , Peroxidasas/deficienciaRESUMEN
Peroxiredoxin 6 (Prdx6; also called antioxidant protein 2, or Aop2) is a candidate gene for Ath1, a locus responsible for the respective susceptibility and resistance of mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) to diet-induced atherosclerosis. To evaluate if Prdx6 underlies Ath1, we compared the diet-induced atherosclerotic lesions in Prdx6 targeted mutant (Prdx6-/-) mice of different genetic backgrounds: B6, 129, and B6;129. PRDX6 protein and mRNA were expressed in normal and atherosclerotic aortas. B6;129 Prdx6-/- macrophages oxidized LDL significantly more than did controls. Plasma lipid hydroperoxide levels were higher in atherogenic diet-fed Prdx6-/- mice with B6;129 and B6 backgrounds than in controls. Prdx6-/- and controls in a 129 genetic background were equally lesion-resistant, and Prdx6-/- and controls in a B6 background were equally lesion-susceptible. In contrast, Prdx6-/- mice in a B6;129 background had significantly larger aortic root lesions than did littermate wild type controls. Therefore, although PRDX6 protein did not affect atherosclerosis susceptibility in either the resistant 129 background or the susceptible B6 background, it may inhibit atherosclerosis in backgrounds with mixed pro- and anti-atherogenic genes. Thus, genetic background plays an important role in modulating atherogenesis in targeted mutant mice. However, we think it is unlikely that Prdx6 underlies Ath1.
Asunto(s)
Arteriosclerosis/genética , Peroxidasas/deficiencia , Animales , Arteriosclerosis/sangre , Predisposición Genética a la Enfermedad , Ratones , Peroxiredoxina VI , PeroxirredoxinasRESUMEN
In the thymus, CD4+ or CD8+ single-positive (SP) thymocytes develop and mature by positive and negative selection or undergo "death by neglect". CD4+ or CD8+ SP then circulate to other lymphoid tissues. We have investigated the role of reactive oxygen species (ROS) in thymocyte development using peroxiredoxin II (PrxII)-null mice. The level ofROS in PrxII-null thymocytes is higher than that in wild-type mice. Deletion of the PrxII gene leads to enlargement of the thymus in young (9 weeks) and old (64 weeks) mice. The increased number ofthymocytes in PrxII-null thymus is related to reduced hypodiploid cell formation. For mice on a normal diet, the ratio of SP to double-positive (DP) thymocytes in thymus of PrxII-null mice is lower than that in wild-type mice. After food restriction, which leads to increased ROS production, this ratio becomes much higher in PrxII-null thymus. The amount of apoptosis, induced by food restriction orby the injection of dexamethasone, is consistently lower in PrxII-null thymocytes than in wild-type thymocytes. In the presence of low serum concentrations, PrxII-deleted T cells proliferate more vigorously after stimulation with concanavalin A. Phytohemagglutinin- or OKT3-stimulated proliferation of human peripheral blood mononuclear cells is also higher in the presence of lower serum concentrations. Collectively, the results suggest for the first time that thymocyte maturations and proliferations are regulated by ROS levels induced by the deletion of PrxII gene in vivo.
Asunto(s)
Peroxidasas/genética , Especies Reactivas de Oxígeno/metabolismo , Timo/metabolismo , Animales , Apoptosis/fisiología , División Celular/fisiología , Eliminación de Gen , Humanos , Ratones , Peroxidasas/deficiencia , Peroxidasas/metabolismo , Peroxirredoxinas , Receptores de Glucocorticoides/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/patologíaRESUMEN
The precise role that individual inflammatory cells and mediators play in the development of gastrointestinal (GI) dysfunction and extraintestinal clinical manifestations of ulcerative colitis (UC) is unknown. In this study, we have used a mouse model of UC to establish a central role for eotaxin and, in turn, eosinophils in the development of the immunopathogenesis of this disease. In this model the administration of dextran sodium sulfate (DSS) induces a prominent colonic eosinophilic inflammation and GI dysfunction (diarrhea with blood and shortening of the colon) that resembles UC in patients. GI dysfunction was associated with evidence of eosinophilic cytolytic degranulation and the release of eosinophil peroxidase (EPO) into the colon lumen. By using IL-5 or eotaxin-deficient mice, we show an important role for eotaxin in eosinophil recruitment into the colon during experimental UC. Furthermore, using EPO-deficient mice and an EPO inhibitor resorcinol we demonstrate that eosinophil-derived peroxidase is critical in the development of GI dysfunction in experimental UC. These findings provide direct evidence of a central role for eosinophils and EPO in GI dysfunction and potentially the immunopathogenesis of UC.
Asunto(s)
Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Eosinófilos/enzimología , Peroxidasas/fisiología , Animales , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Separación Celular , Quimiocina CCL11 , Quimiocinas CC/deficiencia , Quimiocinas CC/genética , Quimiocinas CC/fisiología , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/fisiopatología , Colon/patología , Colon/fisiopatología , Sulfato de Dextran/administración & dosificación , Diarrea/fisiopatología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Peroxidasa del Eosinófilo , Eosinófilos/metabolismo , Eosinófilos/patología , Hemorragia Gastrointestinal/fisiopatología , Inyecciones Intraperitoneales , Interleucina-5/deficiencia , Interleucina-5/genética , Interleucina-5/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peroxidasas/antagonistas & inhibidores , Peroxidasas/deficiencia , Peroxidasas/genética , Resorcinoles/administración & dosificación , Resorcinoles/farmacologíaRESUMEN
Suppression subtractive hybridization performed on Down syndrome (DS) versus control fetal brains revealed differential expression of peroxiredoxin 2 (PRDX2), mapped at 13q12. Peroxiredoxins are antioxidant enzymes involved in protein and lipid protection against oxidative injury and in cellular signalling pathways regulating apoptosis. The under-expression of PRDX2 observed in DS samples was confirmed by real-time PCR (0.73-fold). To test whether decreased expression is associated with enhanced sensitivity of DS neurons to reactive oxygen species, we down-regulated PRDX2 through stable transfections of SH-SY5Y neuroblastoma cells with antisense contructs of the complete PRDX2 coding sequence. In addition, we over-expressed SOD1 and compared the effects of the two genes on cell viability. Cells transfected with either construct showed similar sensitivity to oxidative stress in addition to increased apoptosis under basal conditions and after treatment with oxidative cytotoxic agents. This suggests that the decreased expression of PRDX2 may contribute to the altered redox state in DS at levels comparable to that of the increased expression of SOD1.