RESUMEN
Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. While advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarizes recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. This review lists the disruption of E-cadherin and tight junctions, key signaling pathways, including urokinase type plasminogen activator (uPA), phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT), focal adhesion kinase (FAK), ß-catenin/zinc finger E-box binding homeobox 1 (ZEB-1) and transforming growth factor beta (TGF-ß), together with inactivation of activator protein-1 (AP-1) and suppression of matrix metalloproteinase-9 (MMP-9) activity as key targets and the use of phytochemicals, or natural products, such as those from Agaricus blazei, Albatrellus confluens, Cordyceps militaris, Ganoderma lucidum, Poria cocos and Silybum marianum, together with diet derived fatty acids gamma linolenic acid (GLA) and eicosapentanoic acid (EPA) and inhibitory compounds as useful approaches to target tissue invasion and metastasis as well as other hallmark areas of cancer. Together, these strategies could represent new, inexpensive, low toxicity strategies to aid in the management of cancer metastasis as well as having holistic effects against other cancer hallmarks.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Cadherinas/genética , Humanos , Invasividad Neoplásica/genética , Metástasis de la Neoplasia , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/genéticaRESUMEN
Mitochondrial biogenesis and mitophagy are recognized as critical processes underlying mitochondrial homeostasis. However, the molecular pathway(s) coordinating the balance between these cellular programs is still poorly investigated. Here, we show an induction of the nuclear and mitochondrial peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) during myogenesis, which in turn co-activates the transcription of nuclear and mtDNA-encoded mitochondrial genes. We demonstrate that PGC-1α also buffers oxidative stress occurring during differentiation by promoting the expression of antioxidant enzymes. Indeed, by downregulating PGC-1α, we observed an impairment of antioxidants expression, which was accompanied by a significant reactive oxygen species (ROS) burst and increase of oxidative damage to proteins. In parallel, we detected a decrease of mitochondrial mass and function as well as increased mitophagy through the ROS/FOXO1 pathway. Upon PGC-1α downregulation, we found ROS-dependent nuclear translocation of FOXO1 and transcription of its downstream targets including mitophagic genes such as LC3 and PINK1. Such events were significantly reverted after treatment with the antioxidant Trolox, suggesting that PGC-1α assures mitochondrial integrity by indirectly buffering ROS. Finally, the lack of PGC-1α gave rise to a decrease in MYOG and a strong induction of atrophy-related ubiquitin ligases FBXO32 (FBXO32), indicative of a degenerative process. Overall, our results reveal that in myotubes, PGC-1α takes center place in mitochondrial homeostasis during differentiation because of its ability to avoid ROS-mediated removal of mitochondria.
Asunto(s)
Mitocondrias/metabolismo , Recambio Mitocondrial/genética , Células Musculares/metabolismo , Desarrollo de Músculos/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/genética , Animales , Antioxidantes/farmacología , Diferenciación Celular , Línea Celular , Cromanos/farmacología , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/efectos de los fármacos , Mitofagia , Células Musculares/citología , Células Musculares/efectos de los fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miogenina/genética , Miogenina/metabolismo , Estrés Oxidativo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
The nutrient-sensing lipolytic enzyme adipose triglyceride lipase (ATGL) has a key role in adipose tissue function, and alterations in its activity have been implicated in many age-related metabolic disorders. In adipose tissue reduced blood vessel density is related to hypoxia state, cell death and inflammation. Here we demonstrate that adipocytes of poorly vascularized enlarged visceral adipose tissue (i.e. adipose tissue of old mice) suffer from limited nutrient delivery. In particular, nutrient starvation elicits increased activity of mitochondrial proline oxidase/dehydrogenase (POX/PRODH) that is causal in triggering a ROS-dependent induction of ATGL. We demonstrate that ATGL promotes the expression of genes related to mitochondrial oxidative metabolism (peroxisome proliferator-activated receptor-α, peroxisome proliferator-activated receptor-γ coactivator-1α), thus setting a metabolic switch towards fat utilization that supplies energy to starved adipocytes and prevents cell death, as well as adipose tissue inflammation. Taken together, these results identify ATGL as a stress resistance mediator in adipocytes, restraining visceral adipose tissue dysfunction typical of age-related metabolic disorders.
Asunto(s)
Tejido Adiposo/metabolismo , Apoptosis , Lipasa/metabolismo , Prolina Oxidasa/metabolismo , Células 3T3-L1 , Animales , Dieta , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Inflamación , Lipasa/genética , Ratones , Mitocondrias/metabolismo , PPAR alfa/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Finding new molecular pathways and strategies modulating lipolysis in adipocytes is an attractive goal of the current research. Indeed, it is becoming clear that several human age-related pathologies are caused by adipose tissue expansion and altered lipid metabolism. In the present work, we show that transcription factor forkhead homeobox type protein O1 (FoxO1) is upregulated by nutrient restriction (NR) in adipocytes and exerts the transcriptional control of lipid catabolism via the induction of lysosomal acid lipase (Lipa). An increased autophagy and colocalization of lipid droplets (LDs) with lysosomes was observed implying lipophagy in Lipa-mediated LDs degradation. Interestingly, we found that metformin (Metf), a biguanide drug commonly used to treat type-2 diabetes, exerts effects comparable to that of NR. Actually, it was able to elicit FoxO1-dependent Lipa induction as well as LDs degradation through lipophagy. Moreover, we demonstrate that, during NR or Metf treatment, free fatty acids released by Lipa are directed toward AMP-activated protein kinase-mediated mitochondrial oxidation, thus maintaining energetic homeostasis in adipocytes. In conclusion, our data show that lysosomal-mediated lipid catabolism is activated by NR in adipocytes and give further support to the use of Metf as a NR mimetic to combat age-related diseases associated with altered lipid metabolism.
Asunto(s)
Adipocitos/citología , Autofagia/efectos de los fármacos , Ayuno , Factores de Transcripción Forkhead/metabolismo , Lipólisis/efectos de los fármacos , Metformina/farmacología , Esterol Esterasa/metabolismo , Células 3T3-L1 , Proteínas Quinasas Activadas por AMP , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Adiposidad/efectos de los fármacos , Animales , Regulación hacia Abajo/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Ácidos Grasos/metabolismo , Proteína Forkhead Box O1 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Oxidación-Reducción/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
Endoplasmic reticulum (ER) is the primary site for the synthesis and folding of secreted and membrane-bound proteins. Accumulation of unfolded and misfolded proteins in ER underlies a wide range of human neurodegenerative disorders. Hence, molecules regulating the ER stress response represent potential candidates as drug targets for tackling these diseases. Protein disulphide isomerase (PDI) is a chaperone involved in ER stress pathway, its activity being an important cellular defense against protein misfolding. Here, we demonstrate that human neuroblastoma SH-SY5Y cells overexpressing the reticulon protein 1-C (RTN1-C) reticulon family member show a PDI punctuate subcellular distribution identified as ER vesicles. This represents an event associated with a significant increase of PDI enzymatic activity. We provide evidence that the modulation of PDI localization and activity does not only rely upon ER stress induction or upregulation of its synthesis, but tightly correlates to an alteration in its nitrosylation status. By using different RTN1-C mutants, we demonstrate that the observed effects depend on RTN1-C N-terminal region and on the integrity of the microtubule network. Overall, our results indicate that RTN1-C induces PDI redistribution in ER vesicles, and concomitantly modulates its activity by decreasing the levels of its S-nitrosylated form. Thus RTN1-C represents a promising candidate to modulate PDI function.
Asunto(s)
Estrés del Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Proteínas del Tejido Nervioso/genética , Proteína Disulfuro Isomerasas/genética , Vesículas Transportadoras/metabolismo , Línea Celular Tumoral , Retículo Endoplásmico/ultraestructura , Regulación de la Expresión Génica , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Pliegue de Proteína , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal , Vesículas Transportadoras/ultraestructuraRESUMEN
SIRT1 and PGC-1α are two nutrient sensing master regulators of cellular metabolism and their upregulation is often linked to increased lifespan. SIRT1 and PGC-1α modulate the expression of a set of nuclear genes controlling many metabolic pathways. In recent years mounting evidence has indicated the implication of these proteins in several mitochondrial diseases including neurodegenerative disorders, myopathies and Type II diabetes mellitus. Recently, these proteins have been localized in cytoplasm and mitochondria wherein they target novel substrates opening new insight into their possible function in modulating extranuclear genes and proteins. This review will firstly summarize the nuclear function of SIRT1 and PGC-1α. Then, data from papers demonstrating the presence of SIRT1 and PGC-1α in the cytoplasm and in mitochondria will be outlined so that these extranuclear forms do not remain out of sight. Finally, very recent evidence of the alteration of the pathways governed by SIRT1 and PGC-1α in human mitochondrial diseases will be described and the possible role of their mitochondrial forms will be briefly discussed.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Enfermedades Mitocondriales/metabolismo , Sirtuina 1/metabolismo , Factores de Transcripción/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Sirtuina 1/genéticaRESUMEN
We have studied the effects of superoxide production after Cu,Zn superoxide dismutase (SOD1) down-regulation by RNA interference. We demonstrated that SOD1 depletion induced, only in neuroblastoma cells, a decrease in actin and beta-tubulin content and accumulation of neurofilament light chain and Tau proteins. Alterations of cell morphology and the microfilament network were also observed, together with the up-regulation of the Cdk5/p35 pathway, which is involved in the regulation of actin polymerization. The decrease of filamentous actin was transient and was recovered through the activation of p38/Hsp27 MAPK pathway, as well as after treatment with N-acetyl-L-cysteine. The importance of p38 in the recovery of cytoskeleton was confirmed by experiments carried out in the presence of its inhibitor SB203580, which induced cell death. Our data demonstrate that SOD1 is essential for the preservation of cytoskeleton integrity, by maintaining physiological concentration of reactive oxygen species and inhibiting the activation of the neuronal specific Cdk5/p35 pathway.
Asunto(s)
Citoesqueleto/metabolismo , Neuroblastoma/enzimología , Neuroblastoma/patología , Superóxido Dismutasa/deficiencia , Actinas/metabolismo , Apoptosis , Línea Celular Tumoral , Forma de la Célula , Quinasa 5 Dependiente de la Ciclina/metabolismo , Regulación hacia Abajo , Activación Enzimática , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Neuroblastoma/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Superóxidos/metabolismo , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Polyphenols represent a large class of plant-derived molecules with a general chemical structure that act as potent free radical scavengers. They have long been recognized to possess several therapeutic activities ranging from anti-thrombotic to antioxidant. Moreover, the capability of polyphenols to act as reducing or oxidizing molecules depends on the presence of environmental metals and on the concentrations used. In this work we demonstrated that the stilbene trans-resveratrol was able to commit human breast cancer MCF-7 cells to apoptosis. Mainly, we evidenced a pivotal role of the mitochondria in this phenomenon as cytochrome c release into the cytosol was found after the treatment. We further showed that trans-resveratrol was able to affect cellular redox state. In particular, it induced an early production of ROS and lipid oxidation, and only later compromised the GSH/GSSG ratio. This mode of action was mirrored by a temporally different activation of JNK and p38(MAPK), with the former rapidly induced and the latter weakly activated at long intervals. The results obtained demonstrate a pro-apoptotic activity for trans-resveratrol, and suggest a preferential activation of different classes of MAP kinases in response to different oxidative stimuli (ROS versus GSH/GSSG alteration).
RESUMEN
To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type-1) BHV-1 DNA. An alignment of carboxyl-terminal domains belonging to three proteins encoded by gamma34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type-1 (BHV-1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that UL14 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti-apoptotic function may derive from its heat shock protein-like properties.
Asunto(s)
Apoptosis/genética , Herpesvirus Bovino 1/genética , Reacción en Cadena de la Polimerasa/métodos , Proteínas Virales/genética , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Secuencia de Consenso , Cartilla de ADN/química , Bases de Datos como Asunto , Proteínas del Choque Térmico HSP72/genética , Humanos , Células K562 , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Programas Informáticos , Proteínas Virales/fisiologíaRESUMEN
Modifications of specific amino-acid residues of proteins are fundamental in order to modulate different signaling processes among which the cascade of phosphorylation represents the most effective example. Recently, also, the modification of the redox state of cysteine residues of certain proteins, which is a widespread mechanism in the regulation of protein function, has been proposed to be involved in signaling pathways. Growing evidence shows that some transcription factors could be modulated by both oxidation and phosphorylation. In particular, the pathways regulated by the mitogen activated protein (MAP) kinases represent well-established examples of the cross talk between redox-mediated signaling and phosphorylative cascades. This review will compare the two modes of signal transduction and propose an evolutionary model of a partnership of the two mechanisms in the eukaryotic cell, with redox-mediated signals being more specific and ancestral and phosphorylative signals being more diffuse but predominant in signal propagation.
Asunto(s)
Disulfuros/química , Transducción de Señal/fisiología , Animales , Humanos , Oxidación-Reducción , FosforilaciónRESUMEN
Bax is a cytosolic protein, which in response to stressing apoptotic stimuli, is activated and translocates to mitochondria, thus initiating the intrinsic apoptotic pathway. In spite of many studies and the importance of the issue, the molecular mechanisms that trigger Bax translocation are still obscure. We show by computer simulation that the two cysteine residues of Bax may form disulfide bridges, producing conformational changes that favor Bax translocation. Oxidative, nonapoptogenic treatments produce an up-shift of Bax migration compatible with homodimerization, which is reverted by reducing agents; this is accompanied by translocation to mitochondria. Dimers also appear in pure cytosolic fractions of cell lysates treated with H2O2, showing that Bax dimerization may take place in the cytosol. Bax dimer-enriched lysates support Bax translocation to isolated mitochondria much more efficiently than untreated lysates, indicating that dimerization may promote Bax translocation. The absence of apoptosis in our system allows the demonstration that Bax moves because of oxidations, even in the absence of apoptosis. This provides the first evidence that Bax dimerization and translocation respond to oxidative stimuli, suggesting a novel role for Bax as a sensor of redox imbalance.
Asunto(s)
Apoptosis , Mitocondrias/metabolismo , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/metabolismo , Caspasa 8 , Caspasas/fisiología , Células Cultivadas , Dimerización , Disulfuros/química , Retículo Endoplásmico/fisiología , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Modelos Moleculares , Oxidación-Reducción , Transporte de ProteínasRESUMEN
SH-SY5Y neuroblastoma cells were cultured for up to three serial passages in the presence of the copper chelator triethylene tetramine (Trien). The copper-depleted neuroblastoma cell line obtained showed decreased activities of the copper enzymes Cu, Zn super-oxide dismutase and cytochrome c oxidase with concomitant increases in reactive oxygen species. Mitochondrial antioxidants (Mn superoxide dismutase and Bcl-2)were up-regulated. Overexpression and activation of p53 were early responses, leading to an increase in p21. Eventually, copper-depleted cells detached from the monolayer and underwent apoptosis. Activation of upstream caspase-9, but not caspase-8, suggested that apoptosis proceeds via a mitochondrial pathway, followed by caspase-3 activation. The addition of copper sulfate to the copper-depleted cells restored copper enzymes, normalized antioxidant levels and improved cell viability. We conclude that prolonged copper starvation in these replicating cells leads to mitochondrial damage and oxidative stress and ultimately, apoptosis.
Asunto(s)
Antioxidantes/metabolismo , Apoptosis/fisiología , Cobre/deficiencia , Neuroblastoma/metabolismo , Neuroblastoma/patología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Quelantes/farmacología , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Trientina/farmacología , Células Tumorales CultivadasRESUMEN
SH-SY5Y cells transfected with the enzymatically inactive Cu,Zn superoxide dismutase mutant H46R were more resistant to S-nitrosoglutathione (GSNO)-induced apoptosis. Cytochrome c release from mitochondria, caspase 3 activation, p53 up-regulation, p21 cleavage and Bcl-2 modulation, all involved in the apoptotic process, were significantly less altered with respect to untransfected cells. The H46R resistance to NO was associated with a higher content of reduced glutathione (GSH) and was abolished by blockage of glutathione synthesis. On the other hand, H46R cells were as sensitive as SH-SY5Y cells to puromycin-induced apoptosis; furthermore, they were more susceptible to apoptosis elicited by the superoxide-generating drug paraquat and to cell necrosis provoked by t-butyl hydroperoxide. These results confirm that the level of superoxide dismutase activity is fundamental for protecting cells against oxygen free radical challenge. Its impairment is not detrimental to cells exposed to NO, as long as the overall reducing power represented by GSH is assured. These results are relevant to explain a milder progression of the familial amyotrophic lateral sclerosis disease when associated with the H46R mutation.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Apoptosis/fisiología , Glutatión/análogos & derivados , Glutatión/farmacología , Donantes de Óxido Nítrico/farmacología , Nitrocompuestos/farmacología , Superóxido Dismutasa/genética , Superóxidos/metabolismo , Esclerosis Amiotrófica Lateral/genética , Apoptosis/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Citometría de Flujo , Glutatión/metabolismo , Herbicidas/farmacología , Humanos , Mutación/fisiología , Neuroblastoma , Neuronas/citología , Neuronas/enzimología , Donantes de Óxido Nítrico/metabolismo , Nitrocompuestos/metabolismo , Oxidación-Reducción , Estrés Oxidativo/fisiología , Paraquat/farmacología , Superóxido Dismutasa/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , terc-Butilhidroperóxido/farmacologíaRESUMEN
Treatment of neuroblastoma cells with the copper chelator triethylene tetramine tetrahydrochloride induced intracellular decrease of copper content paralleled by diminished activity of the enzymes Cu, Zn superoxide dismutase, and cytochrome c oxidase. This effect appears to be specific for copper-enzymes and the treatment affects neither viability nor growth capability of cells. However, molecular markers of apoptosis Bcl-2, p53, and caspase-3 were slightly affected in these cells. When copper-deficient cells were challenged with oxidative stress generated by paraquat or puromycin, they underwent a higher degree of apoptosis with respect to copper-adequate control cells. The mechanism underlying paraquat-triggered apoptosis implies dramatic activation of caspase-3 and induction of the transcription factor p53. These results demonstrate that impairment of copper balance predisposes neuronal cells to apoptosis induced by oxidative stress. Overall findings represent a contribution to the comprehension of the link between copper-imbalance and neurodegeneration, which has recently been repeatedly suggested for the most invalidating pathologies of the central nervous system.
Asunto(s)
Apoptosis , Cobre/deficiencia , Neuroblastoma/metabolismo , Neuroblastoma/patología , Estrés Oxidativo , Apoptosis/efectos de los fármacos , Western Blotting , Caspasa 3 , Caspasas/metabolismo , Quelantes/farmacología , Cobre/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Activación Enzimática/efectos de los fármacos , Glutatión/metabolismo , Humanos , Neuroblastoma/enzimología , Estrés Oxidativo/efectos de los fármacos , Paraquat/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Puromicina/farmacología , Superóxido Dismutasa/metabolismo , Trientina/farmacología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Copper plays a key role in brain development, function and survival. Alteration of its homeostasis is suggested to be an aetiological factor in several neurodegenerative diseases. However, the molecular mechanisms relating copper to neurodegeneration are still unknown. In the present report, using morphological analyses of brain sections of mottled/brindled mutant (Mo(br/y)) mice, the animal model of the human genetic copper deficiency associated with neurodegeneration (Menkes' disease), we demonstrated that a high degree of apoptotic cells is present in the neocortex and in the hippocampus. Biochemical characterisation revealed decreased levels of copper content and of the activity of the mitochondrial copper-dependent enzyme cytochrome c oxidase. Copper, zinc-superoxide dismutase activity also shows a slight decrease, while no change was observed for glutathione content. Lower levels of ATP were also found, indicative of a copper-dependent impairment of energy metabolism. Changes appear to be specific for the brain, since no alterations in the activity of liver enzymes were found, although the level of copper was strongly decreased. We also tested biochemical factors involved in cell commitment to apoptosis. The expression of the anti-apoptotic protein Bcl-2, which plays a fundamental role in brain development and morphogenesis, was dramatically decreased and the levels of cytochrome c released from mitochondria into the cytosol were significantly increased. On the basis of these findings, we propose that down-regulation of Bcl-2 can cause neurodegeneration triggered by mitochondrial damage due to copper depletion during brain development in Mo(br/y) mice.
Asunto(s)
Apoptosis , Encéfalo/patología , Síndrome del Pelo Ensortijado/patología , Degeneración Nerviosa/patología , Proteínas Proto-Oncogénicas c-bcl-2 , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Cobre/metabolismo , Modelos Animales de Enfermedad , Complejo IV de Transporte de Electrones/metabolismo , Glutatión/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Etiquetado Corte-Fin in Situ , Masculino , Síndrome del Pelo Ensortijado/metabolismo , Ratones , Ratones Mutantes , Neocórtex/metabolismo , Neocórtex/patología , Degeneración Nerviosa/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione [Cu(I)-GSH] complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme. The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.
Asunto(s)
Cobre/farmacocinética , Superóxido Dismutasa/química , Superóxido Dismutasa/fisiología , Sitios de Unión , Catálisis , Cobre/metabolismo , Escherichia coli/enzimología , Glutatión Transferasa/metabolismo , Concentración de Iones de Hidrógeno , Iones , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Estrés Oxidativo , Plásmidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Factores de TiempoRESUMEN
The apical uptake of 64CuCl2 was investigated in human differentiated intestinal Caco-2 cells grown on permeable supports. At pH 6.0 in the apical compartment, the uptake of copper was linear over the first 6 min and between 10 and 80 microM CuCl2 exhibited non-saturable transport kinetics. In addition, copper uptake was energy-independent, affected by the valency state of copper, preferring Cu(II) over Cu(I), and not influenced by high (10 mM) extracellular calcium. The intracellular distribution of copper was investigated by FPLC at different times of uptake ('pulse') and of 'chase'. Intracellular copper initially bound predominantly to low molecular weight components (i.e., glutathione). and subsequently shifted to higher molecular weight components such as metallothionein and Cu,Zn superoxide dismutase.
Asunto(s)
Cobre/farmacocinética , Mucosa Intestinal/metabolismo , Células CACO-2 , Glutatión/metabolismo , Humanos , Concentración de Iones de HidrógenoRESUMEN
It has previously been demonstrated that CuZn-superoxide dismutase (SOD) is secreted by several human cell lines. This suggests that the circulating enzyme derives from both hemolysis and peripheral tissues as a result of cellular secretion. In the present report, we evaluated the presence of CuZn-SOD in human serum lipoproteins by both enzyme-linked immunosorbent assay and Western blot analysis of immunoprecipitated lipoprotein samples. The distribution of CuZn-SOD activity among the different lipoprotein fractions was also determined by the xanthine/xanthine oxidase method. The results demonstrated that CuZn-SOD is noticeably present in serum lipoproteins and mainly in low and high density lipoproteins (LDL and HDL). Moreover, experiments performed by incubating CuZn-SOD with a lipid emulsion and subsequent separation of the lipid fraction by ultracentrifugation showed that this enzyme associates in a saturable manner with lipids. The CuZn-SOD bound to LDL and HDL could exert a physiological protective role against oxidative damage of these lipoprotein classes that carry out a crucial role in the cholesterol transport.
Asunto(s)
Lipoproteínas/sangre , Lipoproteínas/metabolismo , Superóxido Dismutasa/sangre , Superóxido Dismutasa/metabolismo , Adulto , Transporte Biológico , Western Blotting , Proteínas Portadoras/sangre , Proteínas Portadoras/química , Proteínas Portadoras/clasificación , Proteínas Portadoras/metabolismo , Emulsiones , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lipoproteínas/química , Lipoproteínas/clasificación , Lipoproteínas HDL/sangre , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/sangre , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Masculino , Persona de Mediana Edad , Unión Proteica , Superóxido Dismutasa/análisis , UltracentrifugaciónRESUMEN
Nitric oxide (NO) challenge to human neuroblastoma cells (SH-SY5Y) ultimately results in apoptosis. Tumor suppressor protein p53 and cell cycle inhibitor p21 accumulate as an early sign of S-nitrosoglutathione-mediated toxicity. Cytochrome c release from mitochondria and caspase 3 activation also occurred. Cells transfected with either wild type (WT) or mutant (G93A) Cu, Zn-superoxide dismutase (Cu,Zn-SOD) produced comparable amounts of nitrite/nitrate but showed different degree of apoptosis. G93A cells were the most affected and WT cells the most protected; however, Cu, Zn-SOD content of these two cell lines was 2-fold the SH-SY5Y cells under both resting and treated conditions. We linked decreased susceptibility of the WT cells to higher and more stable Bcl-2 and decreased reactive oxygen species. Conversely, we linked G93A susceptibility to increased reactive oxygen species production since simultaneous administration of S-nitrosoglutathione and copper chelators protects from apoptosis. Furthermore, G93A cells showed a significant decrease of Bcl-2 expression and, as target of NO-derived radicals, showed lower cytochrome c oxidase activity. These results demonstrate that resistance to NO-mediated apoptosis is strictly related to the level and integrity of Cu,Zn-SOD and that the balance between reactive nitrogen and reactive oxygen species regulates neuroblastoma apoptosis.