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1.
Biochim Biophys Acta ; 1852(3): 529-40, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25536029

RESUMEN

Dysfunction of complex I (CI) of the mitochondrial electron transport chain (ETC) features prominently in human pathology. Cell models of ETC dysfunction display adaptive survival responses that still are poorly understood but of relevance for therapy development. Here we comprehensively examined how primary human skin fibroblasts adapt to chronic CI inhibition. CI inhibition triggered transient and sustained changes in metabolism, redox homeostasis and mitochondrial (ultra)structure but no cell senescence/death. CI-inhibited cells consumed no oxygen and displayed minor mitochondrial depolarization, reverse-mode action of complex V, a slower proliferation rate and futile mitochondrial biogenesis. Adaptation was neither prevented by antioxidants nor associated with increased PGC1-α/SIRT1/mTOR levels. Survival of CI-inhibited cells was strictly glucose-dependent and accompanied by increased AMPK-α phosphorylation, which occurred without changes in ATP or cytosolic calcium levels. Conversely, cells devoid of AMPK-α died upon CI inhibition. Chronic CI inhibition did not increase mitochondrial superoxide levels or cellular lipid peroxidation and was paralleled by a specific increase in SOD2/GR, whereas SOD1/CAT/Gpx1/Gpx2/Gpx5 levels remained unchanged. Upon hormone stimulation, fully adapted cells displayed aberrant cytosolic and ER calcium handling due to hampered ATP fueling of ER calcium pumps. It is concluded that CI dysfunction triggers an adaptive program that depends on extracellular glucose and AMPK-α. This response avoids cell death by suppressing energy crisis, oxidative stress induction and substantial mitochondrial depolarization.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Fibroblastos/enzimología , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Estrés Oxidativo , Transducción de Señal , Proteínas Quinasas Activadas por AMP/genética , Animales , Calcio/metabolismo , Línea Celular Transformada , Supervivencia Celular/genética , Cloruros/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Fibroblastos/citología , Humanos , Ratones , Ratones Noqueados , Mitocondrias/genética , Sirtuina 1/genética , Sirtuina 1/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
2.
J Inherit Metab Dis ; 36(6): 929-37, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23361305

RESUMEN

Barth syndrome (BTHS) is an X-linked mitochondrial defect characterised by dilated cardiomyopathy, neutropaenia and 3-methylglutaconic aciduria (3-MGCA). We report on two affected brothers with c.646G > A (p.G216R) TAZ gene mutations. The pathogenicity of the mutation, as indicated by the structure-based functional analyses, was further confirmed by abnormal monolysocardiolipin/cardiolipin ratio in dry blood spots of the patients as well as the occurrence of this mutation in another reported BTHS proband. In both brothers, 2D-echocardiography revealed some features of left ventricular noncompaction (LVNC) despite marked differences in the course of the disease; the eldest child presented with isolated cardiomyopathy from late infancy, whereas the youngest showed severe lactic acidosis without 3-MGCA during the neonatal period. An examination of the patients' fibroblast cultures revealed that extremely low mitochondrial membrane potentials (mtΔΨ about 50 % of the control value) dominated other unspecific mitochondrial changes detected (respiratory chain dysfunction, abnormal ROS production and depressed antioxidant defense). 1) Our studies confirm generalised mitochondrial dysfunction in the skeletal muscle and the fibroblasts of BTHS patients, especially a severe impairment in the mtΔΨ and the inhibition of complex V activity. It can be hypothesised that impaired mtΔΨ and mitochondrial ATP synthase activity may contribute to episodes of cardiac arrhythmia that occurred unexpectedly in BTHS patients. 2) Severe lactic acidosis without 3-methylglutaconic aciduria in male neonates as well as an asymptomatic mild left ventricular noncompaction may characterise the ranges of natural history of Barth syndrome.


Asunto(s)
Síndrome de Barth/complicaciones , Síndrome de Barth/fisiopatología , Potencial de la Membrana Mitocondrial , Síndrome de Barth/diagnóstico , Síndrome de Barth/etiología , Células Cultivadas , Niño , Preescolar , Humanos , Masculino , Músculo Esquelético/patología , Hermanos
3.
Biochim Biophys Acta ; 1797(6-7): 929-38, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20085746

RESUMEN

Reverse electron transfer (RET) from succinate to NAD+ is known to be accompanied by high generation of reactive oxygen species (ROS). In contrast, oxidation of fatty acids by mitochondria, despite being a powerful source of FADH2, does not lead to RET-associated high ROS generation. Here we show that oxidation of carnitine esters of medium- and long-chain fatty acids by rat heart mitochondria is accompanied by neither high level of NADH/NAD+ nor intramitochondrial reduction of acetoacetate to beta-hydroxybutyrate, comparable to those accompanying succinate oxidation, although it produces the same or higher energization of mitochondria as evidenced by high transmembrane potential. Also in contrast to the oxidation of succinate, where conversion of the pH difference between the mitochondrial matrix and the medium into the transmembrane electric potential by addition of nigericin results in a decrease of ROS generation, the same treatment during oxidation of octanoylcarnitine produces a large increase of ROS. Analysis of respiratory chain complexes by Blue Native polyacrylamide gel electrophoresis revealed bands that could tentatively point to supercomplex formation between complexes II and I and complexes II and III. However, no such association could be found between complex I and the electron transferring flavoprotein that participates in fatty acid oxidation. It is speculated that structural association between respective respiratory chain components may facilitate effective reverse electron transfer.


Asunto(s)
Transporte de Electrón , Ácidos Grasos/metabolismo , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/metabolismo , Estrés Oxidativo , Animales , Carnitina/análogos & derivados , Carnitina/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Potencial de la Membrana Mitocondrial , Oxidación-Reducción , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Ácido Succínico/metabolismo
4.
Biochim Biophys Acta ; 1797(6-7): 952-60, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20226758

RESUMEN

p66Shc, the growth factor adaptor protein, can have a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress. We investigated relationships between the extent of p66Shc phosphorylation at Ser36, mitochondrial dysfunctions and an antioxidant defense reactions in fibroblasts derived from five patients with various mitochondrial disorders (two with mitochondrial DNA mutations and three with methylglutaconic aciduria and genetic defects localized, most probably, in nuclear genes). We found that in all these fibroblasts, the extent of p66Shc phosphorylation at Ser36 was significantly increased. This correlated with a substantially decreased level of mitochondrial superoxide dismutase (SOD2) in these cells. This suggest that SOD2 is under control of the Ser36 phosphorylation status of p66Shc protein. As a consequence, an intracellular oxidative stress and accumulation of damages caused by oxygen free radicals are observed in the cells.


Asunto(s)
Enfermedades Mitocondriales/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Estudios de Casos y Controles , Células Cultivadas , ADN Mitocondrial/genética , Femenino , Fibroblastos/metabolismo , Glutaratos/orina , Humanos , Lactante , Recién Nacido , Masculino , Enfermedades Mitocondriales/genética , Modelos Biológicos , Mutación , Estrés Oxidativo , Fosforilación , Serina/química , Proteínas Adaptadoras de la Señalización Shc/química , Piel/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1
5.
Biochem Biophys Res Commun ; 407(4): 772-6, 2011 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-21439941

RESUMEN

Release of reactive oxygen species (ROS), measured as the sum of hydrogen peroxide (H2O2) and superoxide anion radical (O2·â»), from respiring rat heart and skeletal muscle mitochondria was significantly decreased by millimolar concentrations of GTP or GDP. Attempts to differentiate between the two forms of ROS showed that the release of O2·â» rather than that of H2O2 was affected. Meanwhile, intramitochondrial ROS accumulation, measured by inactivation of aconitase, increased. These results suggest that guanine nucleotides inhibit the release of O2·â» from mitochondria. As these nucleotides are known inhibitors of uncoupling proteins (UCPs), it is proposed that UCPs may function as carriers of O2·â», thus enabling its removal from the matrix compartment.


Asunto(s)
Guanosina Difosfato/farmacología , Guanosina Trifosfato/farmacología , Canales Iónicos/metabolismo , Mitocondrias Musculares/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Superóxidos/metabolismo , Animales , Femenino , Peróxido de Hidrógeno/metabolismo , Canales Iónicos/antagonistas & inhibidores , Mitocondrias Musculares/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/antagonistas & inhibidores , Proteína Desacopladora 1
6.
Int J Mol Sci ; 12(8): 5373-89, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21954365

RESUMEN

Reactive oxygen species (ROS) are wieldy accepted as one of the main factors of the aging process. These highly reactive compounds modify nucleic acids, proteins and lipids and affect the functionality of mitochondria in the first case and ultimately of the cell. Any agent or genetic modification that affects ROS production and detoxification can be expected to influence longevity. On the other hand, genetic manipulations leading to increased longevity can be expected to involve cellular changes that affect ROS metabolism. The 66-kDa isoform of the growth factor adaptor Shc (p66Shc) has been recognized as a relevant factor to the oxygen radical theory of aging. The most recent data indicate that p66Shc protein regulates life span in mammals and its phosphorylation on serine 36 is important for the initiation of cell death upon oxidative stress. Moreover, there is strong evidence that apart from aging, p66Shc may be implicated in many oxidative stress-associated pathologies, such as diabetes, mitochondrial and neurodegenerative disorders and tumorigenesis. This article summarizes recent knowledge about the role of p66Shc in aging and senescence and how this protein can influence ROS production and detoxification, focusing on studies performed on skin and skin fibroblasts.


Asunto(s)
Fibroblastos/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Adipocitos/metabolismo , Animales , Antioxidantes/metabolismo , Calcio/metabolismo , Senescencia Celular/genética , Transporte de Electrón , Homeostasis , Humanos , Longevidad , Mitocondrias/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/genética , Transducción de Señal
7.
Postepy Biochem ; 56(2): 165-73, 2010.
Artículo en Polaco | MEDLINE | ID: mdl-20873111

RESUMEN

The p66Shc adaptor protein has been in the spotlight of many researchgroups around the world for over adecade. Experiments conducted inrecent years unraveled its structure and enabled the recognition of basic cellular functions. Despite an undoubtedly tremendous progress in the characterization of p66Shc, mechanisms through which this protein potentially impacts the metabolism of mitochondria, and thus the cellular energetics are still waiting to be elucidated. Particularly interesting and profoundly studied is the concept that p66Shc may be a key component of the cell response to oxidative stress which may effectively contribute to the lifespan of the organism. p66Shc phosphorylation at serine 36 triggers a cascade of events leading to an increase in reactive oxygen species (ROS) production. The widely accepted free radical theory of ageing, proposed by Harman inthe - 1950s, assumes that an uncontrolled increase of ROS may lead to oxidation of fundamental cellular components such as proteins and phospholipids even sometimes premature dand cause DNA damage. Accumulation of such lesions in cells may unfavorably affect the functions of tissues and organs, leading to pathologies oreath of the organism. Although well experimentally established, knowledge regarding the involvement of the p66Shc protein in the production of ROS and its impact on the lifespan of organisms remains insufficient and requires a lot of additional research. Further investigation will permit a better understanding ofthe mechanisms governing the processes o f aging andthe emergence of variouspathologies associated with oxidative stress. This work is an attempt to systematize the existing knowledge about the p66Shc protein structure and functions. Another objective was to draw attention to the most interesting aspects and results of in vivo and in vitro studies in different models in the context of oxidative stress-associated pathologies and in aging.


Asunto(s)
Envejecimiento/fisiología , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Enfermedades Autoinmunes/metabolismo , Daño del ADN , Diabetes Mellitus/metabolismo , Metabolismo Energético/fisiología , Humanos , Hepatopatías Alcohólicas/metabolismo , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Fosforilación , Proteínas Adaptadoras de la Señalización Shc/química , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src
8.
Postepy Biochem ; 54(2): 217-23, 2008.
Artículo en Polaco | MEDLINE | ID: mdl-18807933

RESUMEN

"Blue Native" polyacrylamide gel electrophoresis (BN-PAGE), originally described by Schägger and von Jagow in 1991, is an elegant method to study protein complexes from mitochondrial membranes. BN-PAGE, commonly used in molecular biology to study composition of protein complexes and protein-protein interactions, enables separation of respiratory chain complexes keeping their properties and enzymatic activities unchanged. BN-PAGE, supplemented by other methods, e.g. in gel activity assay, SDS-PAGE (as a first or second dimension) can be successfully adapted for diagnosis of mitochondrial diseases connected with abnormalities of the respiratory chain. Therefore, to make a correct diagnosis of the deficiency of respiratory chain complexes, other methods, as histochemical colorimetric reactions allowing evaluation of the OXPHOS catalytic activity in individual cells and spectrophotometric technique should be used simultaneously with BN-PAGE.


Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Mitocondrias/química , Mitocondrias/metabolismo , Colorantes de Rosanilina/análisis , Coloración y Etiquetado/métodos , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/patología , Proteínas Mitocondriales/análisis , Complejos Multiproteicos/análisis , Colorantes de Rosanilina/química , Solubilidad
9.
Methods Mol Biol ; 1782: 357-381, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29851012

RESUMEN

Mitochondria are considered the main source of reactive oxygen species (ROS) in the cell. For this reason they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. The superoxide anion is the main undesired by-product of mitochondrial oxidative phosphorylation. Its production is triggered by a leak of electrons from the mitochondrial respiratory chain and the reaction of these electrons with O2. Superoxide dismutase (MnSOD, SOD2) from the mitochondrial matrix, as well as superoxide dismutase (Cu/ZnSOD, SOD1) present in small amounts in the mitochondrial intramembrane space, converts superoxide anion to hydrogen peroxide, which can be then converted by catalase to harmless H2O.In the chapter we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depend on the metabolic state of this organelle.


Asunto(s)
Fluorometría/métodos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Cultivo Primario de Células/métodos , Especies Reactivas de Oxígeno/metabolismo , Animales , Encéfalo/citología , Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , Fibroblastos , Colorantes Fluorescentes/química , Fluorometría/instrumentación , Células HeLa , Humanos , Ratones , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Peroxidasa/metabolismo , Cultivo Primario de Células/instrumentación , Piel/citología
10.
J Inorg Biochem ; 101(3): 493-505, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17222910

RESUMEN

Although selenium is taken with diet mainly as selenoamino acids, its hypoglycaemic action on hepatic gluconeogenesis has been studied with the use of inorganic selenium derivatives. The aim of the present investigation was to compare relative efficacies of inorganic and organic selenium compounds in reducing glucose synthesis in hepatocytes and renal tubules, significantly contributing to the glucose homeostasis. In contrast to hepatocytes, both selenite and methylselenocysteine inhibited renal gluconeogenesis by about 40-45% in control rabbits. Selenate did not affect this process, whereas selenomethionine inhibited gluconeogenesis by about 20% in both hepatocytes and renal tubules. In contrast to methylselenocysteine, selenite decreased intracellular ATP content, glutathione reduced/glutathione oxidized (GSH/GSSG) ratio and pyruvate carboxylase, PEPCK and FBPase activities, while methylselenocysteine diminished PEPCK activity due to elevation of intracellular 2-oxoglutarate and GSSG, inhibitors of this enzyme. Experiments in vivo indicate that in 3 of 9 alloxan-diabetic rabbits treated for 14 days with methylselenocysteine (0.182mg/kg body weight) blood glucose level was normalized, whereas in all diabetic rabbits plasma creatinine and urea levels decreased from 2.52+/-0.18 and 87.4+/-9.7 down to 1.63+/-0.11 and 39.0+/-2.8, respectively. In view of these data selenium supplementation might be beneficial for protection against diabetes-induced nephrotoxicity despite selenium accumulation in kidneys and liver.


Asunto(s)
Glucemia/efectos de los fármacos , Gluconeogénesis/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hipoglucemiantes/farmacología , Túbulos Renales/efectos de los fármacos , Compuestos de Selenio/farmacología , Aloxano , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Glucosa-6-Fosfatasa/metabolismo , Hepatocitos/metabolismo , Túbulos Renales/metabolismo , Masculino , Piruvato Carboxilasa/metabolismo , Conejos
11.
Nat Protoc ; 9(2): 312-22, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24434800

RESUMEN

Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.


Asunto(s)
Fraccionamiento Celular/métodos , Membrana Celular/fisiología , Retículo Endoplásmico/fisiología , Técnicas de Preparación Histocitológica/métodos , Hígado/citología , Animales , Centrifugación/métodos , Ratas
12.
Cell Cycle ; 12(4): 674-83, 2013 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-23343770

RESUMEN

The term "mitochondrial permeability transition" (MPT) refers to an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate mitochondrial outer membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade as well as of caspase-independent cell death mechanisms. MPT appears to be mediated by the opening of the so-called "permeability transition pore complex" (PTPC), a poorly characterized and versatile supramolecular entity assembled at the junctions between the inner and outer mitochondrial membranes. In spite of considerable experimental efforts, the precise molecular composition of the PTPC remains obscure and only one of its constituents, cyclophilin D (CYPD), has been ascribed with a crucial role in the regulation of cell death. Conversely, the results of genetic experiments indicate that other major components of the PTPC, such as voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT), are dispensable for MPT-driven MOMP. Here, we demonstrate that the c subunit of the FO ATP synthase is required for MPT, mitochondrial fragmentation and cell death as induced by cytosolic calcium overload and oxidative stress in both glycolytic and respiratory cell models. Our results strongly suggest that, similar to CYPD, the c subunit of the FO ATP synthase constitutes a critical component of the PTPC.


Asunto(s)
Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Neuronas/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Peptidil-Prolil Isomerasa F , Ciclofilinas/química , Ciclofilinas/metabolismo , Células HeLa , Humanos , Mitocondrias/química , Translocasas Mitocondriales de ADP y ATP/química , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/química , Membranas Mitocondriales/química , Poro de Transición de la Permeabilidad Mitocondrial , ATPasas de Translocación de Protón Mitocondriales/química , Neuronas/citología , Estrés Oxidativo , Cultivo Primario de Células , Ratas , Canales Aniónicos Dependientes del Voltaje/química , Canales Aniónicos Dependientes del Voltaje/metabolismo
13.
Int J Biochem Cell Biol ; 45(1): 114-22, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22776741

RESUMEN

Diabetes mellitus is a chronic disease caused by a deficiency in the production of insulin and/or by the effects of insulin resistance. Insulin deficiency leads to hyperglycemia which is the major initiator of diabetic cardiovascular complications escalating with time and driven by many complex biochemical and molecular processes. Four hypotheses, which propose mechanisms of diabetes-associated pathophysiology, are currently considered. Cardiovascular impairment may be caused by an increase in polyol pathway flux, by intracellular advanced glycation end-products formation or increased flux through the hexosamine pathway. The latter of these mechanisms involves activation of the protein kinase C. Cellular and mitochondrial metabolism alterations observed in the course of diabetes are partially associated with an excessive production of reactive oxygen species (ROS). Among many processes and factors involved in ROS production, the 66 kDa isoform of the growth factor adaptor shc (p66Shc protein) is of particular interest. This protein plays a key role in the control of mitochondria-dependent oxidative balance thus it involvement in diabetic complications and other oxidative stress based pathologies is recently intensively studied. In this review we summarize the current understanding of hyperglycemia induced cardiac mitochondrial dysfunction with an emphasis on the oxidative stress and p66Shc protein. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.


Asunto(s)
Hiperglucemia/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Estrés Oxidativo/fisiología , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Animales , Humanos , Hiperglucemia/patología , Miocardio/patología , Especies Reactivas de Oxígeno , Transducción de Señal
14.
Int J Biochem Cell Biol ; 45(1): 141-50, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22885148

RESUMEN

p66Shc is an adaptor protein involved in cell proliferation and differentiation that undergoes phosphorylation at Ser36 in response to oxidative stimuli, consequently inducing a burst of reactive oxygen species (ROS), mitochondrial disruption and apoptosis. Its role during several pathologies suggests that p66Shc mitochondrial signalling can perpetuate a primary mitochondrial defect, thus contributing to the pathophysiology of that condition. Here, we show that in the fibroblasts of neuropathy, ataxia and retinitis pigmentosa (NARP) patients, the p66Shc phosphorylation pathway is significantly induced in response to intracellular oxidative stress related to disrupted ATP synthase activity and mitochondrial membrane hyperpolarisation. We postulate that the increased phosphorylation of p66Shc at Ser36 is partially responsible for further increasing ROS production, resulting in oxidative damage of proteins. Oxidative stress and p66Shc phosphorylation at Ser36 may be mitigated by antioxidant administration or the use of a p66Shc phosphorylation inhibitor. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.


Asunto(s)
Fibroblastos/metabolismo , Miopatías Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Estrés Oxidativo/fisiología , Retinitis Pigmentosa/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Apoptosis/fisiología , Humanos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Mitocondrias/patología , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/patología , Fosforilación Oxidativa , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Proteínas Adaptadoras de la Señalización Shc/genética , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src
15.
Methods Mol Biol ; 810: 183-205, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22057568

RESUMEN

Mitochondria are considered as the main source of reactive oxygen species (ROS) in the cell. For this reason, they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids, and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. The superoxide anion is the main undesired by-product of mitochondrial oxidative phosphorylation. Its production is triggered by a leak of electrons from the mitochondrial respiratory chain and the reaction of these electrons with O(2). Superoxide dismutase (MnSOD, SOD2) from the mitochondrial matrix as well as superoxide dismutase (Cu/ZnSOD, SOD1) present in small amounts in the mitochondrial intramembrane space, convert superoxide anion to hydrogen peroxide, which can be then converted by catalase to harmless H(2)O. In this chapter, we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depends on the metabolic state of this organelle.


Asunto(s)
Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Bencimidazoles/metabolismo , Encéfalo/metabolismo , Calcio/metabolismo , Carbocianinas/metabolismo , Carcinoma de Ehrlich/metabolismo , Fraccionamiento Celular/métodos , Línea Celular Tumoral , Transporte de Electrón , Fibroblastos/metabolismo , Células HeLa , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones , Microscopía Confocal , Consumo de Oxígeno , Fenazinas/metabolismo , Superóxidos/metabolismo
16.
Curr Drug Targets ; 12(6): 827-49, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21269267

RESUMEN

Better understanding of the effect of ageing on mitochondrial metabolism and of the mechanisms of action of various drugs is required to allow optimization of the treatment of many diseases with minimized risk of dangerous impairment of mitochondrial function. Numerous reports show that efficacy of medical treatment depends on the age of treated subjects. This applies particularly to the effect of drugs on various senescence-prone cellular pathways. In this review, we demonstrate how ageing affects various mitochondria-associated pathways and their response to a variety of factors. These factors include registered drugs and other chemicals, and account for diverse consequences which vary depending on the physiological condition. Pharmacological treatments aimed at improving mitochondrial function should thus have in mind the subject age.


Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Mitocondrias/efectos de los fármacos , Enfermedades Mitocondriales/inducido químicamente , Factores de Edad , Envejecimiento , Animales , Humanos , Mitocondrias/metabolismo
17.
Folia Neuropathol ; 49(1): 56-63, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21455844

RESUMEN

UNLABELLED: Association of 3-methylglutaconic aciduria (3-MGCA) with sensorineural deafness and Leigh-like encephalopathy (MEGDEL) was described as a very rare mitochondrial disorder without a known molecular background. We present clinical and biochemical characteristics of a 4.5-year-old girl with a similar association. The clinical course of the disease was as follows: in the neonatal period transient adaptation troubles; at 4-5 mo failure to thrive and hypotonia; at 13 mo: extrapyramidal symptoms, sensorineural deafness, Leigh syndrome on MRI, pigmentary degeneration of retina, episodes of respiratory alkalosis, increased lactate in plasma, urine and brain, and increased excretion of 3-MGCA. Mitochondrial encephalopathy was suspected and muscle biopsy performed. Only mild lipid accumulation was found by muscle histopathology and histochemistry. Unspecific decrease of respiratory chain complexes was revealed by muscle homogenate spectrophotometry. The in-gel activity assay in the patient's muscle confirmed a combined defect of OXPHOS, particularly indicating suppression of mitochondrial ATP synthase (complex V) activity. Measurements of functional mitochondrial bioenergetic parameters in the patient's fibroblasts revealed a decrease in the mitochondrial membrane potential and activity of the mitochondrial respiratory chain. At the same time, a significant increase of ROS production (cytosolic and mitochondrial superoxide and H2O2) with signs of protein damage (protein carbonylation), and decreased antioxidant defence (SOD1 and SOD2) were observed. Additionally, the catalase amount was surprisingly low in comparison with healthy control and other reference 3-MGCA cases (Barth syndrome). CONCLUSION: (1) the natural history of the disease in the presented patient confirms the existence of previously reported clinical phenotype of MEGDEL (2) antioxidant defence impairment due to abnormal catalase metabolism/transport may characterize an unknown basic defect which led to the development of MEGDEL association.


Asunto(s)
Catalasa/metabolismo , Sordera/metabolismo , Fibroblastos/metabolismo , Glutaratos/orina , Enfermedad de Leigh/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Preescolar , Sordera/complicaciones , Sordera/fisiopatología , Femenino , Pérdida Auditiva Sensorineural/complicaciones , Pérdida Auditiva Sensorineural/fisiopatología , Humanos , Enfermedad de Leigh/complicaciones , Enfermedad de Leigh/fisiopatología , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/metabolismo
18.
Aging (Albany NY) ; 2(11): 823-42, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21084727

RESUMEN

The pro-oncogenic transcription factor STAT3 is constitutively activated in a wide variety of tumours that often become addicted to its activity, but no unifying view of a core function determining this widespread STAT3-dependence has yet emerged. We show here that constitutively active STAT3 acts as a master regulator of cell metabolism, inducing aerobic glycolysis and down-regulating mitochondrial activity both in primary fibroblasts and in STAT3-dependent tumour cell lines. As a result, cells are protected from apoptosis and senescence while becoming highly sensitive to glucose deprivation. We show that enhanced glycolysis is dependent on HIF-1α up-regulation, while reduced mitochondrial activity is HIF-1α-independent and likely caused by STAT3-mediated down-regulation of mitochondrial proteins. The induction of aerobic glycolysis is an important component of STAT3 pro-oncogenic activities, since inhibition of STAT3 tyrosine phosphorylation in the tumour cell lines down-regulates glycolysis prior to leading to growth arrest and cell death, both in vitro and in vivo. We propose that this novel, central metabolic role is at the core of the addiction for STAT3 shown by so many biologically different tumours.


Asunto(s)
Transformación Celular Neoplásica , Factor de Transcripción STAT3/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Metabolismo Energético , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Perfilación de la Expresión Génica , Glucólisis/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Mitocondrias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/genética , Transducción de Señal/fisiología
19.
Science ; 330(6008): 1247-51, 2010 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-21030605

RESUMEN

The promyelocytic leukemia (PML) tumor suppressor is a pleiotropic modulator of apoptosis. However, the molecular basis for such a diverse proapoptotic role is currently unknown. We show that extranuclear Pml was specifically enriched at the endoplasmic reticulum (ER) and at the mitochondria-associated membranes, signaling domains involved in ER-to-mitochondria calcium ion (Ca(2+)) transport and in induction of apoptosis. We found Pml in complexes of large molecular size with the inositol 1,4,5-trisphosphate receptor (IP(3)R), protein kinase Akt, and protein phosphatase 2a (PP2a). Pml was essential for Akt- and PP2a-dependent modulation of IP(3)R phosphorylation and in turn for IP(3)R-mediated Ca(2+) release from ER. Our findings provide a mechanistic explanation for the pleiotropic role of Pml in apoptosis and identify a pharmacological target for the modulation of Ca(2+) signals.


Asunto(s)
Apoptosis , Señalización del Calcio , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Homeostasis , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Membranas Intracelulares/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas Nucleares/genética , Fosforilación , Proteína de la Leucemia Promielocítica , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Estrés Fisiológico , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética
20.
Nat Protoc ; 4(11): 1582-90, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19816421

RESUMEN

Many cellular processes require the proper cooperation between mitochondria and the endoplasmic reticulum (ER). Several recent works show that their functional interactions rely on dynamic structural contacts between both organelles. Such contacts, called mitochondria-associated membranes (MAMs), are crucial for the synthesis and intracellular transport of phospholipids, as well as for intracellular Ca(2+) signaling and for the determination of mitochondrial structure. Although several techniques are available to isolate mitochondria, only few are specifically tuned to the isolation of MAM, containing unique regions of ER membranes attached to the outer mitochondrial membrane and mitochondria without contamination from other organelles (i.e., pure mitochondria). Here we provide optimized protocols to isolate these fractions from tissues and cells. These procedures require 4-5 h and can be easily modified and adapted to different tissues and cell types.


Asunto(s)
Fraccionamiento Celular/métodos , Retículo Endoplásmico/ultraestructura , Membranas Intracelulares , Mitocondrias/ultraestructura , Membranas Mitocondriales , Animales , Células Cultivadas , Humanos , Manejo de Especímenes/métodos
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