RESUMO
This article reviews the interactions between nitric oxide (NO) and mitochondrial respiration. Mitochondrial ATP synthesis is responsible for virtually all energy production in mammals, and every other process in living organisms ultimately depends on that energy production. Furthermore, both necrosis and apoptosis, that summarize the main forms of cell death, are intimately linked to mitochondrial integrity. Endogenous and exogenous â¢NO inhibits mitochondrial respiration by different well-studied mechanisms and several nitrogen derivatives. Instantaneously, low concentrations of â¢NO, specifically and reversibly inhibit cytochrome c oxidase in competition with oxygen, in several tissues and cells in culture. Higher concentrations of â¢NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Peroxynitrite can cause opening of the permeability transition pore and opening of this pore causes loss of cytochrome c, which in turn might contribute to peroxynitrite-induced inhibition of respiration. Therefore, the inhibition of cytochrome c oxidase by â¢NO may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen, which depend on reactive nitrogen species formation, pH, proton motriz force and oxygen supply to tissues.
Assuntos
Respiração Celular/fisiologia , Mitocôndrias/metabolismo , Óxido Nítrico/fisiologia , Animais , Bactérias , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Humanos , Ácido Peroxinitroso/fisiologia , PlantasRESUMO
In the Wobbler mouse, a mutation of the Vps54 protein increases oxidative stress in spinal motoneurons, associated to toxic levels of nitric oxide and hyperactivity of nitric oxide synthase (NOS). Progesterone neuroprotection has been reported for several CNS diseases, including the Wobbler mouse neurodegeneration. In the present study, we analyzed progesterone effects on mitochondrial-associated parameters of symptomatic Wobbler mice. The activities of mitochondrial respiratory chain complexes I, II-III and IV and protein levels of mitochondrial and cytosolic NOS were determined in cervical and lumbar cords from control, Wobbler and Wobbler mice receiving a progesterone implant for 18 days. We found a significant reduction of complex I and II-III activities in mitochondria and increased protein levels of mitochondrial, but not cytosolic nNOS, in the cervical cord of Wobbler mice. Progesterone treatment prevented the reduction of complex I in the cervical region and the increased level of mitochondrial nNOS. Wobbler motoneurons also showed accumulation of amyloid precursor protein immunoreactivity and decreased activity and immunostaining of MnSOD. Progesterone treatment avoided these abnormalities. Therefore, administration of progesterone to clinically afflicted Wobblers (i) prevented the abnormal increase of mitochondrial nNOS and normalized respiratory complex I; (ii) decreased amyloid precursor protein accumulation, a sign of axonal degeneration, and (iii) increased superoxide dismutation. Thus, progesterone neuroprotection decreases mitochondriopathy of Wobbler mouse cervical spinal cord.
Assuntos
Doenças Mitocondriais/patologia , Doenças Mitocondriais/prevenção & controle , Complexos Multienzimáticos/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Progesterona/uso terapêutico , Medula Espinal/efeitos dos fármacos , Albinismo Oculocutâneo/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Proteínas de Drosophila , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Camundongos , Camundongos Mutantes , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Doenças Mitocondriais/genética , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Superóxido Dismutase/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
PURPOSE: Our objective was to describe the relationship between sepsis syndrome mortality and cognitive and physical disability in elderly persons. METHODS: A 1-year consecutive cohort study in clinical beds of a university hospital was performed. Variables were severity of sepsis syndrome, organ failure, functional status, age, sex, and positive cultures. Outcomes were in-hospital and 1-year mortalities. RESULTS: The study included 137 patients (>70 years), both sexes. Data from 116 (84.5%) patients were obtainable at 1-year follow-up. Forty-eight (35%) patients presented with sepsis (11/137, 8%) or severe sepsis (37/137, 27%). In-hospital mortality was 15.3% (0% for sepsis and 21.8% if severe) and increased with organ failure (p <.0001). One-year mortality was 54.78% (63/116), mostly related to severe sepsis; predictors were severe organ failure (p <.0001), prior functional status (p =.0005), and Mini-Mental State Examination (p =.03). Prior functional status and organ failure were independent predictors. CONCLUSIONS: In-hospital and 1-year mortality increased with septic syndrome severity, prior functional status, and organ failure.
Assuntos
Avaliação de Resultados em Cuidados de Saúde , Síndrome de Resposta Inflamatória Sistêmica/mortalidade , Idoso , Idoso de 80 Anos ou mais , Argentina/epidemiologia , Estudos de Coortes , Feminino , Mortalidade Hospitalar , Humanos , Masculino , Modelos de Riscos ProporcionaisRESUMO
Activation of ERK1/2 implies the phosphorylation of tyrosine (pTyr) and threonine (pThr) by MEK1/2; both reactions were thought to be cytoplasmic, promoting ERK to reach the nucleus where it activates several transcription factors. In addition, H2O2 concentrations are known to modulate ERK intracellular translocation, which impacts on cellular proliferation. In this context, the objective of this work was to study the sequence of ERK phosphorylation under two redox conditions and to analyze a putative mitochondrial contribution to this process, in LP07 murine lung cells. A time-course of H2O2 administration was used and ERK phosphorylation was analyzed in cytosol, mitochondria and nuclei. At 1µM H2O2, a proliferative redox stimulus, immunoblot revealed a fast and transient increase in cytosol pTyr and a sustained increase in mitochondrial pTyr content. The detection for pThr/pTyrERK (2pERK) showed in cytosol a marked increase at 5 minutes with a fast dephosphorylation after that time, for both H2O2 concentrations. However, at 50 µM H2O2, an anti-proliferative condition, 2pERK was gradually retained in mitochondria. Interestingly, these results were confirmed by in vivo experiments using mice treated with a highly oxidizing agent [H2O2]. By the use of two ERK2 mutant constructions, where Tyr and Thr were replaced by alanine, we confirmed that 2pERK relied almost completely on pThr183. Confocal microscopy confirmed ERK subcellular distribution dependence on the incidence of cytosolic pTyr and mitochondrial pThr at 1µM H2O2. This work shows for the first time, both in vitro and in vivo, an ERK cycle involving a cross-talk between cytosol and mitochondria phosphorylation events, which may play a significant role in cell cycle progression, proliferation or differentiation under two different redox conditions.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Pulmão/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , Animais , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Peróxido de Hidrogênio/metabolismo , Pulmão/citologia , Masculino , Camundongos , Mitocôndrias/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Oxirredução , Fosforilação , Frações Subcelulares/metabolismoRESUMO
Programmed and damage aging theories have traditionally been conceived as stand-alone schools of thought. However, the p66Shc adaptor protein has demonstrated that aging-regulating genes and reactive oxygen species (ROS) are closely interconnected, since its absence modifies metabolic homeostasis by providing oxidative stress resistance and promoting longevity. p66Shc(-/-) mice are a unique opportunity to further comprehend the bidirectional relationship between redox homeostasis and the imbalance of mitochondrial biogenesis and dynamics during aging. This study shows that brain mitochondria of p66Shc(-/-) aged mice exhibit a reduced alteration of redox balance with a decrease in both ROS generation and its detoxification activity. We also demonstrate a strong link between reactive nitrogen species (RNS) and mitochondrial function, morphology, and biogenesis, where low levels of ONOO- formation present in aged p66Shc(-/-) mouse brain prevent protein nitration, delaying the loss of biological functions characteristic of the aging process. Sirt3 modulates age-associated mitochondrial biology and function via lysine deacetylation of target proteins, and we show that its regulation depends on its nitration status and is benefited by the improved NAD+/NADH ratio in aged p66Shc(-/-) brain mitochondria. Low levels of protein nitration and acetylation could cause the metabolic homeostasis maintenance observed during aging in this group, thus increasing its lifespan.
Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Sirtuína 3/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Animais , Homeostase , Camundongos , Camundongos KnockoutRESUMO
This study investigated whether inducible heme oxygenase-1[corrected] (HO-1) [corrected] is targeted to mitochondria and its putative effects on oxidative metabolism in rat liver. Western blot and immune-electron microscopy in whole purified and fractionated organelles showed basal expression of HO-1 protein in both microsomes and mitochondria (inner membrane), accompanied by a parallel HO activity. Inducers of HO-1 increased HO-1 targeting to the inner mitochondrial membrane, which also contained biliverdin reductase, supporting that both enzymes are in the same compartmentalization. Induction of mitochondrial HO-1 was associated with a decrease of mitochondrial heme content and selective reduction of protein expression of cytochrome oxidase (COX) subunit I, which is coded by the mitochondrial genome and synthesized in the mitochondria depending on heme availability; these changes resulted in decreased COX spectrum and activity. Mitochondrial HO-1 induction was also associated with down-regulation of mitochondrial-targeted NO synthase expression and activity, resulting in a reduction of NO-dependent mitochondrial oxidant yield; inhibition of HO-1 activity reverted these effects. In conclusion, we demonstrated for the first time localization of HO-1 protein in mitochondria. It is surmised that mitochondrial HO-1 has important biological roles in regulating mitochondrial heme protein turnover and in protecting against conditions such as hypoxia, neurodegenerative diseases, or sepsis, in which substantially increased mitochondrial NO and oxidant production have been implicated.
Assuntos
Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Mitocôndrias Hepáticas/enzimologia , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Heme Oxigenase (Desciclizante)/análise , Mitocôndrias Hepáticas/metabolismo , Óxido Nítrico Sintase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/análise , Consumo de Oxigênio , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
Intercellular communication is one of the most important events in cell population behavior. In the last decade, tunneling nanotubes (TNTs) have been recognized as a new form of long distance intercellular connection. TNT function is to allow molecular and subcellular structure exchange between neighboring cells via the transfer of molecules and organelles such as calcium ions, prions, viral and bacterial pathogens, small lysosomes and mitochondria. New findings support the concept that mesenchymal stem cells (MSCs) can affect cell microenvironment by the release of soluble factors or the transfer of cellular components to neighboring cells, in a way which significantly contributes to cell regulation and tissue repair, although the underlying mechanisms remain poorly understood. MSCs have many advantages for their implementation in regenerative medicine. The TNTs in these cell types are heterogeneous in both structure and function, probably due to their highly dynamic behavior. In this work we report an extensive and detailed description of types, structure, components, dynamics and functionality of the TNTs bridging neighboring human umbilical cord MSCs obtained from Wharton"s jelly. Characterization studies were carried out through phase contrast, fluorescence, electron microscopy and time lapse images with the aim of describing cells suitable for an eventual regenerative medicine.
Assuntos
Comunicação Celular , Células-Tronco Mesenquimais/metabolismo , Nanotubos/química , Humanos , Células-Tronco Mesenquimais/citologiaRESUMO
Differential expression and activity of constitutive mitochondrial nitric oxide synthase (mtNOS) in the mitochondrial compartment is followed by significant variations in matrix nitric oxide (NO) steady-state concentration. The mitochondrial utilization of NO involves the production of superoxide anion and H(2)O(2), a species freely diffusible outside the mitochondria that participates in the modulation of cell proliferation and apoptosis and in cell transformation and cancer. On these bases, we analyzed the modulation of mtNOS in the frame of cellular redox state in M3, MM3, and P07 murine tumors and their respective cell lines, as compared with normal proliferating and quiescent tissues. The results showed that: (a) tumoral and proliferating mitochondria only retain 10-50% of the activity of complexes I, II-III, and IV and Mn-SOD of quiescent tissues; (b) normal proliferating tissues, like embryonic liver or pregnant mammary gland, have 10-20% of mtNOS expression and activity and mitochondrial H(2)O(2) yield than quiescent nonproliferating tissues; (c) similarly but irrespective of mtNOS expression, tumoral mitochondria have no >5% of mtNOS activity and H(2)O(2) yield of mature tissues; and (d) in opposition to stable tissues, both tumoral and normal proliferating cells exhibit high cyclin D1 expression and low pro-apoptotic p38mitogen-activated protein kinase activity. Dually, H(2)O(2) stimulated tumor cell proliferation (<10 microM) or markedly inhibited it (>10 microM) with parallel variations of cyclin D1, phospho-extracellular-regulated kinase1/2, and phospho-p38mitogen-activated protein kinase. It is surmised that decreased oxidative phosphorylation, defective tumoral mtNOS, and low mitochondrial NO-dependent H(2)O(2) may be a platform to link persistent tumoral growth to embryonic behavior.
Assuntos
Adenocarcinoma/enzimologia , Peróxido de Hidrogênio/metabolismo , Neoplasias Mamárias Experimentais/enzimologia , Mitocôndrias/enzimologia , Óxido Nítrico Sintase/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Ciclina D1/biossíntese , Feminino , Fígado/citologia , Fígado/embriologia , Fígado/enzimologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Gravidez , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Thioredoxin-1 (Trx1) protects the heart from ischemia/reperfusion (I/R) injury. Given that the age at which the first episode of coronary disease takes place has considerably decreased, life at middle-aged (MA) emerges as a new field of study. The aim was determine whether infarct size, Trx1 expression and activity, Akt and GSK-3ß were altered in young (Y) and MA mice overexpressing cardiac Trx1, and in a dominant negative (DN-Trx1) mutant of Trx1. Langendorff-perfused hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion (R). We used 3 and 12 month-old male of wild type (WT), Trx1, and DN-Trx1. Trx1 overexpression reduced infarct size in young mice (WT-Y: 46.8±4.1% vs. Trx1-Y: 27.6±3.5%, p < 0.05). Trx1 activity was reduced by 52.3±3.2% (p < 0.05) in Trx1-MA, accompanied by an increase in nitration by 17.5±0.9%, although Trx1 expression in transgenic mice was similar between young and middle-aged. The expression of p-Akt and p-GSK-3ß increased during reperfusion in Trx1-Y. DN-Trx1 mice showed neither reduction in infarct size nor Akt and GSK-3ß phosphorylation. Our data suggest that the lack of protection in Trx1 middle-aged mice even with normal Trx1 expression may be associated to decreased Trx1 activity, increased nitration and inhibition of p-Akt and p-GSK-3ß.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/fisiologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Estresse Oxidativo , Tiorredoxinas/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Traumatismo por Reperfusão Miocárdica/patologia , Fosforilação , Tiorredoxinas/genéticaRESUMO
OBJECTIVES: To determine clinical characteristics and frequency of leucine-rich repeat kinase 2 gene (LRRK2) mutations in a cohort of patients with Parkinson's disease (PD) from Argentina. BACKGROUND: Variation in the LRRK2 gene represents the most common genetic determinant of PD, only few data are available from Latin-America. DESIGN/METHODS: Informed consent was obtained and all studies were approved by the Institutional Review Boards. Fifty five consecutive PD patients were recruited. A structured interview and neurological examination were used to collect demographic and clinical information. Blood samples were obtained and DNA extracted from patient venous blood. All LRRK2 exons from 25 exon to 51 exon were screened in all patients. RESULTS: Clinical and molecular data of 55 patients with PD were analyzed. Mean age was 68.8±10.6 years. Jewish and Basque ancestries were found positive in 9 and 7 patients, respectively; family history of PD was identified in 16 patients. The G2019S mutation was present in 3 Ashkenazi Jewish subjects (5.45%); all of them reported family history of PD in first-degree relatives. Although Argentina possesses one of the most important Basque communities outside Spain, non R1414G mutation was identified in this cohort. Eleven single polymorphisms (SNP) were identified in this cohort. The mean age at onset was higher in G2019S mutation carriers than non-carriers (66.67 vs 58.78 years). Asymmetrical tremor as initial symptom and non-motor symptoms occurred at similar frequencies in both groups. The G2019S mutation carriers showed a non significant increase in dyskinesias, and 2/3 developed Dopamine Dysregulation Syndrome and visual hallucinations. Systemic disorder identified in G2019S mutation carriers included: celiac disease, hypothyroidism, Hashimoto's Thyroiditis and arterial hypertension. CONCLUSIONS: The prevalence of LRRK2 G2019S mutation in this Argentinean cohort was similar to other international series, with a higher prevalence in Ashkenazi Jewish. The phenotype was indistinguishable from patients with idiopathic PD. Interestingly, we identified immune mediated disorders in two PD patients carrying the G2019S mutation. Within this context, recent studies have identified full-length LRRK2 as a relatively common constituent of many cell types in the immune system including human peripheral blood mononuclear cells. Nevertheless, a casual association could not be excluded and the analysis of more extensive series is required.
Assuntos
Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Argentina , Feminino , Heterozigoto , Humanos , Judeus , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Pessoa de Meia-Idade , Mutação , Doença de Parkinson/etnologia , Doença de Parkinson/fisiopatologia , Linhagem , População BrancaRESUMO
Eukaryotic mitochondria resulted from symbiotic incorporation of α-proteobacteria into ancient archaea species. During evolution, mitochondria lost most of the prokaryotic bacterial genes and only conserved a small fraction including those encoding 13 proteins of the respiratory chain. In this process, many functions were transferred to the host cells, but mitochondria gained a central role in the regulation of cell proliferation and apoptosis, and in the modulation of metabolism; accordingly, defective organelles contribute to cell transformation and cancer, diabetes, and neurodegenerative diseases. Most cell and transcriptional effects of mitochondria depend on the modulation of respiratory rate and on the production of hydrogen peroxide released into the cytosol. The mitochondrial oxidative rate has to remain depressed for cell proliferation; even in the presence of O2, energy is preferentially obtained from increased glycolysis (Warburg effect). In response to stress signals, traffic of pro- and antiapoptotic mitochondrial proteins in the intermembrane space (B-cell lymphoma-extra large, Bcl-2-associated death promoter, Bcl-2 associated X-protein and cytochrome c) is modulated by the redox condition determined by mitochondrial O2 utilization and mitochondrial nitric oxide metabolism. In this article, we highlight the traffic of the different canonical signaling pathways to mitochondria and the contributions of organelles to redox regulation of kinases. Finally, we analyze the dynamics of the mitochondrial population in cell cycle and apoptosis.
Assuntos
Ciclo Celular , Mitocôndrias/fisiologia , Animais , Morte Celular , Proliferação de Células , Humanos , Oxirredução , Fosfotransferases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
The subcellular localization and physiological functions of biomolecules are closely related and thus it is crucial to precisely determine the distribution of different molecules inside the intracellular structures. This is frequently accomplished by fluorescence microscopy with well-characterized markers and posterior evaluation of the signal colocalization. Rigorous study of colocalization requires statistical analysis of the data, albeit yet no single technique has been established as a standard method. Indeed, the few methods currently available are only accurate in images with particular characteristics. Here, we introduce a new algorithm to automatically obtain the true colocalization between images that is suitable for a wide variety of biological situations. To proceed, the algorithm contemplates the individual contribution of each pixel's fluorescence intensity in a pair of images to the overall Pearsons correlation and Manders' overlap coefficients. The accuracy and reliability of the algorithm was validated on both simulated and real images that reflected the characteristics of a range of biological samples. We used this algorithm in combination with image restoration by deconvolution and time-lapse confocal microscopy to address the localization of MEK1 in the mitochondria of different cell lines. Appraising the previously described behavior of Akt1 corroborated the reliability of the combined use of these techniques. Together, the present work provides a novel statistical approach to accurately and reliably determine the colocalization in a variety of biological images.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Sistema de Sinalização das MAP Quinases/fisiologia , Mitocôndrias/metabolismo , Algoritmos , Animais , Biomarcadores , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Células NIH 3T3 , Plasmídeos/metabolismo , Fatores de TempoAssuntos
Endotoxemia/metabolismo , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Animais , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/patologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ácido Peroxinitroso/metabolismo , Distribuição TecidualRESUMO
Phylogenetic studies had shown that evolution of mitochondria occurred in parallel with the maturation of kinases implicated in growth and final size of modern organisms. In the last years, different reports confirmed that MAPKs, Akt, PKA and PKC are present in mitochondria, particularly in the intermembrane space and inner membrane where they meet mitochondrial constitutive upstream activators. Although a priori phosphorylation is the apparent aim of translocation, new perspectives indicate that kinase activation depends on redox status as determined by the mitochondrial production of oxygen species. We observed that the degree of mitochondrial oxidation of ERK Cys(38) and Cys(214) discriminates the kinase to be phosphorylated and determines translocation to the nuclear compartment and proliferation, or accumulation in mitochondria and arrest. Otherwise, transcriptional gene regulation by Akt depends on Cys(60) and Cys(310) oxidation to sulfenic and sulfonic acids. It is concluded that the interactions between kinases and mitochondria control cell signaling pathways and participate in the modulation of cell proliferation and arrest, tissue protection, tumorigenesis and cancer progression.
Assuntos
Mitocôndrias/enzimologia , Mitocôndrias/fisiologia , Proteínas Quinases/fisiologia , Transdução de Sinais/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Humanos , Modelos Biológicos , Infarto do Miocárdio/metabolismo , Neoplasias/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser(473) by mTORC2 and Thr(308) by PDK1. On these bases, we investigated the mechanistic connection of H(2)O(2) yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H(2)O(2) entails the entrance of cytosolic P-Akt1 Ser(473) to mitochondria, where it is further phosphorylated at Thr(308) by constitutive PDK1. Phosphorylation of Thr(308) in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H(2)O(2), Akt1-PDK1 association is disrupted and P-Akt1 Ser(473) accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H(2)O(2) effects on Akt1-PDK interaction depend on the selective oxidation of Cys(310) to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.
Assuntos
Mitocôndrias/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose , Ciclo Celular , Linhagem da Célula , Ácido Cisteico/química , Citosol/metabolismo , Peróxido de Hidrogênio/química , Camundongos , Modelos Biológicos , Células NIH 3T3 , Fosforilação , Ácidos Sulfênicos/químicaRESUMO
Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Sequência de Aminoácidos , Proliferação de Células , Perfilação da Expressão Gênica , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Mitochondria are major cellular sources of hydrogen peroxide (H(2)O(2)), the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H(2)O(2) concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H(2)O(2) due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs) orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H(2)O(2) yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 microM H(2)O(2) increased cell proliferation in ERK1/2-dependent manner whereas high 50 microM H(2)O(2) arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei), the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs) facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H(2)O(2) level. Like this, high H(2)O(2) or directed mutation of redox-sensitive ERK2 Cys(214) impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to increase H(2)O(2) yield should disrupt synchronized MAPK oxidations and the regulation of cell cycle leading cells to remain in a proliferating phenotype.
Assuntos
Mitocôndrias/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias/patologia , Animais , Catálise , Ciclo Celular , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Camundongos , Neoplasias/enzimologia , Oxirredução , Fenótipo , Fosforilação , Transporte Proteico , Transdução de SinaisRESUMO
Mitochondria are the specialized organelles for energy metabolism, but, as a typical example of system biology, they also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or oppositely promote cell arrest and programmed cell death by a limited number of oxidative or nitrosative reactions. These reactions are influenced by matrix nitric oxide (NO) steady-state concentration, either from local production or by gas diffusion to mitochondria from the canonical sources. Likewise, in a range of approximately 30-200 nM, NO turns mitochondrial O(2) utilization down by binding to cytochrome oxidase and elicits a burst of superoxide anion and hydrogen peroxide that diffuses outside mitochondria. Depending on NO levels and antioxidant defenses, more or less H(2)O(2) accumulates in cytosol and nucleus, and the resulting redox grading contributes to dual activation of proliferating and proapoptotic cascades, like ERK1/2 or p38 MAPK. Moreover, these sequential activating pathways participate in rat liver and brain development and in thyroid modulation of mitochondrial metabolism and contribute to hypothyroid phenotype through complex I nitration. On the contrary, lack of NO disrupts pathways like S-nitrosylation or H(2)O(2) production and likewise is a gateway to disease in amyotrophic lateral sclerosis with superoxide dismutase 1 mutations or to cancer proliferation.