Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
1.
Carcinogenesis ; 41(12): 1735-1745, 2020 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-32255484

RESUMO

Functioning mitochondria are crucial for cancer metabolism, but aerobic glycolysis is still considered to be an important pathway for energy production in many tumor cells. Here we show that two well established, classic Hodgkin lymphoma (cHL) cell lines harbor deleterious variants within mitochondrial DNA (mtDNA) and thus exhibit reduced steady-state levels of respiratory chain complexes. However, instead of resulting in the expected bioenergetic defect, these mtDNA variants evoke a retrograde signaling response that induces mitochondrial biogenesis and ultimately results in increased mitochondrial mass as well as function and enhances proliferation in vitro as well as tumor growth in mice in vivo. When complex I assembly was impaired by knockdown of one of its subunits, this led to further increased mitochondrial mass and function and, consequently, further accelerated tumor growth in vivo. In contrast, inhibition of mitochondrial respiration in vivo by the mitochondrial complex I inhibitor metformin efficiently slowed down growth. We conclude that, as a new mechanism, mildly deleterious mtDNA variants in cHL cancer cells cause an increase of mitochondrial mass and enhanced function as a compensatory effect using a retrograde signaling pathway, which provides an obvious advantage for tumor growth.


Assuntos
Carcinogênese/patologia , DNA Mitocondrial/genética , Doença de Hodgkin/patologia , Mutação , Biogênese de Organelas , Animais , Apoptose , Carcinogênese/genética , Carcinogênese/metabolismo , Proliferação de Células , Doença de Hodgkin/genética , Doença de Hodgkin/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação Oxidativa , Células de Reed-Sternberg , Ensaios Antitumorais Modelo de Xenoenxerto
2.
EMBO J ; 35(23): 2566-2583, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27797820

RESUMO

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Animais , Células Cultivadas , Fibroblastos/fisiologia , Camundongos , Camundongos Knockout , Biossíntese de Proteínas
3.
Hum Mol Genet ; 23(23): 6345-55, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25008111

RESUMO

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.


Assuntos
Proteínas Mitocondriais/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Proteínas Mitocondriais/genética , Mutação , Proteínas de Neoplasias/metabolismo , Fosforilação Oxidativa , Polinucleotídeo Adenililtransferase/genética , Cultura Primária de Células , Processamento Pós-Transcricional do RNA , RNA Mitocondrial , Proteínas de Ligação a RNA/metabolismo
4.
EMBO J ; 31(5): 1293-307, 2012 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-22252130

RESUMO

Respiratory chain (RC) complexes are organized into supercomplexes forming 'respirasomes'. The mechanism underlying the interdependence of individual complexes is still unclear. Here, we show in human patient cells that the presence of a truncated COX1 subunit leads to destabilization of complex IV (CIV) and other RC complexes. Surprisingly, the truncated COX1 protein is integrated into subcomplexes, the holocomplex and even into supercomplexes, which however are all unstable. Depletion of the m-AAA protease AFG3L2 increases stability of the truncated COX1 and other mitochondrially encoded proteins, whereas overexpression of wild-type AFG3L2 decreases their stability. Both full-length and truncated COX1 proteins physically interact with AFG3L2. Expression of a dominant negative AFG3L2 variant also promotes stabilization of CIV proteins as well as the assembled complex and rescues the severe phenotype in heteroplasmic cells. Our data indicate that the mechanism underlying pathogenesis in these patients is the rapid clearance of unstable respiratory complexes by quality control pathways, rather than their impaired assembly.


Assuntos
Proteases Dependentes de ATP/metabolismo , Códon sem Sentido , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Células Cultivadas , Ciclo-Oxigenase 1/química , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/química , Estabilidade Enzimática , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica
5.
Clin Sci (Lond) ; 128(12): 895-904, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25626417

RESUMO

Complex I (CI) is the largest of the five multi-subunit complexes constituting the human oxidative phosphorylation (OXPHOS) system. Seven of its catalytic core subunits are encoded by mitochondrial DNA (ND (NADH dehydrogenase)1-6, ND4L (NADH dehydrogenase subunit 4L)), with mutations in all seven having been reported in association with isolated CI deficiency. We investigated two unrelated adult patients presenting with marked exercise intolerance, persistent lactic acidaemia and severe muscle-restricted isolated CI deficiency associated with sub-sarcolemmal mitochondrial accumulation. Screening of the mitochondrial genome detected novel mutations in the MTND1 (NADH dehydrogenase subunit 1) gene, encoding subunit of CI [Patient 1, m.3365T>C predicting p.(Leu20Pro); Patient 2, m.4175G>A predicting p.(Trp290*)] at high levels of mitochondrial DNA heteroplasmy in skeletal muscle. We evaluated the effect of these novel MTND1 mutations on complex assembly showing that CI assembly, although markedly reduced, was viable in the absence of detectable ND1 signal. Real-time PCR and Western blotting showed overexpression of different CI assembly factor transcripts and proteins in patient tissue. Together, our data indicate that the mechanism underlying the expression of the biochemical defect may involve a compensatory response to the novel MTND1 gene mutations, promoting assembly factor up-regulation and stabilization of respiratory chain super-complexes, resulting in partial rescue of the clinical phenotype.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Tolerância ao Exercício/genética , Miopatias Mitocondriais/genética , Mutação , NADH Desidrogenase/genética , Adolescente , DNA Mitocondrial/genética , Teste de Esforço/métodos , Feminino , Humanos , Miopatias Mitocondriais/enzimologia , Músculo Esquelético/enzimologia , Linhagem , Adulto Jovem
6.
J Med Genet ; 49(9): 569-77, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22972948

RESUMO

BACKGROUND: Isolated complex II deficiency is a rare form of mitochondrial disease, accounting for approximately 2% of all respiratory chain deficiency diagnoses. The succinate dehydrogenase (SDH) genes (SDHA, SDHB, SDHC and SDHD) are autosomally-encoded and transcribe the conjugated heterotetramers of complex II via the action of two known assembly factors (SDHAF1 and SDHAF2). Only a handful of reports describe inherited SDH gene defects as a cause of paediatric mitochondrial disease, involving either SDHA (Leigh syndrome, cardiomyopathy) or SDHAF1 (infantile leukoencephalopathy). However, all four SDH genes, together with SDHAF2, have known tumour suppressor functions, with numerous germline and somatic mutations reported in association with hereditary cancer syndromes, including paraganglioma and pheochromocytoma. METHODS AND RESULTS: Here, we report the clinical and molecular investigations of two patients with histochemical and biochemical evidence of a severe, isolated complex II deficiency due to novel SDH gene mutations; the first patient presented with cardiomyopathy and leukodystrophy due to compound heterozygous p.Thr508Ile and p.Ser509Leu SDHA mutations, while the second patient presented with hypotonia and leukodystrophy with elevated brain succinate demonstrated by MR spectroscopy due to a novel, homozygous p.Asp48Val SDHB mutation. Western blotting and BN-PAGE studies confirmed decreased steady-state levels of the relevant SDH subunits and impairment of complex II assembly. Evidence from yeast complementation studies provided additional support for pathogenicity of the SDHB mutation. CONCLUSIONS: Our report represents the first example of SDHB mutation as a cause of inherited mitochondrial respiratory chain disease and extends the SDHA mutation spectrum in patients with isolated complex II deficiency.


Assuntos
Complexo II de Transporte de Elétrons/deficiência , Genes Recessivos/genética , Mutação em Linhagem Germinativa/genética , Leucoencefalopatias/genética , Erros Inatos do Metabolismo/genética , Doenças Mitocondriais/genética , Succinato Desidrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Encéfalo/patologia , Pré-Escolar , Transporte de Elétrons , Complexo II de Transporte de Elétrons/química , Complexo II de Transporte de Elétrons/genética , Feminino , Teste de Complementação Genética , Humanos , Lactente , Recém-Nascido , Leucoencefalopatias/complicações , Imageamento por Ressonância Magnética , Masculino , Erros Inatos do Metabolismo/complicações , Erros Inatos do Metabolismo/enzimologia , Doenças Mitocondriais/complicações , Doenças Mitocondriais/enzimologia , Dados de Sequência Molecular , Músculo Esquelético/patologia , Mutação/genética , Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/química
7.
Anal Biochem ; 389(1): 1-5, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19285029

RESUMO

Isolation of mitochondria by current methods relies mainly on their physicochemical properties. Here we describe an alternative approach to obtain functional mitochondria from human cells in a fast, reproducible, and standardized procedure. The new approach is based on superparamagnetic microbeads conjugated to anti-TOM22 antibody. The bead conjugates label the cytoplasmic part of the human mitochondrial membrane protein TOM22 and, thus, allow for a gentle isolation of mitochondria in a high gradient magnetic field. By comparing the MACS (magnetic cell separation) approach with mitochondria isolation methods using differential centrifugation and ultracentrifugation we demonstrate that the MACS approach provides the highest yield of isolated mitochondria. The quality, enrichment, and purity of mitochondria isolated with this protocol are comparable to mitochondria obtained using the ultracentrifuge method, and a typical separation procedure takes only approximately 1 to 2h from initial cell homogenization. Mitochondria isolated with the new approach are sufficient for protein import, blue native gel electrophoresis, and other mitochondrial assays.


Assuntos
Separação Imunomagnética/métodos , Magnetismo , Microesferas , Mitocôndrias , Anticorpos/química , Citometria de Fluxo , Humanos , Proteínas de Membrana/imunologia , Proteínas Mitocondriais/imunologia
8.
J Invest Dermatol ; 138(1): 132-140, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28867657

RESUMO

Accumulation of large-scale mitochondrial DNA (mtDNA) deletions and chronic, subclinical inflammation are concomitant during skin aging, thus raising the question of a causal link. To approach this, we generated mice expressing a mutant mitochondrial helicase (K320E-TWINKLE) in the epidermis to accelerate the accumulation of mtDNA deletions in this skin compartment. Mice displayed low amounts of large-scale deletions and a dramatic depletion of mtDNA in the epidermis and showed macroscopic signs of severe skin inflammation. The mtDNA alterations led to an imbalanced stoichiometry of mitochondrial respiratory chain complexes, inducing a unique combination of cytokine expression, causing a severe inflammatory phenotype, with massive immune cell infiltrates already before birth. Altogether, these data unraveled a previously unknown link between an imbalanced stoichiometry of the mitochondrial respiratory chain complexes and skin inflammation and suggest that severe respiratory chain dysfunction, as observed in few cells leading to a mosaic in aged tissues, might be involved in the development of chronic subclinical inflammation.


Assuntos
DNA Helicases/metabolismo , DNA Mitocondrial/metabolismo , Dermatite/imunologia , Epiderme/imunologia , Mitocôndrias/imunologia , Proteínas Mitocondriais/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , DNA Helicases/genética , Dermatite/genética , Dermatite/patologia , Modelos Animais de Doenças , Transporte de Elétrons/genética , Transporte de Elétrons/imunologia , Embrião de Mamíferos , Epiderme/patologia , Feminino , Humanos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/imunologia , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , Cultura Primária de Células , Envelhecimento da Pele/genética , Envelhecimento da Pele/imunologia
9.
Free Radic Biol Med ; 42(4): 499-509, 2007 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17275682

RESUMO

Ultraviolet A (UVA), the long wavelength part of the sun's ultraviolet radiation, elicits its harmful effects through production of reactive oxygen species. In this study, we have tested the hypothesis that the mitochondrial electron transport chain, the main source of reactive oxygen species in cells, importantly contributes to UVA-induced cell damage. Model cell lines completely lacking a mitochondrial electron transport chain (rho(0)-cells) were not protected against UVA-induced cell death. Also, primary human fibroblasts and keratinocytes with induced depletion of electron transport chain activity were not better protected against UVA-induced cell death. On the other hand, diphenyleneiodonium and resiniferatoxin, inhibitors of plasma membrane oxidases, protected primary human fibroblasts against UVA, as potently as the lipid peroxidation chain breaker Trolox. These data indicate that plasma membrane electron transport systems, but not the mitochondrial electron transport chain, play a major role in UVA-induced cell death.


Assuntos
Morte Celular/efeitos da radiação , Mitocôndrias/efeitos da radiação , Raios Ultravioleta , Transporte de Elétrons , Citometria de Fluxo , Células HeLa , Humanos , Mitocôndrias/metabolismo , Consumo de Oxigênio , Espécies Reativas de Oxigênio
10.
Cell Metab ; 25(3): 698-712, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28132808

RESUMO

Ca2+ signals were reported to control lipid homeostasis, but the Ca2+ channels and pathways involved are largely unknown. Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca2+ channel ORAI1. We show that SOCE-deficient mice accumulate pathological amounts of lipid droplets in the liver, heart, and skeletal muscle. Cells from patients with loss-of-function mutations in STIM1 or ORAI1 show a similar phenotype, suggesting a cell-intrinsic role for SOCE in the regulation of lipid metabolism. SOCE is crucial to induce mobilization of fatty acids from lipid droplets, lipolysis, and mitochondrial fatty acid oxidation. SOCE regulates cyclic AMP production and the expression of neutral lipases as well as the transcriptional regulators of lipid metabolism, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), and peroxisome proliferator-activated receptor α (PPARα). SOCE-deficient cells upregulate lipophagy, which protects them from lipotoxicity. Our data provide evidence for an important role of SOCE in lipid metabolism.


Assuntos
Cálcio/metabolismo , Lipólise/genética , Transcrição Gênica , Adenilil Ciclases/metabolismo , Animais , Ácidos Graxos/metabolismo , Células HEK293 , Humanos , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Camundongos , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Oxirredução , PPAR alfa/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/genética , Regulação para Cima/genética
11.
J Cell Biol ; 211(5): 1057-75, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26644517

RESUMO

Keratin intermediate filaments (KIFs) protect the epidermis against mechanical force, support strong adhesion, help barrier formation, and regulate growth. The mechanisms by which type I and II keratins contribute to these functions remain incompletely understood. Here, we report that mice lacking all type I or type II keratins display severe barrier defects and fragile skin, leading to perinatal mortality with full penetrance. Comparative proteomics of cornified envelopes (CEs) from prenatal KtyI(-/-) and KtyII(-/-)(K8) mice demonstrates that absence of KIF causes dysregulation of many CE constituents, including downregulation of desmoglein 1. Despite persistence of loricrin expression and upregulation of many Nrf2 targets, including CE components Sprr2d and Sprr2h, extensive barrier defects persist, identifying keratins as essential CE scaffolds. Furthermore, we show that KIFs control mitochondrial lipid composition and activity in a cell-intrinsic manner. Therefore, our study explains the complexity of keratinopathies accompanied by barrier disorders by linking keratin scaffolds to mitochondria, adhesion, and CE formation.


Assuntos
Epiderme/metabolismo , Queratinas/metabolismo , Lipídeos/química , Mitocôndrias/metabolismo , Animais , Adesão Celular , Membrana Celular/metabolismo , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteoma/metabolismo , Fatores de Transcrição/metabolismo
12.
EMBO Mol Med ; 6(2): 183-93, 2014 02.
Artigo em Inglês | MEDLINE | ID: mdl-24413189

RESUMO

Disorders of the mitochondrial genome cause a wide spectrum of disease, these present mainly as neurological and/or muscle related pathologies. Due to the intractability of the human mitochondrial genome there are currently no effective treatments for these disorders. The majority of the pathogenic mutations lie in the genes encoding mitochondrial tRNAs. Consequently, the biochemical deficiency is due to mitochondrial protein synthesis defects, which manifest as aberrant cellular respiration and ATP synthesis. It has previously been reported that overexpression of mitochondrial aminoacyl tRNA synthetases has been effective, in cell lines, at partially suppressing the defects resulting from mutations in their cognate mt-tRNAs. We now show that leucyl tRNA synthetase is able to partially rescue defects caused by mutations in non-cognate mt-tRNAs. Further, a C terminal peptide alone can enter mitochondria and interact with the same spectrum of mt-tRNAs as the entire synthetase, in intact cells. These data support the possibility that a small peptide could correct at least the biochemical defect associated with many mt-tRNA mutations, inferring a novel therapy for these disorders.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Mitocôndrias/genética , Mutação/genética , RNA de Transferência de Leucina/genética , Supressão Genética , Aminoacil-tRNA Sintetases/química , Proliferação de Células , Humanos , Mitocôndrias/enzimologia , Fosforilação Oxidativa , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína
13.
EMBO Mol Med ; 6(5): 624-39, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24648500

RESUMO

In the normal quiescent vasculature, only 0.01% of endothelial cells (ECs) are proliferating. However, this proportion increases dramatically following the angiogenic switch during tumor growth or wound healing. Recent evidence suggests that this angiogenic switch is accompanied by a metabolic switch. Here, we show that proliferating ECs increasingly depend on mitochondrial oxidative phosphorylation (OxPhos) for their increased energy demand. Under growth conditions, ECs consume three times more oxygen than quiescent ECs and work close to their respiratory limit. The increased utilization of the proton motif force leads to a reduced mitochondrial membrane potential in proliferating ECs and sensitizes to mitochondrial uncoupling. The benzoquinone embelin is a weak mitochondrial uncoupler that prevents neoangiogenesis during tumor growth and wound healing by exhausting the low respiratory reserve of proliferating ECs without adversely affecting quiescent ECs. We demonstrate that this can be exploited therapeutically by attenuating tumor growth in syngenic and xenograft mouse models. This novel metabolic targeting approach might be clinically valuable in controlling pathological neoangiogenesis while sparing normal vasculature and complementing cytostatic drugs in cancer treatment.


Assuntos
Benzoquinonas/farmacologia , Respiração Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Desacopladores/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Camundongos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Desacopladores/uso terapêutico , Cicatrização/efeitos dos fármacos
15.
Mol Cell Biol ; 28(7): 2446-59, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18227154

RESUMO

To further understand pathways coordinating the expression of nuclear genes encoding mitochondrial proteins, we studied mitochondrial biogenesis during differentiation of myoblasts to myotubes. This energy-demanding process was accompanied by a fivefold increase of ATP turnover, covered by an eightfold increase of mitochondrial activity. While no change in mitochondrial DNA copy number was observed, mRNAs as well as proteins for nucleus-encoded cytochrome c, cytochrome c oxidase subunit IV, and mitochondrial transcription factor A (TFAM) increased, together with total cellular RNA and protein levels. Detailed analysis of the cytochrome c promoter by luciferase reporter, binding affinity, and electrophoretic mobility shift assays as well as mutagenesis studies revealed a critical role for cyclic AMP responsive element binding protein 1 (CREB-1) for promoter activation. Expression of two CREB-1 isoforms was observed by using specific antibodies and quantitative reverse transcription-PCR, and a shift from phosphorylated CREB-1Delta in myoblasts to phosphorylated CREB-1alpha protein in myotubes was shown, while mRNA ratios remained unchanged. Chromatin immunoprecipitation assays confirmed preferential binding of CREB-1alpha in situ to the cytochrome c promoter in myotubes. Overexpression of constitutively active and dominant-negative forms supported the key role of CREB-1 in regulating the expression of genes encoding mitochondrial proteins during myogenesis and probably also in other situations of enhanced mitochondrial biogenesis.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Citocromos c/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Mitocôndrias Musculares/metabolismo , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Fator 1 Ativador da Transcrição/genética , Fator 1 Ativador da Transcrição/metabolismo , Animais , Diferenciação Celular , Células Cultivadas/metabolismo , Citocromos c/biossíntese , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Genes Reporter , Humanos , Camundongos , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Desenvolvimento Muscular/genética , Consumo de Oxigênio , Fosforilação , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , Ratos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima
16.
J Invest Dermatol ; 127(5): 1084-93, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17185981

RESUMO

The energy metabolism of the epidermis has been the subject of controversy; thus we characterized the mitochondrial phenotype of human primary keratinocytes and fibroblasts, in cell culture and in human skin sections. We found that keratinocytes respire as much as fibroblasts, however, maximal activities of the respiratory chain (RC) complexes were 2- to 5-fold lower, whereas expression levels of RC proteins were similar. Maximal activities of aconitase and isocitrate dehydrogenase, two mitochondrial enzymes especially vulnerable to superoxide, were lower than in fibroblasts. Indeed, superoxide anion levels were much higher in keratinocytes, and keratinocytes displayed higher lipid peroxidation levels and a lower reduced glutathione/oxidized glutathione ratio, indicating enhanced oxidative stress. Although superoxide dismutase activity and especially expression of the mitochondrial superoxide dismutase, Mn-SOD, were drastically lower in keratinocytes, explaining the high superoxide levels, glutathione peroxidase activity and protein were almost undetectable in fibroblasts. Catalase activity and hydrogen peroxide levels were similar. In summary, we could show that keratinocytes actively use the mitochondrial RC not only for adenosine 5' triphosphate synthesis but also for the accumulation of superoxide anions, even at the expense of mitochondrial functional capacity, indicating that superoxide-driven mitochondrial impairment might be a prerequisite for keratinocyte differentiation.


Assuntos
Queratinócitos/metabolismo , Mitocôndrias/fisiologia , Superóxido Dismutase/fisiologia , Superóxidos/metabolismo , Aconitato Hidratase/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Criança , Transporte de Elétrons/fisiologia , Metabolismo Energético/fisiologia , Células Epidérmicas , Epiderme/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Isocitrato Desidrogenase/metabolismo , Queratinócitos/citologia , Peroxidação de Lipídeos/fisiologia , Masculino , Estresse Oxidativo/fisiologia
17.
Exp Cell Res ; 313(14): 3076-89, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17509565

RESUMO

Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca(2+) homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A>G and 3302A>G in tRNA(Leu(UUR)), as well as Rho(0) cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP+ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases.


Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Transporte de Elétrons/fisiologia , Glicólise/fisiologia , Mitocôndrias/metabolismo , Aminoácidos/metabolismo , Antioxidantes/metabolismo , Linhagem Celular , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Potenciais da Membrana/fisiologia , Oxirredução , Estresse Oxidativo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa