RESUMO
The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.
RESUMO
Chemotherapy can cause cachexia, which consists of weight loss associated with muscle atrophy. The exact mechanisms underlying this skeletal muscle toxicity are largely unknown and co-therapies to attenuate chemotherapy-induced side effects are lacking. By using a rat model of cisplatin-induced cachexia, we here characterized the mitochondrial homeostasis in tibialis anterior cachectic muscle and evaluated the potential beneficial effects of the growth hormone secretagogues (GHS) hexarelin and JMV2894 in this setting. We found that cisplatin treatment caused a decrease in mitochondrial biogenesis (PGC-1α, NRF-1, TFAM, mtDNA, ND1), mitochondrial mass (Porin and Citrate synthase activity) and fusion index (MFN2, Drp1), together with changes in the expression of autophagy-related genes (AKT/FoxO pathway, Atg1, Beclin1, LC3AII, p62) and enhanced ROS production (PRX III, MnSOD). Importantly, JMV2894 and hexarelin are capable to antagonize this chemotherapy-induced mitochondrial dysfunction. Thus, our findings reveal a key-role played by mitochondria in the mechanism responsible for GHS beneficial effects in skeletal muscle, strongly indicating that targeting mitochondrial dysfunction might be a promising area of research in developing therapeutic strategies to prevent or limit muscle wasting in cachexia.
Assuntos
Caquexia/induzido quimicamente , Cisplatino/efeitos adversos , Hormônio do Crescimento/farmacologia , Indóis/farmacologia , Mitocôndrias/patologia , Músculo Esquelético/metabolismo , Oligopeptídeos/farmacologia , Piperidinas/farmacologia , Secretagogos/farmacologia , Triazóis/farmacologia , Animais , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Caquexia/patologia , Modelos Animais de Doenças , Proteína Forkhead Box O3/metabolismo , Hormônio do Crescimento/administração & dosagem , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Biogênese de Organelas , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Secretagogos/administração & dosagemRESUMO
We previously reported the ability of dietary supplementation with acetyl-l-carnitine (ALCAR) to prevent age-related decreases of mitochondrial biogenesis in skeletal muscle and liver of old rats. Here, we investigate the effects of ALCAR supplementation in cerebral hemispheres and cerebellum of old rats by analyzing several parameters linked to mitochondrial biogenesis, mitochondrial dynamics and antioxidant defenses. We measured the level of the coactivators PGC-1α and PGC-1ß and of the factors regulating mitochondrial biogenesis, finding an age-related decrease of PGC-1ß, whereas PGC-1α level was unvaried. Twenty eight-month old rats supplemented with ALCAR for one and two months showed increased levels of both factors. Accordingly, the expression of the two transcription factors NRF-1 and TFAM followed the same trend of PGC-1ß. The level of mtDNA, ND1 and the activity of citrate synthase, were decreased with aging and increased following ALCAR treatment. Furthermore, ALCAR counteracted the age-related increase of deleted mtDNA. We also analyzed the content of proteins involved in mitochondrial dynamics (Drp1, Fis1, OPA1 and MNF2) and found an age-dependent increase of MFN2 and of the long form of OPA1. ALCAR treatment restored the content of the two proteins to the level of the young rats. No changes with aging and ALCAR were observed for Drp1 and Fis1. ALCAR reduced total cellular levels of oxidized PRXs and counteracted the age-related decrease of PRX3 and SOD2. Overall, our findings indicate a systemic positive effect of ALCAR dietary treatment and a tissue specific regulation of mitochondrial homeostasis in brain of old rats. Moreover, it appears that ALCAR acts as a nutrient since in most cases its effects were almost completely abolished one month after treatment suspension. Dietary supplementation of old rats with this compound seems a valuable approach to prevent age-related mitochondrial dysfunction and might ultimately represent a strategy to delay age-associated negative consequences in mitochondrial homeostasis.
Assuntos
Acetilcarnitina/farmacologia , Envelhecimento/metabolismo , Antioxidantes/metabolismo , Encéfalo/efeitos dos fármacos , Suplementos Nutricionais , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Biogênese de Organelas , Fatores Etários , Envelhecimento/genética , Envelhecimento/patologia , Animais , Encéfalo/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Mutação , Estresse Oxidativo/efeitos dos fármacos , Ratos Endogâmicos F344 , Fatores de Transcrição/metabolismoRESUMO
Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM.
Assuntos
DNA Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Ouriços-do-Mar/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Transferência Ressonante de Energia de Fluorescência , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
GST fusion proteins expressed in bacteria often tend to form aggregates and are inefficiently purified by standard procedures, which employ a mixture of detergents that compromise the binding efficiency to the affinity resin and the biological activity of the recombinant proteins. Moreover, the binding to the resin is negatively affected by the molecular weight of the fusion protein. Here we report a simple and efficient method to purify active large GST-tagged proteins, which uses high ionic strength buffer to solubilize the protein aggregates in a bacterial lysate. Affinity-chromatography purification is achieved by adopting two columns connected in series, which facilitate the binding of large GST fused molecules. This approach was applied to purify the 180-kDa GST-tagged mitochondrial RNA polymerase. We also report conditions for simple and efficient GST tag removal from the eluted protein. Finally we demonstrate that the recombinant enzyme is capable to catalyze RNA synthesis.
Assuntos
Glutationa Transferase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Cromatografia de Afinidade , Glutationa Transferase/isolamento & purificação , Concentração Osmolar , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber's hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber's hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies.
Assuntos
DNA Mitocondrial/genética , Renovação Mitocondrial/genética , Atrofia Óptica Hereditária de Leber/diagnóstico , Atrofia Óptica Hereditária de Leber/genética , Penetrância , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Adulto JovemRESUMO
Sea urchin mtDNA is transcribed via a different mechanism compared to vertebrates. To gain information on the apparatus of sea urchin mitochondrial transcription we have characterized the DNA binding properties of the mitochondrial transcription factor A (TFAM). The protein contains two HMG box domains but, differently from vertebrates, displays a very short C-terminal tail. Phylogenetic analysis showed that the distribution of tail length is mixed in the different lineages, indicating that it is a trait that undergoes rapid changes during evolution. Homology modeling suggests that the protein adopts the same configuration of the human counterpart and possibly a similar mode of binding to DNA. DNase I footprinting showed that TFAM specifically contacts mtDNA at a fixed distance from three AT-rich consensus sequences that were supposed to act as transcriptional initiation sites. Bound sequences are homologous and contain an inverted repeat motif, which resembles that involved in the intercalation of human TFAM in LSP DNA. The here reported data indicate that sea urchin TFAM specifically binds mtDNA. The protein could intercalate residues at the DNA inverted motif and, despite its short tail, might have a role in mitochondrial transcription.
Assuntos
DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ouriços-do-Mar/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Pegada de DNA , Proteínas de Ligação a DNA/química , Variação Genética , Proteínas Mitocondriais/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/químicaRESUMO
DREF [DRE (DNA replication-related element)-binding factor] controls the transcription of numerous genes in Drosophila, many involved in nuclear DNA (nDNA) replication and cell proliferation, three in mitochondrial DNA (mtDNA) replication and two in mtDNA transcription termination. In this work, we have analysed the involvement of DREF in the expression of the known remaining genes engaged in the minimal mtDNA replication (d-mtDNA helicase) and transcription (the activator d-mtTFB2) machineries and of a gene involved in mitochondrial mRNA translation (d-mtTFB1). We have identified their transcriptional initiation sites and DRE sequences in their promoter regions. Gel-shift and chromatin immunoprecipitation assays demonstrate that DREF interacts in vitro and in vivo with the d-mtDNA helicase and d-mtTFB2, but not with the d-mtTFB1 promoters. Transient transfection assays in Drosophila S2 cells with mutated DRE motifs and truncated promoter regions show that DREF controls the transcription of d-mtDNA helicase and d-mtTFB2, but not that of d-mtTFB1. RNA interference of DREF in S2 cells reinforces these results showing a decrease in the mRNA levels of d-mtDNA helicase and d-mtTFB2 and no changes in those of the d-mtTFB1. These results link the genetic regulation of nuclear DNA replication with the genetic control of mtDNA replication and transcriptional activation in Drosophila.
Assuntos
DNA Helicases/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Animais , Western Blotting , Núcleo Celular , Imunoprecipitação da Cromatina , DNA Helicases/metabolismo , Proteínas de Drosophila/genética , Ensaio de Desvio de Mobilidade Eletroforética , Luciferases , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Aging markedly affects mitochondrial biogenesis and functions particularly in tissues highly dependent on the organelle's bioenergetics capability such as the brain's frontal cortex. Calorie restriction (CR) diet is, so far, the only intervention able to delay or prevent the onset of several age-related alterations in different organisms. We determined the contents of mitochondrial transcription factor A (TFAM), mitochondrial DNA (mtDNA), and the 4.8-kb mtDNA deletion in the frontal cortex from young (6-month-old) and aged (26-month-old), ad libitum-fed (AL) and calorie-restricted (CR), rats. We found a 70 % increase in TFAM amount, a 25 % loss in mtDNA content, and a 35 % increase in the 4.8-kb deletion content in the aged AL animals with respect to the young rats. TFAM-specific binding to six mtDNA regions was analyzed by mtDNA immunoprecipitation and semiquantitative polymerase chain reaction (PCR), showing a marked age-related decrease. Quantitative real-time PCR at two subregions involved in mtDNA replication demonstrated, in aged AL rats, a remarkable decrease (60-70 %) of TFAM-bound mtDNA. The decreased TFAM binding is a novel finding that may explain the mtDNA loss in spite of the compensatory TFAM increased amount. In aged CR rats, TFAM amount increased and mtDNA content decreased with respect to young rats' values, but the extent of the changes was smaller than in aged AL rats. Attenuation of the age-related effects due to the diet in the CR animals was further evidenced by the unchanged content of the 4.8-kb deletion with respect to that of young animals and by the partial prevention of the age-related decrease in TFAM binding to mtDNA.
Assuntos
Envelhecimento/genética , Restrição Calórica , DNA Mitocondrial/metabolismo , Lobo Frontal/metabolismo , Fatores de Transcrição/metabolismo , Envelhecimento/metabolismo , Animais , Western Blotting , Córtex Cerebral/metabolismo , Dano ao DNA , Replicação do DNA , DNA Mitocondrial/genética , Modelos Animais de Doenças , Deleção de Genes , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genéticaRESUMO
The MTERF protein family comprises members from Metazoans and plants. All the Metazoan MTERF proteins characterized to date, including the mitochondrial transcription termination factors, play a key role in mitochondrial gene expression. In this study we report the characterization of Drosophila MTERF5 (D-MTERF5), a mitochondrial protein existing only in insects, probably originated from a duplication event of the transcription termination factor DmTTF. D-MTERF5 knock-down in D.Mel-2 cells alters transcript levels with an opposite pattern to that produced by DmTTF knock-down. D-MTERF5 is able to interact with mtDNA at the same sites contacted by DmTTF, but only in the presence of the termination factor. We propose that the two proteins participate in the transcription termination process, with D-MTERF5 engaged in relieving the block exerted by DmTTF. This hypothesis is supported also by D-MTERF5 homology modeling, which suggests that this protein contains protein-protein interaction domains. Co-regulation by DREF (DNA Replication-related Element binding Factor) of D-MTERF5 and DmTTF implies that expression of the two factors needs to be co-ordinated to ensure fine modulation of Drosophila mitochondrial transcription.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Drosophila , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Proteínas Mitocondriais/genética , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: An increase in mitochondrial DNA (mtDNA) content and mitochondrial biogenesis associated with the activation of PGC-1α signalling pathway was previously reported in type I endometrial cancer. The aim of this study has been to evaluate if mtDNA content and the citrate synthase (CS) activity, an enzyme marker of mitochondrial mass, increase in progression from control endometrium to hyperplasia to type I endometrial carcinoma. RESULTS: Given that no statistically significant change in mtDNA content and CS activity in endometrium taken from different phases of the menstrual cycle or in menopause was found, these samples were used as control. Our research shows, for the first time, that mtDNA content and citrate synthase activity increase in hyperplastic endometrium compared to control tissues, even if their levels remain lower compared to cancer tissue. In particular, mtDNA content increases seem to precede increases in CS activity. No statistically significant change in mtDNA content and in CS activity was found in relation to different histopathological conditions such as grade, myometrial invasion and stage. CONCLUSION: MtDNA content and citrate synthase activity increases in pre-malignant lesions could be a potential molecular marker for progression from hyperplasia to carcinoma.
Assuntos
DNA Mitocondrial/análise , Hiperplasia Endometrial/metabolismo , Neoplasias do Endométrio/metabolismo , Endométrio/metabolismo , Tamanho do Órgão , Citrato (si)-Sintase/metabolismo , Progressão da Doença , Hiperplasia Endometrial/enzimologia , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/enzimologia , Neoplasias do Endométrio/patologia , Endométrio/enzimologia , Feminino , HumanosRESUMO
The behavior of the peroxisome proliferator-activated receptor-γ coactivators PGC-1α/PGC-ß-dependent mitochondrial biogenesis signaling pathway, as well as the level of some antioxidant enzymes and proteins involved in mitochondrial dynamics in the liver of old rats before and after 2 months of acetyl-L-carnitine (ALCAR) supplementation, was tested. The results reveal that ALCAR treatment is able to reverse the age-associated decline of PGC-1α, PGC-1ß, nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 (ND1), and cytochrome c oxidase subunit IV (COX IV) protein levels, of mitochondrial DNA (mtDNA) content, and of citrate synthase activity. Moreover, it partially reverses the mitochondrial superoxide dismutase 2 (SOD2) decline and reduces the cellular content of oxidized peroxiredoxins. These data demonstrate that ALCAR treatment is able to promote in the old rat liver a new mitochondrial population that can contribute to the cellular oxidative stress reduction. Furthermore, a remarkable decline of Drp1 and of Mfn2 proteins is reported here for the first time, suggesting a reduced mitochondrial dynamics in aging liver with no effect of ALCAR treatment.
Assuntos
Acetilcarnitina/metabolismo , Envelhecimento , Mitocôndrias/metabolismo , PPAR gama/metabolismo , Peroxirredoxinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Autofagia , DNA Mitocondrial/metabolismo , Suplementos Nutricionais , Fígado/metabolismo , Masculino , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Ratos , Ratos Endogâmicos F344 , Superóxido Dismutase/metabolismo , Fatores de TempoRESUMO
The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.
Assuntos
RNA Polimerases Dirigidas por DNA/isolamento & purificação , Glutationa Transferase/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , RNA Polimerases Dirigidas por DNA/biossíntese , RNA Polimerases Dirigidas por DNA/genética , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Humanos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Oxidative stress has a central role in aging and in several age-linked diseases such as neurodegenerative diseases, diabetes and cancer. Mitochondria, as the main cellular source and target of reactive oxygen species (ROS) in aging, are recognized as very important players in the above reported diseases. Impaired mitochondrial oxidative phosphorylation has been reported in several aging tissues. Defective mitochondria are not only responsible of bioenergetically less efficient cells but also increase ROS production further contributing to tissues oxidative stress. Acetyl-L-carnitine (ALCAR) is a biomolecule able to limit age-linked mitochondrial decay in brain, liver, heart and skeletal muscles by increasing mitochondrial efficiency. Here the global changes induced by aging and by ALCAR supplementation to old rat on the mitochondrial proteome of rat liver has been analyzed by means of the two-dimensional polyacrylamide gel electrophoresis. Mass spectrometry has been used to identify the differentially expressed proteins. A significant age-related change occurred in 31 proteins involved in several metabolisms. ALCAR supplementation altered the levels of 26 proteins. In particular, ALCAR reversed the age-related alterations of 10 mitochondrial proteins relative to mitochondrial cristae morphology, to the oxidative phosphorylation and antioxidant systems, to urea cycle, to purine biosynthesis.
Assuntos
Acetilcarnitina/farmacologia , Envelhecimento/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteoma/efeitos dos fármacos , Fatores Etários , Envelhecimento/fisiologia , Animais , Eletroforese em Gel Bidimensional , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Proteoma/análise , Proteoma/metabolismo , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The age-related decay of mitochondrial function is a major contributor to the aging process. We tested the effects of 2-month-daily acetyl-L-carnitine (ALCAR) supplementation on mitochondrial biogenesis in the soleus muscle of aged rats. This muscle is heavily dependent on oxidative metabolism. Mitochondrial (mt) DNA content, citrate synthase activity, transcript levels of some nuclear- and mitochondrial-coded genes (cytochrome c oxidase subunit IV [COX-IV], 16S rRNA, COX-I) and of some factors involved in the mitochondrial biogenesis signaling pathway (peroxisome proliferator-activated receptor gamma [PPARgamma] coactivator-1alpha [PGC-1alpha], mitochondrial transcription factor A mitochondrial [TFAM], mitochondrial transcription factor 2B [TFB2]), as well as the protein content of PGC-1alpha were determined. The results suggest that the ALCAR treatment in old rats activates PGC-1alpha-dependent mitochondrial biogenesis, thus partially reverting the age-related mitochondrial decay.
Assuntos
Acetilcarnitina/farmacologia , Envelhecimento/efeitos dos fármacos , Suplementos Nutricionais , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Proteínas de Ligação a RNA/fisiologia , Fatores de Transcrição/fisiologia , Acetilcarnitina/administração & dosagem , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Citrato (si)-Sintase/metabolismo , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Mitocondriais/efeitos dos fármacos , Masculino , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/ultraestrutura , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Ligação a RNA/agonistas , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Endogâmicos F344 , Fatores de Transcrição/agonistas , Fatores de Transcrição/metabolismoRESUMO
The effect of acetyl-L-carnitine (ALCAR) supplementation to 3-month-old rats in normal-loading and unloading conditions has been here investigated by a combined morphological, biochemical and transcriptional approach to test whether ALCAR might cause a remodeling of the metabolic/contractile phenotype of soleus muscle. Morphological assessment demonstrated an increase of type I oxidative fiber content and cross-sectional area in ALCAR-treated animals both in normal-loading and in unloading conditions. ALCAR prevented loss of mitochondrial mass in unloaded animals whereas no ALCAR-dependent increase of mitochondrial mass occurred in normal-loaded muscle. Validated microarray analysis delineated an ALCAR-induced maintenance of a slow-oxidative expression program only in unloaded soleus muscle. Indeed, the muscle adjustment of the expression profile of factors underlying mitochondrial oxidative metabolism, protein turnover, fiber type differentiation and an adaptation of voltage-gated ion channel expression was distinguishable with respect to the loading status. This selectivity may suggest a key role of muscle loading status in the manifestation of ALCAR effects. The results extend to a broader level of biological informations the previous notion on ALCAR positive effect in rat soleus muscle during unloading and point to a role of ALCAR for the maintenance of its slow-oxidative fiber character.
Assuntos
Acetilcarnitina/farmacologia , Elevação dos Membros Posteriores/fisiologia , Músculo Esquelético/fisiologia , Animais , Citrato (si)-Sintase/biossíntese , Feminino , Perfilação da Expressão Gênica , Mitocôndrias Musculares/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos WistarRESUMO
The MTERF family is a large protein family, identified in metazoans and plants, which consists of four subfamilies, MTERF1, 2, 3 and 4. Mitochondrial localisation was predicted for the vast majority of MTERF family members and demonstrated for the characterised MTERF proteins. The main structural feature of MTERF proteins is the presence of a modular architecture, based on repetitions of a 30-residue module, the mTERF motif, containing leucine zipper-like heptads. The MTERF family includes transcription termination factors: human mTERF, sea urchin mtDBP and Drosophila DmTTF. In addition to terminating transcription, they are involved in transcription initiation and in the control of mtDNA replication. This multiplicity of functions seems to flank differences in the gene organisation of mitochondrial genomes. MTERF2 and MTERF3 play antithetical roles in controlling mitochondrial transcription: that is, mammalian and Drosophila MTERF3 act as negative regulators, whereas mammalian MTERF2 functions as a positive regulator. Both proteins contact mtDNA in the promoter region, perhaps establishing interactions, either mutual or with other factors. Regulation of MTERF gene expression in human and Drosophila depends on nuclear transcription factors NRF-2 and DREF, respectively, and proceeds through pathways which appear to discriminate between factors positively or negatively acting in mitochondrial transcription. In this emerging scenario, it appears that MTERF proteins act to coordinate mitochondrial transcription.
RESUMO
In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.
Assuntos
DNA Mitocondrial/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação da Expressão Gênica , Proteínas/genética , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação/genética , Western Blotting , Imunoprecipitação da Cromatina , DNA Helicases/genética , DNA Polimerase beta/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Fator de Transcrição de Proteínas de Ligação GA/genética , Células HeLa , Humanos , Proteínas Mitocondriais , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
PGC-1alpha-dependent pathway of mitochondrial biogenesis was investigated for the first time in type I endometrial cancer and in normal endometrium. In cancer endometrial tissue the citrate synthase activity, the mitochondrial DNA content and the TFAM level were found doubled compared to control endometrial tissue. Moreover, a 1.6- and 1.8-fold increase, respectively, of NRF-1 and PGG-1alpha expression was found. This study demonstrates, for the first time, that the increased mitochondrial biogenesis in type I endometrial cancer is associated to the upregulation of PGC-1alpha signalling pathway.
Assuntos
Carcinoma/metabolismo , DNA Mitocondrial/metabolismo , Neoplasias do Endométrio/metabolismo , Proteínas de Choque Térmico/metabolismo , Mitocôndrias/metabolismo , Fatores de Transcrição/metabolismo , Proliferação de Células , Citrato (si)-Sintase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Humanos , Proteínas Mitocondriais/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Characterization of the basic transcription machinery of mammalian mitochondrial DNA has been greatly supported by the availability of pure recombinant mitochondrial RNA polymerase (mtRNAP) and accessory factors, which allowed to develop a reconstituted in vitro transcription system. This chapter outlines a general strategy that makes use of a minimal promoter-independent transcription assay to study mitochondrial transcription termination in animal systems. We used such a system to investigate the transcription termination properties of the sea urchin factor mtDBP, however, it is applicable to the study of transcription termination in a variety of organisms, provided that the pure mtRNAP and the transcription termination factor are available.The assay here described contains the recombinant proteins mtRNAP and mtDBP, both expressed in insect cells, and a template consisting of a 3'-tailed DNA construct bearing the sequence bound by mtDBP. Transcription by the RNA polymerase produces run-off and terminated molecules, the size of the latter being consistent with RNA chain arrest in correspondence of the mtDBP-DNA complex. Transcription termination is protein-dependent as addition of increasing amounts of mtDBP to the assay causes a decrease in the intensity of the run-off and the gradual appearance of short-terminated molecules. Furthermore, we report a method, based on pulse-chase experiments, which allows us to distinguish between the true termination and the pausing events.