RESUMO
Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3'-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ10. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ10 biosynthesis.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Regulação da Expressão Gênica/fisiologia , Fosforilação Oxidativa , Consumo de Oxigênio/fisiologia , Ubiquinona/biossíntese , Regiões 3' não Traduzidas/fisiologia , Proteína Semelhante a ELAV 1/genética , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Ubiquinona/genéticaRESUMO
The mitochondrial H(+)-ATP synthase is a bottleneck component in the provision of metabolic energy by oxidative phosphorylation. The expression of its catalytic subunit (ß-F1-ATPase) is stringently controlled at post-transcriptional levels during oncogenesis, the cell cycle and in development. Here we show that miR-127-5p targets the 3'UTR of ß-F1-ATPase mRNA (ß-mRNA) significantly reducing its translational efficiency without affecting ß-mRNA abundance. Despite the reduced expression of ß-F1-ATPase in most human carcinomas, we observed no expression of miR-127-5p in different human cancer cell lines, minimizing the potential role of miR-127-5p as a regulator of the bioenergetic activity of mitochondria in cancer. In contrast, miR-127-5p is highly over-expressed in the human fetal liver. Consistent with previous findings in the rat, the expression of ß-F1-ATPase in the human liver also seems to be controlled at post-transcriptional levels during development, what might suggest a role for miR-127-5p in controlling ß-mRNA translation and thus in defining the bioenergetic activity of human liver mitochondria. Moreover, immunolocalization techniques and subcellular fractionation experiments using different antibodies against ß-F1-ATPase reveal that the ectopic expression of ß-F1-ATPase at the cell surface of the hepatocytes and HepG2 cells is negligible or stands for scrutiny.
Assuntos
Regiões 3' não Traduzidas/genética , MicroRNAs/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Biossíntese de Proteínas/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Feto/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/enzimologia , Hepatócitos/ultraestrutura , Humanos , Fígado/embriologia , Fígado/metabolismo , Fígado/ultraestrutura , MicroRNAs/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
A distinctive metabolic trait of tumors is their enforced aerobic glycolysis. This phenotype was first reported by Otto Warburg, who suggested that the increased glucose consumption of cancer cells under aerobic conditions might result from an impaired bioenergetic activity of their mitochondria. A central player in defining the bioenergetic activity of the cell is the mitochondrial H(+)-ATP synthase. The expression of its catalytic subunit ß-F1-ATPase is tightly regulated at post-transcriptional levels during mammalian development and in the cell cycle. Moreover, the down-regulation of ß-F1-ATPase is a hallmark of most human carcinomas. In this review we summarize our present understanding of the molecular mechanisms that participate in promoting the "abnormal" aerobic glycolysis of prevalent human carcinomas. The role of the ATPase Inhibitor Factor 1 (IF1) and of Ras-GAP SH3 binding protein 1 (G3BP1), controlling the activity of the H(+)-ATP synthase and the translation of ß-F1-ATPase mRNA respectively in cancer cells is emphasized. Furthermore, we underline the role of mitochondrial dysfunction as a pivotal player of tumorigenesis.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Humanos , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , ATPases Mitocondriais Próton-Translocadoras/genética , Modelos Biológicos , Neoplasias/genética , Fenótipo , Processamento de Proteína Pós-Traducional/genéticaRESUMO
The post-transcriptional regulation of nuclear mRNAs that encode core components of mitochondria has relevant implications in cell physiology. The mRNA that encodes the catalytic subunit of the mitochondrial H(+)-ATP synthase subunit beta (ATP5B, beta-F1-ATPase) is localized in a large ribonucleoprotein (RNP) complex (beta-F1-RNP), which is subjected to stringent translational control during development and the cell cycle, and in carcinogenesis. Because downregulation of beta-F1-ATPase is a conserved feature of most prevalent human carcinomas, we have investigated the molecular composition of the human beta-F1-RNP. By means of an improved affinity-chromatography procedure and protein sequencing we have identified nine RNA-binding proteins (RNABPs) of the beta-F1-RNP. Immunoprecipitation assays of Ras-GAP SH3 binding protein 1 (G3BP1) and fluorescent in-situ hybridization of mRNA indicate a direct interaction of the endogenous G3BP1 with mRNA of beta-F1-ATPase (beta-F1 mRNA). RNA-bridged trimolecular fluorescence complementation (TriFC) assays confirm the interaction of G3BP1 with the 3'-UTR of beta-F1 mRNA in cytoplasmic RNA-granules. Confocal and high-resolution immunoelectron-microscopy experiments suggest that the beta-F1-RNP is sorted to the periphery of mitochondria. Molecular and functional studies indicate that the interaction of G3BP1 with beta-F1 mRNA inhibits its translation at the initiation level, supporting a role for G3BP1 in the glycolytic switch that occurs in cancer.
Assuntos
Proteínas de Transporte/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , RNA Mensageiro/genética , Ribonucleoproteínas/metabolismo , Regiões 3' não Traduzidas , Proteínas de Transporte/genética , DNA Helicases , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Imunoprecipitação , Espectrometria de Massas , Mitocôndrias/genética , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica , Biossíntese de Proteínas , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genéticaRESUMO
Down-regulation of beta-F1-ATPase (the catalytic subunit of the mitochondrial H+-ATP synthase) is a hallmark of many human tumours. The expression level of beta-F1-ATPase provides a marker of the prognosis of cancer patients, as well as of the tumour response to chemotherapy. However, the mechanisms that participate in down-regulating its expression in human tumours remain unknown. In the present study, we have investigated the expression of beta-F1-ATPase mRNA (termed beta-mRNA) in breast, colon and lung adenocarcinomas and squamous carcinomas of the lung. Despite the down-regulation of the protein, tumour beta-mRNA levels remained either unchanged (breast and lung adenocarcinomas) or significantly increased (colon and squamous lung carcinomas) when compared with paired normal tissues, suggesting a specific translation-masking event for beta-mRNA in human cancer. Consistently, we show using cell-free translation assays that a large fraction (approximately 70%) of protein extracts derived from breast and lung adenocarcinomas specifically repress the translation of beta-mRNA. We show that the 3'UTR (3' untranslated region) of human beta-mRNA is a relevant cis-acting element required for efficient translation of the transcript. However, an RNA chimaera bearing the 3'UTR of human beta-mRNA does not recapitulate the inhibitory effect of tumour extracts on beta-mRNA translation. Overall, the findings of the present study support the hypothesis that down-regulation of the bioenergetic activity of mitochondria in human tumours is exerted by translation silencing of beta-mRNA.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/genética , Neoplasias/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Western Blotting , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Estadiamento de Neoplasias , Neoplasias/enzimologia , Neoplasias/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Extratos de Tecidos/farmacologiaRESUMO
Recently, the inevitable metabolic reprogramming experienced by cancer cells as a result of the onset of cellular proliferation has been added to the list of hallmarks of the cancer cell phenotype. Proliferation is bound to the synchronous fluctuation of cycles of an increased glycolysis concurrent with a restrained oxidative phosphorylation. Mitochondria are key players in the metabolic cycling experienced during proliferation because of their essential roles in the transduction of biological energy and in defining the life-death fate of the cell. These two activities are molecularly and functionally integrated and are both targets of commonly altered cancer genes. Moreover, energetic metabolism of the cancer cell also affords a target to develop new therapies because the activity of mitochondria has an unquestionable tumor suppressor function. In this review, we summarize most of these findings paying special attention to the opportunity that translation of energetic metabolism into the clinics could afford for the management of cancer patients. More specifically, we emphasize the role that mitochondrial beta-F1-ATPase has as a marker for the prognosis of different cancer patients as well as in predicting the tumor response to therapy.
Assuntos
Proliferação de Células , Genes Supressores de Tumor , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/patologia , ATPases Translocadoras de Prótons/genética , Metabolismo Energético , Humanos , Neoplasias/metabolismo , Fosforilação OxidativaRESUMO
The cancer cell phenotype has been summarized in six hallmarks [D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (1) (2000) 57-70]. Following the conceptual trait established in that review towards the comprehension of cancer, herein we summarize the basis of an underlying principle that is fulfilled by cancer cells and tumors: its avidity for glucose. Our purpose is to push forward that the metabolic reprogramming that operates in the cancer cell represents a seventh hallmark of the phenotype that offers a vast array of possibilities for the future treatment of the disease. We summarize the metabolic pathways that extract matter and energy from glucose, paying special attention to the concerted regulation of these pathways by the ATP mass-action ratio. The molecular and functional evidences that support the high glucose uptake and the "abnormal" aerobic glycolysis of the carcinomas are detailed discussing also the role that some oncogenes and tumor suppressors have in these pathways. We overview past and present evidences that sustain that mitochondria of the cancer cell are impaired, supporting the original Warburg's formulation that ascribed the high glucose uptake of cancer cells to a defective mitochondria. A simple proteomic approach designed to assess the metabolic phenotype of cancer, i.e., its bioenergetic signature, molecularly and functionally supports Warburg's hypothesis. Furthermore, we discuss the clinical utility that the bioenergetic signature might provide. Glycolysis is presented as the "selfish" pathway used for cellular proliferation, providing both the metabolic precursors and the energy required for biosynthetic purposes, in the context of a plethora of substrates. The glucose avidity of carcinomas is thus presented as the result of both the installment of glycolysis for cellular proliferation and of the impairment of mitochondrial activity in the cancer cell. At the end, the repression of mitochondrial activity affords the cancer cell with a cell-death resistant phenotype making them prone to malignant growth.
Assuntos
Glicólise , Mitocôndrias/fisiologia , Neoplasias/metabolismo , Animais , Proliferação de Células , Metabolismo Energético , Genes Neoplásicos , Glucose/metabolismo , Humanos , Neoplasias/patologia , Fosforilação OxidativaRESUMO
The 26S proteasome of eukaryotic cells mediates ubiquitin-dependent as well as ubiquitin-independent degradation of proteins in many regulatory processes as well as in protein quality control. The proteasome itself is a dynamic complex with varying compositions and interaction partners. Studies in Saccharomyces cerevisiae have revealed that expression of proteasome subunit genes is coordinately controlled by the Rpn4 transcriptional activator. The cellular level of Rpn4 itself is subject to a complex regulation, which, aside of a transcriptional control of its gene, intriguingly involves ubiquitin-dependent as well as ubiquitin-independent control of its stability by the proteasome. A novel study by Ju et al. [D. Ju, H. Yu, X. Wang, Y. Xie, Ubiquitin-mediated degradation of Rpn4 is controlled by a phosphorylation-dependent ubiquitylation signal, Biochim. Biophys. Acta (in press), doi:10.1016/j.bbamcr.2007.04.012] now revealed another level of complexity by showing that phosphorylation of a specific serine residue in Rpn4 is required for its efficient targeting by the Ubr2 ubiquitin ligase.