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1.
Nucleic Acids Res ; 46(19): 10340-10352, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30053103

RESUMO

Fine regulation of the phosphatase and tensin homologue (PTEN) phosphatase dosage is critical for homeostasis and tumour suppression. The 3'-untranslated region (3'-UTR) of Pten mRNA was extensively linked to post-transcriptional regulation by microRNAs (miRNAs). In spite of this critical regulatory role, alternative 3'-UTRs of Pten have not been systematically characterized. Here, we reveal an important diversity of Pten mRNA isoforms generated by alternative polyadenylation sites. Several 3'-UTRs are co-expressed and their relative expression is dynamically regulated. In spite of encoding multiple validated miRNA-binding sites, longer isoforms are largely refractory to miRNA-mediated silencing, are more stable and contribute to the bulk of PTEN protein and signalling functions. Taken together, our results warrant a mechanistic re-interpretation of the post-transcriptional mechanisms involving Pten mRNAs and raise concerns on how miRNA-binding sites are being validated.


Assuntos
MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Poliadenilação/genética , Isoformas de RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Homeostase , Camundongos , Células NIH 3T3 , Estabilidade de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética
2.
Genome Res ; 26(5): 636-48, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26984228

RESUMO

The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5' TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5' TOP motif but that 5' UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5' UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5' UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5' UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5' UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5' UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells.


Assuntos
Regiões 5' não Traduzidas/fisiologia , Fator de Iniciação 4E em Eucariotos/metabolismo , Biossíntese de Proteínas/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Apoptose/fisiologia , Feminino , Humanos , Células MCF-7
3.
Cell Rep ; 43(9): 114695, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39250314

RESUMO

MicroRNAs (miRNAs) play crucial roles in physiological functions and disease, but the regulation of their nuclear biogenesis remains poorly understood. Here, BioID on Drosha, the catalytic subunit of the microprocessor complex, reveals its proximity to splicing factor proline- and glutamine (Q)-rich (SFPQ), a multifunctional RNA-binding protein (RBP) involved in forming paraspeckle nuclear condensates. SFPQ depletion impacts both primary and mature miRNA expression, while other paraspeckle proteins (PSPs) or the paraspeckle scaffolding RNA NEAT1 do not, indicating a paraspeckle-independent role. Comprehensive transcriptomic analyses show that SFPQ loss broadly affects RNAs and miRNA host gene (HG) expression, influencing both their transcription and the stability of their products. Notably, SFPQ protects the oncogenic miR-17∼92 polycistron from degradation by the nuclear exosome targeting (NEXT)-exosome complex and is tightly linked with its overexpression across a broad variety of cancers. Our findings reveal a dual role for SFPQ in regulating miRNA HG transcription and stability, as well as its significance in cancers.


Assuntos
Núcleo Celular , MicroRNAs , Fator de Processamento Associado a PTB , Humanos , MicroRNAs/metabolismo , MicroRNAs/genética , Fator de Processamento Associado a PTB/metabolismo , Fator de Processamento Associado a PTB/genética , Núcleo Celular/metabolismo , Transcrição Gênica , Ribonuclease III/metabolismo , Ribonuclease III/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Células HeLa
4.
iScience ; 26(4): 106314, 2023 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-37009228

RESUMO

Skin plays central roles in systemic physiology, and it undergoes significant functional changes during aging. Members of the peroxisome proliferator-activated receptor-gamma coactivator (PGC-1) family (PGC-1s) are key regulators of the biology of numerous tissues, yet we know very little about their impact on skin functions. Global gene expression profiling and gene silencing in keratinocytes uncovered that PGC-1s control the expression of metabolic genes as well as that of terminal differentiation programs. Glutamine emerged as a key substrate promoting mitochondrial respiration, keratinocyte proliferation, and the expression of PGC-1s and terminal differentiation programs. Importantly, gene silencing of PGC-1s reduced the thickness of a reconstructed living human epidermal equivalent. Exposure of keratinocytes to a salicylic acid derivative potentiated the expression of PGC-1s and terminal differentiation genes and increased mitochondrial respiration. Overall, our results show that the PGC-1s are essential effectors of epidermal physiology, revealing an axis that could be targeted in skin conditions and aging.

5.
Biomater Sci ; 10(21): 6077-6115, 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36097955

RESUMO

Exogenously delivered mRNA-based drugs are emerging as a new class of therapeutics with the potential to treat several diseases. Over the last decade, advancements in the design of non-viral delivery tools have enabled mRNA to be evaluated for several therapeutic purposes including protein replacement therapies, gene editing, and vaccines. However, in vivo delivery of mRNA to targeted organs and cells remains a critical challenge. Evaluation of the biodistribution of mRNA vehicles is of utmost importance for the development of effective pharmaceutical candidates. In this review, we discuss the recent advances in the design of nanoparticles loaded with mRNA and extrapolate the key factors influencing their biodistribution following administration. Finally, we highlight the latest developments in the preclinical and clinical translation of mRNA therapeutics for protein supplementation therapy.


Assuntos
Nanopartículas , Vacinas , RNA Mensageiro , Distribuição Tecidual , Preparações Farmacêuticas
6.
Cancer Lett ; 541: 215738, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35594996

RESUMO

Mitochondria are specialized metabolic and immune organelles that have important roles in tumor progression, metastasis, and response to chemotherapy and immunotherapy. Mitochondrial biogenesis and functions are under the control of the peroxisome-proliferator activated receptor-gamma (PGC-1) transcriptional coactivators. Recent research unveiled the role of PGC-1α in bolstering mitochondrial oxidative functions and in the suppression of metastasis in melanoma, but the role of PGC-1s in tumor immunology remains elusive. Herein, we show that low PGC-1s expression in human melanoma tumors is associated with increased expression of a repertoire of immunosuppressive (CD73, PD-L2, Galectin-9) and pro-inflammatory (IL-8, TNF, IL-1ß) transcripts, and that experimental depletion of PGC-1ß recapitulates this signature in human melanoma cell lines. The depletion of PGC-1ß reduces the expression of HSPA9, impairs mitochondrial activity, and leads to cell cycle arrest. Using pharmacological and gene silencing approaches, we further show that MEK1/2 and IRF-1 mediate the observed immune transcriptional response. Overall, this research suggests that mitochondrial biogenesis modulators can modulate tumor progression, immune evasion, and response to therapeutics through transcriptional control of immune pathways.


Assuntos
Melanoma , Mitocôndrias , Biogênese de Organelas , Proteínas de Ligação a RNA , Expressão Gênica/imunologia , Humanos , Fator Regulador 1 de Interferon , Melanoma/genética , Melanoma/metabolismo , Mitocôndrias/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
7.
J Virol ; 82(8): 3984-96, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18272581

RESUMO

The IkappaB kinase-related kinases, TBK1 and IKKi, were recently shown to be responsible for the C-terminal phosphorylation of IRF-3. However, the identity of the phosphoacceptor site(s) targeted by these two kinases remains unclear. Using a biological assay based on the IRF-3-mediated production of antiviral cytokines, we demonstrate here that all Ser/Thr clusters of IRF-3 are required for its optimal transactivation capacity. In vitro kinase assays using full-length His-IRF-3 as a substrate combined with mass spectrometry analysis revealed that serine 402 and serine 396 are directly targeted by TBK1. Analysis of Ser/Thr-to-Ala mutants revealed that the S396A mutation, located in cluster II, abolished IRF-3 homodimerization, CBP association, and nuclear accumulation. However, production of antiviral cytokines was still present in IRF-3 S396A-expressing cells. Interestingly, mutation of serine 339, which is involved in IRF-3 stability, also abrogated CBP association and dimerization without affecting gene transactivation as long as serine 396 remained available for phosphorylation. Complementation of IRF-3-knockout mouse embryonic fibroblasts also revealed a compensatory mechanism of serine 339 and serine 396 in the ability of IRF-3 to induce expression of the interferon-stimulated genes ISG56 and ISG54. These data lead us to reconsider the current model of IRF-3 activation. We propose that conventional biochemical assays used to measure IRF-3 activation are not sensitive enough to detect the small fraction of IRF-3 needed to elicit a biological response. Importantly, our study establishes a molecular link between the role of serine 339 in IRF-3 homodimerization, CBP association, and its destabilization.


Assuntos
Fator Regulador 3 de Interferon/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Substituição de Aminoácidos/genética , Animais , Antivirais/metabolismo , Linhagem Celular , Núcleo Celular/química , Células Cultivadas , Chlorocebus aethiops , Citocinas/biossíntese , Dimerização , Fibroblastos , Deleção de Genes , Teste de Complementação Genética , Humanos , Fator Regulador 3 de Interferon/genética , Espectrometria de Massas , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Fatores de Transcrição/biossíntese
8.
Sci Rep ; 9(1): 12903, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501473

RESUMO

In subretinal inflammation, activated mononuclear phagocytes (MP) play a key role in the progression of retinopathies. Little is known about the mechanism involved in the loss of photoreceptors leading to vision impairment. Studying retinal damage induced by photo-oxidative stress, we observed that cluster of differentiation 36 (CD36)-deficient mice featured less subretinal MP accumulation and attenuated photoreceptor degeneration. Moreover, treatment with a CD36-selective azapeptide ligand (MPE-001) reduced subretinal activated MP accumulation in wild type mice and preserved photoreceptor layers and function as assessed by electroretinography in a CD36-dependent manner. The azapeptide modulated the transcriptome of subretinal activated MP by reducing pro-inflammatory markers. In isolated MP, MPE-001 induced dissociation of the CD36-Toll-like receptor 2 (TLR2) oligomeric complex, decreasing nuclear factor-kappa B (NF-κB) and NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. In addition, MPE-001 caused an aerobic metabolic shift in activated MP, involving peroxisome proliferator-activated receptor-γ (PPAR-γ) activation, which in turn mitigated inflammation. Accordingly, PPAR-γ inhibition blocked the cytoprotective effect of MPE-001 on photoreceptor apoptosis elicited by activated MP. By altering activated MP metabolism, MPE-001 decreased immune responses to alleviate subsequent inflammation-dependent neuronal injury characteristic of various vision-threatening retinal disorders.


Assuntos
Antígenos CD36/metabolismo , Metabolismo Energético/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Retinite/etiologia , Retinite/metabolismo , Animais , Biomarcadores , Citocinas/metabolismo , Suscetibilidade a Doenças , Inflamassomos/metabolismo , Mediadores da Inflamação/metabolismo , Ligantes , Metaboloma , Metabolômica/métodos , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Ligação Proteica , Retinite/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismo
9.
Front Oncol ; 8: 75, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29629336

RESUMO

Metabolic reprogramming confers cancer cells the ability to grow and survive under nutrient-depleted or stressful microenvironments. The amplification of oncogenes, the loss of tumor suppressors, as well as context- and lineage-specific determinants can converge and profoundly affect the metabolic status of cancer cells. Cumulating evidences suggest that highly glycolytic cells under the influence of oncogenes such as BRAF, or evolving in hypoxic microenvironments, will promote metastasis through modulation of multiple steps of tumorigenesis such as the epithelial-to-mesenchymal transition (EMT). On the contrary, increased reliance on mitochondrial respiration is associated with hyperplasic rather than metastatic disease. The PGC-1α transcriptional coactivator, a master regulator of mitochondrial biogenesis, has recently been shown to exert antimetastatic effects in cancer, notably through inhibition of EMT. Besides, PGC-1α has the opposite role in specific cancer subtypes, in which it appears to provide growth advantages. Thus, the regulation and role of PGC-1α in cancer is not univocal, and its use as a prognostic marker appears limited given its highly dynamic nature and its multifaceted regulation by transcriptional and posttranslational mechanisms. Herein, we expose key oncogenic and lineage-specific modules that finely regulate PGC-1α to promote or dampen the metastatic process. We propose a unifying model based on the systematic analysis of its controversial implication in cancer from cell proliferation to EMT and metastasis. This short review will provide a good understanding of current challenges associated with the study of PGC-1α.

10.
Aging Cell ; 17(6): e12830, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30192051

RESUMO

Plant extracts containing salicylates are probably the most ancient remedies to reduce fever and ease aches of all kind. Recently, it has been shown that salicylates activate adenosine monophosphate-activated kinase (AMPK), which is now considered as a promising target to slow down aging and prevent age-related diseases in humans. Beneficial effects of AMPK activation on lifespan have been discovered in the model organism Caenorhabditis elegans (C. elegans). Indeed, salicylic acid and acetylsalicylic acid extend lifespan in worms by activating AMPK and the forkhead transcription factor DAF-16/FOXO. Here, we investigated whether another salicylic acid derivative 5-octanoyl salicylic acid (C8-SA), developed as a controlled skin exfoliating ingredient, had similar properties using C. elegans as a model. We show that C8-SA increases lifespan of C. elegans and that a variety of pathways and genes are required for C8-SA-mediated lifespan extension. C8-SA activates AMPK and inhibits TOR both in nematodes and in primary human keratinocytes. We also show that C8-SA can induce both autophagy and the mitochondrial unfolded protein response (UPRmit ) in nematodes. This induction of both processes is fully required for lifespan extension in the worm. In addition, we found that the activation of autophagy by C8-SA fails to occur in worms with compromised UPRmit , suggesting a mechanistic link between these two processes. Mutants that are defective in the mitochondrial unfolded protein response exhibit constitutive high autophagy levels. Taken together, these data therefore suggest that C8-SA positively impacts longevity in worms through induction of autophagy and the UPRmit .


Assuntos
Autofagia/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Longevidade/efeitos dos fármacos , Mitocôndrias/metabolismo , Ácido Salicílico/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Caenorhabditis elegans/efeitos dos fármacos , Restrição Calórica , Insulina/metabolismo , Mitocôndrias/efeitos dos fármacos , Mutação/genética , Transdução de Sinais/efeitos dos fármacos
11.
Cancer Res ; 78(9): 2191-2204, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29440170

RESUMO

Metabolic reprogramming is a hallmark of cancer that includes increased glucose uptake and accelerated aerobic glycolysis. This phenotype is required to fulfill anabolic demands associated with aberrant cell proliferation and is often mediated by oncogenic drivers such as activated BRAF. In this study, we show that the MAPK-activated p90 ribosomal S6 kinase (RSK) is necessary to maintain glycolytic metabolism in BRAF-mutated melanoma cells. RSK directly phosphorylated the regulatory domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), an enzyme that catalyzes the synthesis of fructose-2,6-bisphosphate during glycolysis. Inhibition of RSK reduced PFKFB2 activity and glycolytic flux in melanoma cells, suggesting an important role for RSK in BRAF-mediated metabolic rewiring. Consistent with this, expression of a phosphorylation-deficient mutant of PFKFB2 decreased aerobic glycolysis and reduced the growth of melanoma in mice. Together, these results indicate that RSK-mediated phosphorylation of PFKFB2 plays a key role in the metabolism and growth of BRAF-mutated melanomas.Significance: RSK promotes glycolytic metabolism and the growth of BRAF-mutated melanoma by driving phosphorylation of an important glycolytic enzyme. Cancer Res; 78(9); 2191-204. ©2018 AACR.


Assuntos
Melanoma/genética , Fosfofrutoquinase-2/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proliferação de Células/genética , Reprogramação Celular/genética , Glucose/metabolismo , Glicólise/genética , Células HeLa , Humanos , Melanoma/metabolismo , Melanoma/patologia , Fosforilação
12.
Cancer Res ; 78(17): 4826-4838, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29930100

RESUMO

The ShcA adaptor protein transduces oncogenic signals downstream of receptor tyrosine kinases. We show here that breast tumors engage the ShcA pathway to increase their metabolism. ShcA signaling enhanced glucose catabolism through glycolysis and oxidative phosphorylation, rendering breast cancer cells critically dependent on glucose. ShcA signaling simultaneously increased the metabolic rate and flexibility of breast cancer cells by inducing the PGC-1α transcriptional coactivator, a central regulator of mitochondrial metabolism. Breast tumors that engaged ShcA signaling were critically dependent on PGC-1α to support their increased metabolic rate. PGC-1α deletion drastically delayed breast tumor onset in an orthotopic mouse model, highlighting a key role for PGC-1α in tumor initiation. Conversely, reduced ShcA signaling impaired both the metabolic rate and flexibility of breast cancer cells, rendering them reliant on mitochondrial oxidative phosphorylation. This metabolic reprogramming exposed a targetable metabolic vulnerability, leading to a sensitization of breast tumors to inhibitors of mitochondrial complex I (biguanides). Genetic inhibition of ShcA signaling in the Polyoma virus middle T (MT) breast cancer mouse model sensitized mammary tumors to biguanides during the earliest stages of breast cancer progression. Tumor initiation and growth were selectively and severely impaired in MT/ShcA-deficient animals. These data demonstrate that metabolic reprogramming is a key component of ShcA signaling and serves an unappreciated yet vital role during breast cancer initiation and progression. These data further unravel a novel interplay between ShcA and PGC-1α in the coordination of metabolic reprogramming and demonstrate the sensitivity of breast tumors to drugs targeting oxidative phosphorylation.Significance: This study uncovers a previously unrecognized mechanism that links aberrant RTK signaling with metabolic perturbations in breast cancer and exposes metabolic vulnerabilities that can be targeted by inhibitors of oxidative phosphorylation. Cancer Res; 78(17); 4826-38. ©2018 AACR.


Assuntos
Neoplasias da Mama/genética , Neoplasias Mamárias Animais/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Animais , Biguanidas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/virologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Polyomavirus/patogenicidade , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo
13.
Cell Metab ; 28(6): 817-832.e8, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30244971

RESUMO

There is increasing interest in therapeutically exploiting metabolic differences between normal and cancer cells. We show that kinase inhibitors (KIs) and biguanides synergistically and selectively target a variety of cancer cells. Synthesis of non-essential amino acids (NEAAs) aspartate, asparagine, and serine, as well as glutamine metabolism, are major determinants of the efficacy of KI/biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine, and serine synthesis by modulating mRNA translation, while ablation of 4E-BP1/2 substantially decreases sensitivity of breast cancer and melanoma cells to KI/biguanide combinations. Efficacy of the KI/biguanide combinations is also determined by HIF-1α-dependent perturbations in glutamine metabolism, which were observed in VHL-deficient renal cancer cells. This suggests that cancer cells display metabolic plasticity by engaging non-redundant adaptive mechanisms, which allows them to survive therapeutic insults that target cancer metabolism.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aminoácidos/metabolismo , Animais , Biguanidas/farmacologia , Proteínas de Ciclo Celular , Fatores de Iniciação em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Células K562 , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
JCI Insight ; 2(13)2017 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-28679948

RESUMO

Magnesium (Mg2+) plays pleiotropic roles in cellular biology, and it is essentially required for all living organisms. Although previous studies demonstrated intracellular Mg2+ levels were regulated by the complex of phosphatase of regenerating liver 2 (PRL2) and Mg2+ transporter of cyclin M (CNNMs), physiological functions of PRL2 in whole animals remain unclear. Interestingly, Mg2+ was recently identified as a regulator of circadian rhythm-dependent metabolism; however, no mechanism was found to explain the clock-dependent Mg2+ oscillation. Herein, we report PRL2 as a missing link between sex and metabolism, as well as clock genes and daily cycles of Mg2+ fluxes. Our results unveil that PRL2-null animals displayed sex-dependent alterations in body composition, and expression of PRLs and CNNMs were sex- and circadian time-dependently regulated in brown adipose tissues. Consistently, PRL2-KO mice showed sex-dependent alterations in thermogenesis and in circadian energy metabolism. These physiological changes were associated with an increased rate of uncoupled respiration with lower intracellular Mg2+ in PRL2-KO cells. Moreover, PRL2 deficiency causes inhibition of the ATP citrate lyase axis, which is involved in fatty acid synthesis. Overall, our findings support that sex- and circadian-dependent PRL2 expression alter intracellular Mg2+ levels, which accordingly controls energy metabolism status.

15.
Cell Rep ; 21(1): 1-9, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978464

RESUMO

Reactive oxygen species (ROS) are continuously produced as a by-product of mitochondrial metabolism and eliminated via antioxidant systems. Regulation of mitochondrially produced ROS is required for proper cellular function, adaptation to metabolic stress, and bypassing cellular senescence. Here, we report non-canonical regulation of the cellular energy sensor AMP-activated protein kinase (AMPK) by mitochondrial ROS (mROS) that functions to maintain cellular metabolic homeostasis. We demonstrate that mitochondrial ROS are a physiological activator of AMPK and that AMPK activation triggers a PGC-1α-dependent antioxidant response that limits mitochondrial ROS production. Cells lacking AMPK activity display increased mitochondrial ROS levels and undergo premature senescence. Finally, we show that AMPK-PGC-1α-dependent control of mitochondrial ROS regulates HIF-1α stabilization and that mitochondrial ROS promote the Warburg effect in cells lacking AMPK signaling. These data highlight a key function for AMPK in sensing and resolving mitochondrial ROS for stress resistance and maintaining cellular metabolic balance.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Homeostase/genética , Redes e Vias Metabólicas/genética , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases Ativadas por AMP/deficiência , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Senescência Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Transgênicos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/deficiência , Cultura Primária de Células , Estabilidade Proteica , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo , Proteína Desacopladora 3/genética , Proteína Desacopladora 3/metabolismo , Glutationa Peroxidase GPX1
16.
Methods Mol Biol ; 1458: 273-90, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27581029

RESUMO

The tumor microenvironment is a complex and heterogeneous milieu in which cancer cells undergo metabolic reprogramming to fuel their growth. Cancer cell lines grown in vitro using traditional culture methods represent key experimental models to gain a mechanistic understanding of tumor biology. This protocol describes the use of gas chromatography-mass spectrometry (GC-MS) to assess metabolic changes in cancer cells grown under varied levels of oxygen and nutrients that may better mimic the tumor microenvironment. Intracellular metabolite changes, metabolite uptake and release, as well as stable isotope ((13)C) tracer analyses are done in a single experimental setup to provide an integrated understanding of metabolic adaptation. Overall, this chapter describes some essential tools and methods to perform comprehensive metabolomics analyses.


Assuntos
Metaboloma , Metabolômica , Neoplasias/metabolismo , Neoplasias/patologia , Microambiente Tumoral , Linhagem Celular Tumoral , Células Cultivadas , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hipóxia/metabolismo , Metabolômica/métodos , Fluxo de Trabalho
17.
Cell Rep ; 14(4): 920-931, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26804918

RESUMO

Reprogramming of cellular metabolism plays a central role in fueling malignant transformation, and AMPK and the PGC-1α/ERRα axis are key regulators of this process. The intersection of gene-expression and binding-event datasets for breast cancer cells shows that activation of AMPK significantly increases the expression of PGC-1α/ERRα and promotes the binding of ERRα to its cognate sites. Unexpectedly, the data also reveal that ERRα, in concert with PGC-1α, negatively regulates the expression of several one-carbon metabolism genes, resulting in substantial perturbations in purine biosynthesis. This PGC-1α/ERRα-mediated repression of one-carbon metabolism promotes the sensitivity of breast cancer cells and tumors to the anti-folate drug methotrexate. These data implicate the PGC-1α/ERRα axis as a core regulatory node of folate cycle metabolism and further suggest that activators of AMPK could be used to modulate this pathway in cancer.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Metotrexato/farmacologia , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Ácido Fólico/metabolismo , Humanos , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Purinas/biossíntese , Receptor ERRalfa Relacionado ao Estrogênio
18.
Nat Commun ; 7: 12156, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27402251

RESUMO

Despite the initial benefits of treating HER2-amplified breast cancer patients with the tyrosine kinase inhibitor lapatinib, resistance inevitably develops. Here we report that lapatinib induces the degradation of the nuclear receptor ERRα, a master regulator of cellular metabolism, and that the expression of ERRα is restored in lapatinib-resistant breast cancer cells through reactivation of mTOR signalling. Re-expression of ERRα in resistant cells triggers metabolic adaptations favouring mitochondrial energy metabolism through increased glutamine metabolism, as well as ROS detoxification required for cell survival under therapeutic stress conditions. An ERRα inverse agonist counteracts these metabolic adaptations and overcomes lapatinib resistance in a HER2-induced mammary tumour mouse model. This work reveals a molecular mechanism by which ERRα-induced metabolic reprogramming promotes survival of lapatinib-resistant cancer cells and demonstrates the potential of ERRα inhibition as an effective adjuvant therapy in poor outcome HER2-positive breast cancer.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Mamárias Experimentais/tratamento farmacológico , Quinazolinas/uso terapêutico , Receptores de Estrogênio/genética , Animais , Neoplasias da Mama/metabolismo , Sobrevivência Celular , Humanos , Lapatinib , Células MCF-7 , Neoplasias Mamárias Experimentais/metabolismo , Vírus do Tumor Mamário do Camundongo , Camundongos , Receptor ErbB-2/metabolismo , Infecções por Retroviridae , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Infecções Tumorais por Vírus , Receptor ERRalfa Relacionado ao Estrogênio
19.
Cell Cycle ; 14(4): 473-80, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25590164

RESUMO

Protein synthesis is one of the most energy consuming processes in the cell. The mammalian/mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that integrates a multitude of extracellular signals and intracellular cues to drive growth and proliferation. mTOR activity is altered in numerous pathological conditions, including metabolic syndrome and cancer. In addition to its well-established role in regulating mRNA translation, emerging studies indicate that mTOR modulates mitochondrial functions. In mammals, mTOR coordinates energy consumption by the mRNA translation machinery and mitochondrial energy production by stimulating synthesis of nucleus-encoded mitochondria-related proteins including TFAM, mitochondrial ribosomal proteins and components of complexes I and V. In this review, we highlight findings that link mTOR, mRNA translation and mitochondrial functions.


Assuntos
Proliferação de Células/fisiologia , Mitocôndrias/fisiologia , Modelos Biológicos , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Humanos
20.
Metabolites ; 4(2): 166-83, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24957021

RESUMO

Mitochondria are a focal point in metabolism, given that they play fundamental roles in catabolic, as well as anabolic reactions. Alterations in mitochondrial functions are often studied in whole cells, and metabolomics experiments using 13C-labeled substrates, coupled with mass isotopomer distribution analyses, represent a powerful approach to study global changes in cellular metabolic activities. However, little is known regarding the assessment of metabolic activities in isolated mitochondria using this technology. Studies on isolated mitochondria permit the evaluation of whether changes in cellular metabolic activities are due to modifications in the intrinsic properties of the mitochondria. Here, we present a streamlined approach to accurately determine 13C, as well as 12C enrichments in isolated mitochondria from mammalian tissues or cultured cells by GC/MS. We demonstrate the relevance of this experimental approach by assessing the effects of drugs perturbing mitochondrial functions on the mass isotopomer enrichment of metabolic intermediates. Furthermore, we investigate 13C and 12C enrichments in mitochondria isolated from cancer cells given the emerging role of metabolic alterations in supporting tumor growth. This original method will provide a very sensitive tool to perform metabolomics studies on isolated mitochondria.

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