RESUMO
The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Proteoma/análise , Transcriptoma , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Cisteína/metabolismo , Receptor Nuclear Órfão DAX-1/metabolismo , Redes Reguladoras de Genes , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Ligantes , Neoplasias Pulmonares/metabolismoRESUMO
Across eukaryotic species, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Mitochondrial dysfunction activates an unfolded protein response (UPR(mt)), a stress signaling mechanism designed to ensure mitochondrial homeostasis. Perturbation of mitochondria during larval development in C. elegans not only delays aging but also maintains UPR(mt) signaling, suggesting an epigenetic mechanism that modulates both longevity and mitochondrial proteostasis throughout life. We identify the conserved histone lysine demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of lifespan in response to mitochondrial dysfunction across species. Reduction of function of the demethylases potently suppresses longevity and UPR(mt) induction, while gain of function is sufficient to extend lifespan in a UPR(mt)-dependent manner. A systems genetics approach in the BXD mouse reference population further indicates conserved roles of the mammalian orthologs in longevity and UPR(mt) signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging downstream of mitochondrial perturbations.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Animais , Caenorhabditis elegans/genética , Longevidade , Camundongos , Mitocôndrias/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Resposta a Proteínas não DobradasRESUMO
Cells constantly adapt their metabolism to meet their energy needs and respond to nutrient availability. Eukaryotes have evolved a very sophisticated system to sense low cellular ATP levels via the serine/threonine kinase AMP-activated protein kinase (AMPK) complex. Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP generation and decrease ATP consumption. In the past decade, the discovery of numerous new AMPK substrates has led to a more complete understanding of the minimal number of steps required to reprogramme cellular metabolism from anabolism to catabolism. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and autophagy. Recent studies have revealed that one ancestral function of AMPK is to promote mitochondrial health, and multiple newly discovered targets of AMPK are involved in various aspects of mitochondrial homeostasis, including mitophagy. This Review discusses how AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/fisiologia , Mitocôndrias/metabolismo , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/fisiologia , Animais , Autofagia , Metabolismo Energético , Homeostase , Humanos , Mitocôndrias/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The evolution of AMPK and its homologs enabled exquisite responsivity and control of cellular energetic homeostasis. Recent work has been critical in establishing the mechanisms that determine AMPK activity, novel targets of AMPK action, and the distribution of AMPK-mediated control networks across the cellular landscape. The role of AMPK as a hub of metabolic control has led to intense interest in pharmacologic activation as a therapeutic avenue for a number of disease states, including obesity, diabetes, and cancer. As such, critical work on the compartmentalization of AMPK, its downstream targets, and the systems it influences has progressed in recent years. The variegated distribution of AMPK-mediated control of metabolic homeostasis has revealed key insights into AMPK in normal biology and future directions for AMPK-based therapeutic strategies.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Citoplasma/metabolismo , Metabolismo Energético , Homeostase , Humanos , Mitocôndrias/metabolismo , Domínios Proteicos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Despite being the frontline therapy for type 2 diabetes, the mechanisms of action of the biguanide drug metformin are still being discovered. In particular, the detailed molecular interplays between the AMPK and the mTORC1 pathway in the hepatic benefits of metformin are still ill defined. Metformin-dependent activation of AMPK classically inhibits mTORC1 via TSC/RHEB, but several lines of evidence suggest additional mechanisms at play in metformin inhibition of mTORC1. Here we investigated the role of direct AMPK-mediated serine phosphorylation of RAPTOR in a new RaptorAA mouse model, in which AMPK phospho-serine sites Ser722 and Ser792 of RAPTOR were mutated to alanine. Metformin treatment of primary hepatocytes and intact murine liver requires AMPK regulation of both RAPTOR and TSC2 to fully inhibit mTORC1, and this regulation is critical for both the translational and transcriptional response to metformin. Transcriptionally, AMPK and mTORC1 were both important for regulation of anabolic metabolism and inflammatory programs triggered by metformin treatment. The hepatic transcriptional response in mice on high-fat diet treated with metformin was largely ablated by AMPK deficiency under the conditions examined, indicating the essential role of this kinase and its targets in metformin action in vivo.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Metformina/farmacologia , Proteína Regulatória Associada a mTOR/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Genótipo , Hipoglicemiantes/farmacologia , Inflamação , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metabolismo/efeitos dos fármacos , Metformina/uso terapêutico , Camundongos , Fosforilação/efeitos dos fármacos , Proteína Regulatória Associada a mTOR/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.
Assuntos
Drosophila melanogaster/metabolismo , Metabolismo Energético , Insulina/metabolismo , Transdução de Sinais , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ingestão de Alimentos , Fatores de Transcrição Forkhead/metabolismo , Histona Desacetilases/metabolismo , Lipase/metabolismo , Metabolismo dos Lipídeos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Triglicerídeos/metabolismoRESUMO
Class IIa histone deacetylases (HDACs) are signal-dependent modulators of transcription with established roles in muscle differentiation and neuronal survival. We show here that in liver, class IIa HDACs (HDAC4, 5, and 7) are phosphorylated and excluded from the nucleus by AMPK family kinases. In response to the fasting hormone glucagon, class IIa HDACs are rapidly dephosphorylated and translocated to the nucleus where they associate with the promoters of gluconeogenic enzymes such as G6Pase. In turn, HDAC4/5 recruit HDAC3, which results in the acute transcriptional induction of these genes via deacetylation and activation of FOXO family transcription factors. Loss of class IIa HDACs in murine liver results in inhibition of FOXO target genes and lowers blood glucose, resulting in increased glycogen storage. Finally, suppression of class IIa HDACs in mouse models of type 2 diabetes ameliorates hyperglycemia, suggesting that inhibitors of class I/II HDACs may be potential therapeutics for metabolic syndrome.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Histona Desacetilases/metabolismo , Proteínas Quinases Ativadas por AMP , Acetilação , Animais , Núcleo Celular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteína Forkhead Box O1 , Glucagon/metabolismo , Gluconeogênese , Homeostase , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
Replicative crisis is a senescence-independent process that acts as a final barrier against oncogenic transformation by eliminating pre-cancerous cells with disrupted cell cycle checkpoints1. It functions as a potent tumour suppressor and culminates in extensive cell death. Cells rarely evade elimination and evolve towards malignancy, but the mechanisms that underlie cell death in crisis are not well understood. Here we show that macroautophagy has a dominant role in the death of fibroblasts and epithelial cells during crisis. Activation of autophagy is critical for cell death, as its suppression promoted bypass of crisis, continued proliferation and accumulation of genome instability. Telomere dysfunction specifically triggers autophagy, implicating a telomere-driven autophagy pathway that is not induced by intrachromosomal breaks. Telomeric DNA damage generates cytosolic DNA species with fragile nuclear envelopes that undergo spontaneous disruption. The cytosolic chromatin fragments activate the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway and engage the autophagy machinery. Our data suggest that autophagy is an integral component of the tumour suppressive crisis mechanism and that loss of autophagy function is required for the initiation of cancer.
Assuntos
Autofagia , Carcinogênese/genética , Carcinogênese/patologia , Proliferação de Células , Instabilidade Cromossômica , Autofagia/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromatina/patologia , Instabilidade Cromossômica/genética , Dano ao DNA/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Proteínas de Membrana/metabolismo , Membrana Nuclear/patologia , Nucleotidiltransferases/metabolismo , Telômero/genética , Telômero/patologiaRESUMO
AMPK is a highly conserved master regulator of metabolism, which restores energy balance during metabolic stress both at the cellular and physiological levels. The identification of numerous AMPK targets has helped explain how AMPK restores energy homeostasis. Recent advancements illustrate novel mechanisms of AMPK regulation, including changes in subcellular localization and phosphorylation by non-canonical upstream kinases. Notably, the therapeutic potential of AMPK is widely recognized and heavily pursued for treatment of metabolic diseases such as diabetes, but also obesity, inflammation, and cancer. Moreover, the recently solved crystal structure of AMPK has shed light both into how nucleotides activate AMPK and, importantly, also into the sites bound by small molecule activators, thus providing a path for improved drugs.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo Energético , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/química , Animais , Autofagia , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática , Ativadores de Enzimas/uso terapêutico , Humanos , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/enzimologia , Doenças Metabólicas/patologia , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Mitofagia , Modelos Moleculares , Fosforilação , Conformação Proteica , Proteólise , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismoRESUMO
Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK(-/-) EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diferenciação Celular , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Lisossomos/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células-Tronco Embrionárias , Endoderma/patologia , Camundongos , Mutação , Transdução de Sinais/genética , Via de Sinalização Wnt/genéticaRESUMO
Nature Reviews Molecular Cell Biology celebrated its 10-year anniversary during this past year with a series of specially commissioned articles. To complement this, here we have asked researchers from across the field for their insights into how molecular cell biology research has evolved during this past decade, the key concepts that have emerged and the most promising interfaces that have developed. Their comments highlight the broad impact that particular advances have had, some of the basic understanding that we still require, and the collaborative approaches that will be essential for driving the field forward.
Assuntos
Biologia Celular/história , Biologia Molecular/história , Biologia Molecular/tendências , Biologia Celular/tendências , História do Século XX , História do Século XXI , Biologia Molecular/métodosRESUMO
The benefits of endurance exercise on general health make it desirable to identify orally active agents that would mimic or potentiate the effects of exercise to treat metabolic diseases. Although certain natural compounds, such as reseveratrol, have endurance-enhancing activities, their exact metabolic targets remain elusive. We therefore tested the effect of pathway-specific drugs on endurance capacities of mice in a treadmill running test. We found that PPARbeta/delta agonist and exercise training synergistically increase oxidative myofibers and running endurance in adult mice. Because training activates AMPK and PGC1alpha, we then tested whether the orally active AMPK agonist AICAR might be sufficient to overcome the exercise requirement. Unexpectedly, even in sedentary mice, 4 weeks of AICAR treatment alone induced metabolic genes and enhanced running endurance by 44%. These results demonstrate that AMPK-PPARdelta pathway can be targeted by orally active drugs to enhance training adaptation or even to increase endurance without exercise.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/metabolismo , PPAR delta/agonistas , Resistência Física/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleotídeos/farmacologia , Tiazóis/farmacologia , Proteínas Quinases Ativadas por AMP , Administração Oral , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/farmacologia , Animais , Biomimética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Condicionamento Físico Animal , Ribonucleotídeos/administração & dosagemRESUMO
Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation, how ULK1 promotes autophagy remains poorly understood. Here, we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered 15 phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.
Assuntos
Autofagia/fisiologia , Benzamidas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/farmacologia , Sequência de Aminoácidos , Animais , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Benzamidas/química , Domínio Catalítico/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Sequência Consenso , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Dados de Sequência Molecular , Fosforilação , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Pirimidinas/química , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The serine/threonine kinase LKB1 is a tumor suppressor whose loss is associated with increased metastatic potential. In an effort to define biochemical signatures of metastasis associated with LKB1 loss, we discovered that the epithelial-to-mesenchymal transition transcription factor Snail1 was uniquely upregulated upon LKB1 deficiency across cell types. The ability of LKB1 to suppress Snail1 levels was independent of AMPK but required the related kinases MARK1 and MARK4. In a screen for substrates of these kinases involved in Snail regulation, we identified the scaffolding protein DIXDC1. Similar to loss of LKB1, DIXDC1 depletion results in upregulation of Snail1 in a FAK-dependent manner, leading to increased cell invasion. MARK1 phosphorylation of DIXDC1 is required for its localization to focal adhesions and ability to suppress metastasis in mice. DIXDC1 is frequently downregulated in human cancers, which correlates with poor survival. This study defines an AMPK-independent phosphorylation cascade essential for LKB1-dependent control of metastatic behavior.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Invasividade Neoplásica/genética , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares , Camundongos , Proteínas dos Microfilamentos/genética , Invasividade Neoplásica/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Recent advances in genome editing technologies have enabled the insertion of epitope tags at endogenous loci with relative efficiency. We describe an approach for investigation of protein interaction dynamics of the AMP-activated kinase complex AMPK using a catalytic subunit AMPKα2 (PRKAA2 gene) as the bait, based on CRISPR/Cas9-mediated genome editing coupled to stable isotope labeling in cell culture, multidimensional protein identification technology, and computational and statistical analyses. Furthermore, we directly compare this genetic epitope tagging approach to endogenous immunoprecipitations of the same gene under homologous conditions to assess differences in observed interactors. Additionally, we directly compared each enrichment strategy in the genetically modified cell-line with two separate endogenous antibodies. For each approach, we analyzed the interaction profiles of this protein complex under basal and activated states, and after implementing the same analytical, computational, and statistical analyses, we found that high-confidence protein interactors vary greatly with each method and between commercially available endogenous antibodies.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genômica/métodos , Mapeamento de Interação de Proteínas/métodos , Anticorpos , Células Cultivadas , Cromatografia de Afinidade , Edição de Genes , Células HEK293 , Humanos , Imunoprecipitação , Marcação por Isótopo , Espectrometria de MassasRESUMO
The Liver Kinase B1 (LKB1) tumor suppressor acts as a metabolic energy sensor to regulate AMP-activated protein kinase (AMPK) signaling and is commonly mutated in various cancers, including non-small cell lung cancer (NSCLC). Tumor cells deficient in LKB1 may be uniquely sensitized to metabolic stresses, which may offer a therapeutic window in oncology. To address this question we have explored how functional LKB1 impacts the metabolism of NSCLC cells using 13C metabolic flux analysis. Isogenic NSCLC cells expressing functional LKB1 exhibited higher flux through oxidative mitochondrial pathways compared to those deficient in LKB1. Re-expression of LKB1 also increased the capacity of cells to oxidize major mitochondrial substrates, including pyruvate, fatty acids, and glutamine. Furthermore, LKB1 expression promoted an adaptive response to energy stress induced by anchorage-independent growth. Finally, this diminished adaptability sensitized LKB1-deficient cells to combinatorial inhibition of mitochondrial complex I and glutaminase. Together, our data implicate LKB1 as a major regulator of adaptive metabolic reprogramming and suggest synergistic pharmacological strategies for mitigating LKB1-deficient NSCLC tumor growth.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Metabolismo Energético , Neoplasias Pulmonares/metabolismo , Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Células A549 , Quinases Proteína-Quinases Ativadas por AMP , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Calcineurina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Longevidade/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Inibidores de Calcineurina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Regulação para Baixo , Metabolismo Energético , Ativação Enzimática , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Longevidade/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/química , Transativadores/deficiência , Transativadores/genética , Fatores de Transcrição/biossíntese , Transcrição GênicaRESUMO
One of the major metabolic changes associated with cellular transformation is enhanced nutrient utilization, which supports tumor progression by fueling both energy production and providing biosynthetic intermediates for growth. The liver kinase B1 (LKB1) is a serine/threonine kinase and tumor suppressor that couples bioenergetics to cell-growth control through regulation of mammalian target of rapamycin (mTOR) activity; however, the influence of LKB1 on tumor metabolism is not well defined. Here, we show that loss of LKB1 induces a progrowth metabolic program in proliferating cells. Cells lacking LKB1 display increased glucose and glutamine uptake and utilization, which support both cellular ATP levels and increased macromolecular biosynthesis. This LKB1-dependent reprogramming of cell metabolism is dependent on the hypoxia-inducible factor-1α (HIF-1α), which accumulates under normoxia in LKB1-deficient cells and is antagonized by inhibition of mTOR complex I signaling. Silencing HIF-1α reverses the metabolic advantages conferred by reduced LKB1 signaling and impairs the growth and survival of LKB1-deficient tumor cells under low-nutrient conditions. Together, our data implicate the tumor suppressor LKB1 as a central regulator of tumor metabolism and growth control through the regulation of HIF-1α-dependent metabolic reprogramming.
Assuntos
Metabolismo Energético/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Redes e Vias Metabólicas/genética , Proteínas Serina-Treonina Quinases/deficiência , Quinases Proteína-Quinases Ativadas por AMP , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Fibroblastos , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Redes e Vias Metabólicas/fisiologia , Camundongos , Complexos Multiproteicos/metabolismo , Consumo de Oxigênio/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover a conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status.
Assuntos
Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases Ativadas por AMP , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Apoptose , Ciclo Celular , Linhagem Celular , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos , Complexos Multienzimáticos/genética , Complexos Multiproteicos , Biblioteca de Peptídeos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteômica , Proteína Regulatória Associada a mTOR , Serina/metabolismo , Especificidade por Substrato , Serina-Treonina Quinases TOR , Fatores de Transcrição/antagonistas & inibidoresRESUMO
Quantification of proteomes by mass spectrometry has proven to be useful to study human pathology recapitulated in cellular or animal models of disease. Enriching and quantifying newly synthesized proteins (NSPs) at set time points by mass spectrometry has the potential to identify important early regulatory or expression changes associated with disease states or perturbations. NSP can be enriched from proteomes by employing pulsed introduction of the noncanonical amino acid, azidohomoalanine (AHA). We demonstrate that pulsed introduction of AHA in the feed of mice can label and identify NSP from multiple tissues. Furthermore, we quantitate differences in new protein expression resulting from CRE-LOX initiated knockout of LKB1 in mouse livers. Overall, the PALM strategy allows for the first time in vivo labeling of mouse tissues to differentiate protein synthesis rates at discrete time points.