RESUMEN
Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a 13C labeling approach, called 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.
Asunto(s)
Adenosina , Isótopos de Carbono , Guanosina/análogos & derivados , Procesamiento Postranscripcional del ARN , ARN , Adenosina/química , Adenosina/metabolismo , Adenosina/farmacología , Isótopos de Carbono/química , Isótopos de Carbono/farmacología , Guanosina/química , Guanosina/metabolismo , Guanosina/farmacología , Marcaje Isotópico , ARN/química , ARN/metabolismo , Espectrometría de Masas en TándemRESUMEN
The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it is unclear whether mechanistic target of rapamycin (mTOR)-4EBP1/2 is the exclusive translation regulator of this group of genes, and furthermore, systematic searches for novel translation modulators have been immensely challenging because of difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for translation modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translation effects in a manner that is dependent on GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a noncanonical, mTOR-independent mode for translation regulation of ribosomal proteins.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Complejos Multiproteicos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/biosíntesis , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Portadoras/genética , Línea Celular Transformada , Línea Celular Tumoral , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Complejos Multiproteicos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas Ribosómicas/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Inactivation of the retinoblastoma tumor suppressor (pRB) alters the expression of a myriad of genes. To understand the altered cellular environment that these changes create, we took advantage of the Drosophila model system and used targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile the metabolic changes that occur when RBF1, the fly ortholog of pRB, is removed. We show that RBF1-depleted tissues and larvae are sensitive to fasting. Depletion of RBF1 causes major changes in nucleotide synthesis and glutathione metabolism. Under fasting conditions, these changes interconnect, and the increased replication demand of RBF1-depleted larvae is associated with the depletion of glutathione pools. In vivo (13)C isotopic tracer analysis shows that RBF1-depleted larvae increase the flux of glutamine toward glutathione synthesis, presumably to minimize oxidative stress. Concordantly, H(2)O(2) preferentially promoted apoptosis in RBF1-depleted tissues, and the sensitivity of RBF1-depleted animals to fasting was specifically suppressed by either a glutamine supplement or the antioxidant N-acetyl-cysteine. Effects of pRB activation/inactivation on glutamine catabolism were also detected in human cell lines. These results show that the inactivation of RB proteins causes metabolic reprogramming and that these consequences of RBF/RB function are present in both flies and human cell lines.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Glutamina/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Daño del ADN , Ayuno/metabolismo , Glutatión/biosíntesis , Humanos , Larva , Mutación , Nucleótidos/biosíntesis , Estrés Oxidativo , Proteína de Retinoblastoma , Estrés FisiológicoRESUMEN
Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.
Asunto(s)
Hipoxia de la Célula , Glutamina/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Lipogénesis , Acetilcoenzima A/biosíntesis , Acetilcoenzima A/metabolismo , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linfocitos T CD8-positivos/citología , Carbono/metabolismo , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Células Cultivadas , Ciclo del Ácido Cítrico , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isocitrato Deshidrogenasa/deficiencia , Isocitrato Deshidrogenasa/genética , Ácidos Cetoglutáricos/metabolismo , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Oxidación-Reducción , Oxígeno/metabolismo , Ácido Palmítico/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Cancer and proliferating cells exhibit an increased demand for glutamine-derived carbons to support anabolic processes. In addition, reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) was recently shown to be a major source of citrate synthesis from glutamine. The role of NAD(P)H/NAD(P)(+) cofactors in coordinating glucose and glutamine utilization in the tricarboxylic acid (TCA) cycle is not well understood, with the source(s) of NADPH for the reductive carboxylation reaction remaining unexplored. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial enzyme that transfers reducing equivalents from NADH to NADPH. Here, we show that knockdown of NNT inhibits the contribution of glutamine to the TCA cycle and activates glucose catabolism in SkMel5 melanoma cells. The increase in glucose oxidation partially occurred through pyruvate carboxylase and rendered NNT knockdown cells more sensitive to glucose deprivation. Importantly, knocking down NNT inhibits reductive carboxylation in SkMel5 and 786-O renal carcinoma cells. Overexpression of NNT is sufficient to stimulate glutamine oxidation and reductive carboxylation, whereas it inhibits glucose catabolism in the TCA cycle. These observations are supported by an impairment of the NAD(P)H/NAD(P)(+) ratios. Our findings underscore the role of NNT in regulating central carbon metabolism via redox balance, calling for other mechanisms that coordinate substrate preference to maintain a functional TCA cycle.
Asunto(s)
Ciclo del Ácido Cítrico/fisiología , Glucosa/metabolismo , NADP Transhidrogenasa AB-Específica/metabolismo , NADP/metabolismo , NAD/metabolismo , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Glucosa/genética , Ratones , Ratones Desnudos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , NAD/genética , NADP/genética , NADP Transhidrogenasa AB-Específica/genética , Oxidación-ReducciónRESUMEN
Introduction: CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in local tissues can result in significant exposure of cytotoxic T cells to the lactate metabolite. Lactate has been known to act as an immunosuppressor, at least in part due to its association with tissue acidosis. Methods: To dissect the role of the lactate anion, independently of pH, we performed phenotypical and metabolic assays, high-throughput RNA sequencing, and mass spectrometry, on primary cultures of murine or human CD8+ T cells exposed to high doses of pH-neutral sodium lactate. Results: The lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and can displace glucose as a carbon source. Activation in the presence of sodium lactate significantly alters the CD8+ T cell transcriptome, including the expression key effector differentiation markers such as granzyme B and interferon-gamma. Discussion: Our studies reveal novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells.
Asunto(s)
Ácido Láctico , Transcriptoma , Ratones , Humanos , Animales , Ácido Láctico/metabolismo , Lactato de Sodio/metabolismo , Linfocitos T CD8-positivos/metabolismo , Linfocitos T Citotóxicos/metabolismoRESUMEN
Oncogenes such as K-ras mediate cellular and metabolic transformation during tumorigenesis. To analyze K-Ras-dependent metabolic alterations, we employed ¹³C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD) of ¹5N-labeled glutamine, and transcriptomic profiling in mouse fibroblast and human carcinoma cell lines. Stable isotope-labeled glucose and glutamine tracers and computational determination of intracellular fluxes indicated that cells expressing oncogenic K-Ras exhibited enhanced glycolytic activity, decreased oxidative flux through the tricarboxylic acid (TCA) cycle, and increased utilization of glutamine for anabolic synthesis. Surprisingly, a non-canonical labeling of TCA cycle-associated metabolites was detected in both transformed cell lines. Transcriptional profiling detected elevated expression of several genes associated with glycolysis, glutamine metabolism, and nucleotide biosynthesis upon transformation with oncogenic K-Ras. Chemical perturbation of enzymes along these pathways further supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle. These results provide evidence for a role of oncogenic K-Ras in the metabolic reprogramming of cancer cells.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Ciclo del Ácido Cítrico , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Glucosa/metabolismo , Glutamina/metabolismo , Glucólisis , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , Animales , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Fibroblastos/citología , Citometría de Flujo , Cromatografía de Gases y Espectrometría de Masas , Perfilación de la Expresión Génica , Humanos , Marcaje Isotópico , Ratones , Células 3T3 NIH , Neoplasias/genética , Neoplasias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
Exercise has a wide range of systemic effects. In animal models, repeated exertion reduces malignant tumor progression, and clinically, exercise can improve outcome for cancer patients. The etiology of the effects of exercise on tumor progression are unclear, as are the cellular actors involved. We show here that in mice, exercise-induced reduction in tumor growth is dependent on CD8+ T cells, and that metabolites produced in skeletal muscle and excreted into plasma at high levels during exertion in both mice and humans enhance the effector profile of CD8+ T-cells. We found that activated murine CD8+ T cells alter their central carbon metabolism in response to exertion in vivo, and that immune cells from trained mice are more potent antitumor effector cells when transferred into tumor-bearing untrained animals. These data demonstrate that CD8+ T cells are metabolically altered by exercise in a manner that acts to improve their antitumoral efficacy.
Exercise affects almost all tissues in the body, and scientists have found that being physically active can reduce the risk of several types of cancer as well as improving outcomes for cancer patients. However, it is still unknown how exercise exerts its protective effects. One of the hallmarks of cancer is the ability of cancer cells to evade detection by the immune system, which can in some cases stop the body from eliminating tumor cells. Rundqvist et al. used mice to investigate how exercise helps the immune system act against tumor progression. They found that when mice exercised, tumor growth was reduced, and this decrease in growth depended on the levels of a specific type of immune cell, the CD8+ T cell, circulating in the blood. Additionally, Rundqvist et al. found that CD8+ T cells were made more effective by molecules that muscles released into the blood during exercise. Isolating immune cells after intense exercise showed that these super-effective CD8+ T cells alter how they use molecules for energy production after exertion. Next, immune cells from mice that had exercised frequently were transferred into mice that had not exercised, where they were more effective against tumor cells than the immune cells from untrained mice. These results demonstrate that CD8+ T cells are altered by exercise to improve their effectiveness against tumors. The ability of T cells to identify and eliminate cancer cells is essential to avoid tumor growth, and is one of the foundations of current immune therapy treatments. Exercise could improve the outcome of these treatments by increasing the activation of the immune system, making tumor-fighting cells more effective.
Asunto(s)
Carcinogénesis , Condicionamiento Físico Animal , Linfocitos T Citotóxicos/fisiología , Animales , Línea Celular Tumoral , Femenino , RatonesRESUMEN
Nutrient deprivation inhibits mRNA translation through mTOR and eIF2α signaling, but it is unclear how the translational program is controlled to reflect the degree of a metabolic stress. In a model of breast cellular transformation, various forms of nutrient deprivation differentially affect the rate of protein synthesis and its recovery over time. Genome-wide translational profiling of glutamine-deprived cells reveals a rapid upregulation of mRNAs containing uORFs and downregulation of ribosomal protein mRNAs, which are followed by selective translation of cytokine and inflammatory mRNAs. Transcription and translation of inflammatory and cytokine genes are stimulated in response to diverse metabolic stresses and depend on eIF2α phosphorylation, with the extent of stimulation correlating with the decrease in global protein synthesis. In accord with the inflammatory stimulus, glutamine deprivation stimulates the migration of transformed cells. Thus, pro-inflammatory gene expression is coupled to metabolic stress, and this can affect cancer cell behavior upon nutrient limitation.
Asunto(s)
Desnutrición/genética , Biosíntesis de Proteínas/genética , Estrés Fisiológico/genética , Humanos , Transducción de SeñalRESUMEN
Many cancer-associated mutations that deregulate cellular metabolic responses to hypoxia also reprogram carbon metabolism to promote utilization of glutamine. In renal cell carcinoma (RCC), cells deficient in the von Hippel-Lindau (VHL) tumor suppressor gene use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate (αKG). Glutamine can also generate aspartate, the carbon source for pyrimidine biosynthesis, and glutathione for redox balance. Here we have shown that VHL-/- RCC cells rely on RC-derived aspartate to maintain de novo pyrimidine biosynthesis. Glutaminase 1 (GLS1) inhibitors depleted pyrimidines and increased ROS in VHL-/- cells but not in VHL+/+ cells, which utilized glucose oxidation for glutamate and aspartate production. GLS1 inhibitor-induced nucleoside depletion and ROS enhancement led to DNA replication stress and activation of an intra-S phase checkpoint, and suppressed the growth of VHL-/- RCC cells. These effects were rescued by administration of glutamate, αKG, or nucleobases with N-acetylcysteine. Further, we observed that the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib synergizes with GLS1 inhibitors to suppress the growth of VHL-/- cells in vitro and in vivo. This work describes a mechanism that explains the sensitivity of RCC tumor growth to GLS1 inhibitors and supports the development of therapeutic strategies for targeting VHL-deficient RCC.
Asunto(s)
Glutaminasa/antagonistas & inhibidores , Neoplasias Renales/tratamiento farmacológico , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Animales , Carcinoma de Células Renales , Glutamatos/genética , Glutamatos/metabolismo , Glutaminasa/genética , Glutaminasa/metabolismo , Glutamina/genética , Glutamina/metabolismo , Humanos , Neoplasias Renales/enzimología , Neoplasias Renales/genética , Neoplasias Renales/patología , Ratones , Ratones Desnudos , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase S del Ciclo Celular/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glutamine has recently emerged as a key substrate to support cancer cell proliferation, and the quantification of its metabolic flux is essential to understand the mechanisms by which this amino acid participates in the metabolic rewiring that sustains the survival and growth of neoplastic cells. Glutamine metabolism involves two major routes, glutaminolysis and reductive carboxylation, both of which begin with the deamination of glutamine to glutamate and the conversion of glutamate into α-ketoglutarate. In glutaminolysis, α-ketoglutarate is oxidized via the tricarboxylic acid cycle and decarboxylated to pyruvate. In reductive carboxylation, α-ketoglutarate is reductively converted into isocitrate, which is isomerized to citrate to supply acetyl-CoA for de novo lipogenesis. Here, we describe methods to quantify the metabolic flux of glutamine through these two routes, as well as the contribution of glutamine to lipid synthesis. Examples of how these methods can be applied to study metabolic pathways of oncological relevance are provided.
Asunto(s)
Bioquímica/métodos , Ácido Glutámico/análisis , Glutamina/análisis , Glutamina/metabolismo , Neoplasias/metabolismo , Isótopos de Carbono , Hipoxia de la Célula , Células Cultivadas , Cromatografía de Gases y Espectrometría de Masas , Ácido Glutámico/metabolismo , Humanos , Lípidos/biosíntesisRESUMEN
Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [(1-13)C]glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts.