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1.
Nat Commun ; 12(1): 2029, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795689

RESUMO

Mucosal-associated invariant T (MAIT) cells have important functions in immune responses against pathogens and in diseases, but mechanisms controlling MAIT cell development and effector lineage differentiation remain unclear. Here, we report that IL-2/IL-15 receptor ß chain and inducible costimulatory (ICOS) not only serve as lineage-specific markers for IFN-γ-producing MAIT1 and IL-17A-producing MAIT17 cells, but are also important for their differentiation, respectively. Both IL-2 and IL-15 induce mTOR activation, T-bet upregulation, and subsequent MAIT cell, especially MAIT1 cell, expansion. By contrast, IL-1ß induces more MAIT17 than MAIT1 cells, while IL-23 alone promotes MAIT17 cell proliferation and survival, but synergizes with IL-1ß to induce strong MAIT17 cell expansion in an mTOR-dependent manner. Moreover, mTOR is dispensable for early MAIT cell development, yet pivotal for MAIT cell effector differentiation. Our results thus show that mTORC2 integrates signals from ICOS and IL-1ßR/IL-23R to exert a crucial role for MAIT17 differentiation, while the IL-2/IL-15R-mTORC1-T-bet axis ensures MAIT1 differentiation.


Assuntos
Citocinas/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Ativação Linfocitária/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/imunologia , Células T Invariáveis Associadas à Mucosa/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Interleucina-15/imunologia , Interleucina-15/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células T Invariáveis Associadas à Mucosa/citologia , Células T Invariáveis Associadas à Mucosa/metabolismo , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismo
2.
Nat Commun ; 12(1): 1876, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767183

RESUMO

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.


Assuntos
/patologia , Ciclo do Ácido Cítrico/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Benzamidas/farmacologia , Linhagem Celular , Chlorocebus aethiops , Glucose/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Pulmão/metabolismo , Pulmão/virologia , Morfolinas/farmacologia , Naftiridinas/farmacologia , Pirimidinas/farmacologia , Piruvato Carboxilase/biossíntese , Células Vero , Replicação Viral/efeitos dos fármacos
3.
Nat Commun ; 12(1): 1589, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707434

RESUMO

Glutathione peroxidase 4 (GPX4) utilizes glutathione (GSH) to detoxify lipid peroxidation and plays an essential role in inhibiting ferroptosis. As a selenoprotein, GPX4 protein synthesis is highly inefficient and energetically costly. How cells coordinate GPX4 synthesis with nutrient availability remains unclear. In this study, we perform integrated proteomic and functional analyses to reveal that SLC7A11-mediated cystine uptake promotes not only GSH synthesis, but also GPX4 protein synthesis. Mechanistically, we find that cyst(e)ine activates mechanistic/mammalian target of rapamycin complex 1 (mTORC1) and promotes GPX4 protein synthesis at least partly through the Rag-mTORC1-4EBP signaling axis. We show that pharmacologic inhibition of mTORC1 decreases GPX4 protein levels, sensitizes cancer cells to ferroptosis, and synergizes with ferroptosis inducers to suppress patient-derived xenograft tumor growth in vivo. Together, our results reveal a regulatory mechanism to coordinate GPX4 protein synthesis with cyst(e)ine availability and suggest using combinatorial therapy of mTORC1 inhibitors and ferroptosis inducers in cancer treatment.


Assuntos
Cisteína/metabolismo , Cistina/metabolismo , Ferroptose/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Glutationa/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Neoplasias/patologia
4.
Nat Commun ; 12(1): 1055, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594058

RESUMO

mTORC1, a central controller of cell proliferation in response to growth factors and nutrients, is dysregulated in cancer. Whereas arginine activates mTORC1, it is overridden by high expression of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1). Because cancer cells often encounter low levels of nutrients, an alternative mechanism might exist to regulate CASTOR1 expression. Here we show K29-linked polyubiquitination and degradation of CASTOR1 by E3 ubiquitin ligase RNF167. Furthermore, AKT phosphorylates CASTOR1 at S14, significantly increasing its binding to RNF167, and hence its ubiquitination and degradation, while simultaneously decreasing its affinity to MIOS, leading to mTORC1 activation. Therefore, AKT activates mTORC1 through both TSC2- and CASTOR1-dependent pathways. Several cell types with high CASTOR1 expression are insensitive to arginine regulation. Significantly, AKT and RNF167-mediated CASTOR1 degradation activates mTORC1 independent of arginine and promotes breast cancer progression. These results illustrate a mTORC1 regulating mechanism and identify RNF167 as a therapeutic target for mTORC1-dysregulated diseases.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Arginina/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Cinética , Lisina/metabolismo , Camundongos Nus , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação/efeitos dos fármacos
5.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557396

RESUMO

HIV-1 infects T cells, but the most frequent AIDS-related lymphomas are of B-cell origin. Molecular mechanisms of HIV-1-induced oncogenic transformation of B cells remain largely unknown. HIV-1 Tat protein may participate in this process by penetrating and regulating gene expression in B cells. Both immune and cancer cells can reprogram communications between extracellular signals and intracellular signaling pathways via the Akt/mTORC1 pathway, which plays a key role in the cellular response to various stimuli including viral infection. Here, we investigated the role of HIV-1 Tat on the modulation of the Akt/mTORC1 pathway in B cells. We found that HIV-1 Tat activated the Akt/mTORC1 signaling pathway; this leads to aberrant activation of activation-induced cytidine deaminase (AICDA) due to inhibition of the AICDA transcriptional repressors c-Myb and E2F8. These perturbations may ultimately lead to an increased genomic instability and proliferation that might cause B cell malignancies.


Assuntos
Linfócitos B/patologia , Citidina Desaminase/metabolismo , Dano ao DNA , Regulação da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Citidina Desaminase/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
6.
Nat Genet ; 53(1): 16-26, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33414552

RESUMO

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.


Assuntos
Neoplasias Colorretais/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Regiões 5' não Traduzidas/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Carcinogênese/patologia , Proliferação de Células , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Glutamina/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Estimativa de Kaplan-Meier , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/metabolismo , Metástase Neoplásica , Oncogenes , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
7.
Nat Commun ; 12(1): 245, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431855

RESUMO

Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors.


Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Transcriptoma/genética , Resultado do Tratamento
8.
Am J Physiol Endocrinol Metab ; 320(3): E551-E565, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33427053

RESUMO

Extrauterine growth restriction in premature infants is largely attributed to reduced lean mass accretion and is associated with long-term morbidities. Previously, we demonstrated that prematurity blunts the feeding-induced stimulation of translation initiation signaling and protein synthesis in skeletal muscle of neonatal pigs. The objective of the current study was to determine whether the blunted feeding response is mediated by reduced responsiveness to insulin, amino acids, or both. Pigs delivered by cesarean section preterm (PT; 103 days, n = 25) or at term (T; 112 days, n = 26) were subject to euinsulinemic-euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps four days after delivery. Indices of mechanistic target of rapamycin complex 1 (mTORC1) signaling and fractional protein synthesis rates were measured after 2 h. Although longissimus dorsi (LD) muscle protein synthesis increased in response to both INS and AA, the increase was 28% lower in PT than in T. Upstream of mTORC1, Akt phosphorylation, an index of insulin signaling, was increased with INS but was 40% less in PT than in T. The abundances of mTOR·RagA and mTOR·RagC, indices of amino acid signaling, increased with AA but were 25% less in PT than in T. Downstream of mTORC1, eIF4E·eIF4G abundance was increased by both INS and AA but attenuated by prematurity. These results suggest that preterm birth blunts both insulin- and amino acid-induced activation of mTORC1 and protein synthesis in skeletal muscle, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the high prevalence of extrauterine growth restriction in prematurity.NEW & NOTEWORTHY Extrauterine growth faltering is a major complication of premature birth, but the underlying cause is poorly understood. Our results demonstrate that preterm birth blunts both the insulin-and amino acid-induced activation of mTORC1-dependent translation initiation and protein synthesis in skeletal muscle, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the reduced accretion of lean mass and extrauterine growth restriction of premature infants.


Assuntos
Aminoácidos/farmacologia , Insulina/farmacologia , Músculo Esquelético/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Nascimento Prematuro/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Músculo Esquelético/metabolismo , Gravidez , Transdução de Sinais/efeitos dos fármacos , Suínos
9.
Am J Physiol Heart Circ Physiol ; 320(3): H980-H990, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33416457

RESUMO

Perinatal hypoxia induces permanent structural and functional changes in the lung and its pulmonary circulation that are associated with the development of pulmonary hypertension (PH) in later life. The mechanistic target of the rapamycin (mTOR) pathway is vital for fetal lung development and is implicated in hypoxia-associated PH, yet its involvement in the developmental programming of PH remains unclear. Pregnant C57/BL6 dams were placed in hyperbaric (760 mmHg) or hypobaric chambers during gestation (505 mmHg, day 15 through postnatal day 4) or from weaning through adulthood (420 mmHg, postnatal day 21 through 8 wk). Pulmonary hemodynamics and right ventricular systolic pressure (RVSP) were measured at 8 wk. mTOR pathway proteins were assessed in fetal (day 18.5) and adult lung (8 wk). Perinatal hypoxia induced PH during adulthood, even in the absence of a sustained secondary hypoxic exposure, as indicated by reduced pulmonary artery acceleration time (PAAT) and peak flow velocity through the pulmonary valve, as well as greater RVSP, right ventricular (RV) wall thickness, and RV/left ventricular (LV) weight. Such effects were independent of increased blood viscosity. In fetal lung homogenates, hypoxia reduced the expression of critical downstream mTOR targets, most prominently total and phosphorylated translation repressor protein (4EBP1), as well as vascular endothelial growth factor, a central regulator of angiogenesis in the fetal lung. In contrast, adult offspring of hypoxic dams tended to have elevated p4EBP1 compared with controls. Our data suggest that inhibition of mTORC1 activity in the fetal lung as a result of gestational hypoxia may interrupt pulmonary vascular development and thereby contribute to the developmental programming of PH.NEW & NOTEWORTHY We describe the first study to evaluate a role for the mTOR pathway in the developmental programming of pulmonary hypertension. Our findings suggest that gestational hypoxia impairs mTORC1 activation in the fetal lung and may impede pulmonary vascular development, setting the stage for pulmonary vascular disease in later life.


Assuntos
Hipóxia Fetal/complicações , Hipertensão Pulmonar/etiologia , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neovascularização Fisiológica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Hipóxia Fetal/metabolismo , Hipóxia Fetal/fisiopatologia , Idade Gestacional , Hemodinâmica , Oxigenação Hiperbárica , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Camundongos Endogâmicos C57BL , Fosforilação , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Circulação Pulmonar , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Função Ventricular Direita , Pressão Ventricular
10.
Mol Cell ; 81(2): 386-397.e7, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33340488

RESUMO

In tumors, nutrient availability and metabolism are known to be important modulators of growth signaling. However, it remains elusive whether cancer cells that are growing out in the metastatic niche rely on the same nutrients and metabolic pathways to activate growth signaling as cancer cells within the primary tumor. We discovered that breast-cancer-derived lung metastases, but not the corresponding primary breast tumors, use the serine biosynthesis pathway to support mTORC1 growth signaling. Mechanistically, pyruvate uptake through Mct2 supported mTORC1 signaling by fueling serine biosynthesis-derived α-ketoglutarate production in breast-cancer-derived lung metastases. Consequently, expression of the serine biosynthesis enzyme PHGDH was required for sensitivity to the mTORC1 inhibitor rapamycin in breast-cancer-derived lung tumors, but not in primary breast tumors. In summary, we provide in vivo evidence that the metabolic and nutrient requirements to activate growth signaling differ between the lung metastatic niche and the primary breast cancer site.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Mamárias Experimentais/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Fosfoglicerato Desidrogenase/genética , Serina/biossíntese , Animais , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Ácidos Cetoglutáricos/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Fosfoglicerato Desidrogenase/metabolismo , Ácido Pirúvico/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirolimo/farmacologia
11.
Mol Cell ; 81(2): 398-407.e4, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33340489

RESUMO

Mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and proliferation by sensing fluctuations in environmental cues such as nutrients, growth factors, and energy levels. The Rag GTPases (Rags) serve as a critical module that signals amino acid (AA) availability to modulate mTORC1 localization and activity. Recent studies have demonstrated how AAs regulate mTORC1 activity through Rags. Here, we uncover an unconventional pathway that activates mTORC1 in response to variations in threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. TARS2 interacts with inactive Rags, particularly GTP-RagC, leading to increased GTP loading of RagA. mTORC1 activity in cells lacking TARS2 is resistant to Thr repletion, showing that TARS2 is necessary for Thr-dependent mTORC1 activation. The requirement of TARS2, but not cytoplasmic threonyl-tRNA synthetase TARS, for this effect demonstrates an additional layer of complexity in the regulation of mTORC1 activity.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mitocôndrias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Treonina-tRNA Ligase/genética , Treonina/metabolismo , Regulação da Expressão Gênica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de Sinais , Treonina-tRNA Ligase/antagonistas & inibidores , Treonina-tRNA Ligase/metabolismo
12.
Nature ; 590(7845): 315-319, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33328636

RESUMO

Effective pharmacotherapy for major depressive disorder remains a major challenge, as more than 30% of patients are resistant to the first line of treatment (selective serotonin reuptake inhibitors)1. Sub-anaesthetic doses of ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist2,3, provide rapid and long-lasting antidepressant effects in these patients4-6, but the molecular mechanism of these effects remains unclear7,8. Ketamine has been proposed to exert its antidepressant effects through its metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK)9. The antidepressant effects of ketamine and (2R,6R)-HNK in rodents require activation of the mTORC1 kinase10,11. mTORC1 controls various neuronal functions12, particularly through cap-dependent initiation of mRNA translation via the phosphorylation and inactivation of eukaryotic initiation factor 4E-binding proteins (4E-BPs)13. Here we show that 4E-BP1 and 4E-BP2 are key effectors of the antidepressant activity of ketamine and (2R,6R)-HNK, and that ketamine-induced hippocampal synaptic plasticity depends on 4E-BP2 and, to a lesser extent, 4E-BP1. It has been hypothesized that ketamine activates mTORC1-4E-BP signalling in pyramidal excitatory cells of the cortex8,14. To test this hypothesis, we studied the behavioural response to ketamine and (2R,6R)-HNK in mice lacking 4E-BPs in either excitatory or inhibitory neurons. The antidepressant activity of the drugs is mediated by 4E-BP2 in excitatory neurons, and 4E-BP1 and 4E-BP2 in inhibitory neurons. Notably, genetic deletion of 4E-BP2 in inhibitory neurons induced a reduction in baseline immobility in the forced swim test, mimicking an antidepressant effect. Deletion of 4E-BP2 specifically in inhibitory neurons also prevented the ketamine-induced increase in hippocampal excitatory neurotransmission, and this effect concurred with the inability of ketamine to induce a long-lasting decrease in inhibitory neurotransmission. Overall, our data show that 4E-BPs are central to the antidepressant activity of ketamine.


Assuntos
Antidepressivos/farmacologia , Fator de Iniciação 4E em Eucariotos/metabolismo , Ketamina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transtorno Depressivo Maior/tratamento farmacológico , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Ketamina/análogos & derivados , Ketamina/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Mutação , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Neurônios/classificação , Neurônios/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Transmissão Sináptica/efeitos dos fármacos
13.
Int J Mol Sci ; 22(1)2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-33375025

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. Dysregulation in the mTORC1 network underlies a wide array of pathological states, including metabolic diseases, neurological disorders, and cancer. Tumor cells are characterized by uncontrolled growth and proliferation due to a reduced dependency on exogenous growth factors. The genetic events underlying this property, such as mutations in the PI3K-Akt and Ras-Erk signaling networks, lead to constitutive activation of mTORC1 in nearly all human cancer lineages. Aberrant activation of mTORC1 has been shown to play a key role for both anabolic tumor growth and resistance to targeted therapeutics. While displaying a growth factor-independent mTORC1 activity and proliferation, tumors cells remain dependent on exogenous nutrients such as amino acids (AAs). AAs are an essential class of nutrients that are obligatory for the survival of any cell. Known as the building blocks of proteins, AAs also act as essential metabolites for numerous biosynthetic processes such as fatty acids, membrane lipids and nucleotides synthesis, as well as for maintaining redox homeostasis. In most tumor types, mTORC1 activity is particularly sensitive to intracellular AA levels. This dependency, therefore, creates a targetable vulnerability point as cancer cells become dependent on AA transporters to sustain their homeostasis. The following review will discuss the role of AA transporters for mTORC1 signaling in cancer cells and their potential as therapeutic drug targets.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neoplasias/metabolismo , Transdução de Sinais/fisiologia , Sistemas de Transporte de Aminoácidos/genética , Animais , Proliferação de Células/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mutação , Neoplasias/genética , Neoplasias/patologia , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
14.
Nat Commun ; 11(1): 5133, 2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33046706

RESUMO

Cathepsin D (CTSD) is a lysosomal protease and a marker of poor prognosis in breast cancer. However, the cells responsible for this association and the function of CTSD in cancer are still incompletely understood. By using a conditional CTSD knockout mouse crossed to the transgenic MMTV-PyMT breast cancer model we demonstrate that CTSD deficiency in the mammary epithelium, but not in myeloid cells, blocked tumor development in a cell-autonomous manner. We show that lack of CTSD impaired mechanistic Target of Rapamycin Complex 1 (mTORC1) signaling and induced reversible cellular quiescence. In line, CTSD-deficient tumors started to grow with a two-month delay and quiescent Ctsd-/- tumor cells re-started proliferation upon long-term culture. This was accompanied by rewiring of oncogenic gene expression and signaling pathways, while mTORC1 signaling remained permanently disabled in CTSD-deficient cells. Together, these studies reveal a tumor cell-autonomous effect of CTSD deficiency, and establish a pivotal role of this protease in the cellular response to oncogenic stimuli.


Assuntos
Neoplasias da Mama/metabolismo , Catepsina D/genética , Epitélio/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Neoplasias da Mama/genética , Catepsina D/deficiência , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais
15.
Science ; 370(6514): 351-356, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060361

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.


Assuntos
Aminoácidos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Lisossomos/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas R-SNARE/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Pinocitose , Proteólise
16.
Nat Commun ; 11(1): 4510, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908143

RESUMO

With human median lifespan extending into the 80s in many developed countries, the societal burden of age-related muscle loss (sarcopenia) is increasing. mTORC1 promotes skeletal muscle hypertrophy, but also drives organismal aging. Here, we address the question of whether mTORC1 activation or suppression is beneficial for skeletal muscle aging. We demonstrate that chronic mTORC1 inhibition with rapamycin is overwhelmingly, but not entirely, positive for aging mouse skeletal muscle, while genetic, muscle fiber-specific activation of mTORC1 is sufficient to induce molecular signatures of sarcopenia. Through integration of comprehensive physiological and extensive gene expression profiling in young and old mice, and following genetic activation or pharmacological inhibition of mTORC1, we establish the phenotypically-backed, mTORC1-focused, multi-muscle gene expression atlas, SarcoAtlas (https://sarcoatlas.scicore.unibas.ch/), as a user-friendly gene discovery tool. We uncover inter-muscle divergence in the primary drivers of sarcopenia and identify the neuromuscular junction as a focal point of mTORC1-driven muscle aging.


Assuntos
Envelhecimento/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fibras Musculares Esqueléticas/patologia , Junção Neuromuscular/patologia , Sarcopenia/patologia , Envelhecimento/efeitos dos fármacos , Animais , Linhagem Celular , Modelos Animais de Doenças , Eletromiografia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Microdissecção e Captura a Laser , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Mioblastos , Junção Neuromuscular/efeitos dos fármacos , Técnicas de Patch-Clamp , RNA-Seq , Sarcopenia/genética , Sarcopenia/fisiopatologia , Sarcopenia/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/administração & dosagem
17.
Proc Natl Acad Sci U S A ; 117(41): 25560-25570, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32989144

RESUMO

Deregulated global mRNA translation is an emerging feature of cancer cells. Oncogenic transformation in colorectal cancer (CRC) is driven by mutations in APC, KRAS, SMAD4, and TP53, known as the adenoma-carcinoma sequence (ACS). Here we introduce each of these driver mutations into intestinal organoids to show that they are modulators of global translational capacity in intestinal epithelial cells. Increased global translation resulting from loss of Apc expression was potentiated by the presence of oncogenic Kras G12D Knockdown of Smad4 further enhanced global translation efficiency and was associated with a lower 4E-BP1-to-eIF4E ratio. Quadruple mutant cells with additional P53 loss displayed the highest global translational capacity, paralleled by high proliferation and growth rates, indicating that the proteome is heavily geared toward cell division. Transcriptional reprogramming facilitating global translation included elevated ribogenesis and activation of mTORC1 signaling. Accordingly, interfering with the mTORC1/4E-BP/eIF4E axis inhibited the growth potential endowed by accumulation of multiple drivers. In conclusion, the ACS is characterized by a strongly altered global translational landscape in epithelial cells, exposing a therapeutic potential for direct targeting of the translational apparatus.


Assuntos
Adenoma/genética , Carcinoma/genética , Mutação/ética , Biossíntese de Proteínas/genética , Adenoma/metabolismo , Animais , Carcinoma/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Células HEK293 , Humanos , Intestinos/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Transgênicos , Organoides/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos
18.
Nat Commun ; 11(1): 4706, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943618

RESUMO

Yeast physiology is temporally regulated, this becomes apparent under nutrient-limited conditions and results in respiratory oscillations (YROs). YROs share features with circadian rhythms and interact with, but are independent of, the cell division cycle. Here, we show that YROs minimise energy expenditure by restricting protein synthesis until sufficient resources are stored, while maintaining osmotic homeostasis and protein quality control. Although nutrient supply is constant, cells sequester and store metabolic resources via increased transport, autophagy and biomolecular condensation. Replete stores trigger increased H+ export which stimulates TORC1 and liberates proteasomes, ribosomes, chaperones and metabolic enzymes from non-membrane bound compartments. This facilitates translational bursting, liquidation of storage carbohydrates, increased ATP turnover, and the export of osmolytes. We propose that dynamic regulation of ion transport and metabolic plasticity are required to maintain osmotic and protein homeostasis during remodelling of eukaryotic proteomes, and that bioenergetic constraints selected for temporal organisation that promotes oscillatory behaviour.


Assuntos
Metabolismo Energético/fisiologia , Células Eucarióticas/fisiologia , Proteostase/fisiologia , Autofagia/fisiologia , Reatores Biológicos , Ritmo Circadiano , Glicogênio/metabolismo , Resposta ao Choque Térmico , Ionomicina , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metabolômica , Chaperonas Moleculares , Concentração Osmolar , Pressão Osmótica , Oxigênio/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteoma , Proteômica , Ribossomos , Leveduras/fisiologia
19.
Nat Commun ; 11(1): 4681, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943626

RESUMO

Although advanced lipidomics technology facilitates quantitation of intracellular lipid components, little is known about the regulation of lipid metabolism in cancer cells. Here, we show that disruption of the Gdpd3 gene encoding a lysophospholipase D enzyme significantly decreased self-renewal capacity in murine chronic myelogenous leukaemia (CML) stem cells in vivo. Sophisticated lipidomics analyses revealed that Gdpd3 deficiency reduced levels of certain lysophosphatidic acids (LPAs) and lipid mediators in CML cells. Loss of Gdpd3 also activated AKT/mTORC1 signalling and cell cycle progression while suppressing Foxo3a/ß-catenin interaction within CML stem cell nuclei. Strikingly, CML stem cells carrying a hypomorphic mutation of Lgr4/Gpr48, which encodes a leucine-rich repeat (LRR)-containing G-protein coupled receptor (GPCR) acting downstream of Gdpd3, displayed inadequate disease-initiating capacity in vivo. Our data showing that lysophospholipid metabolism is required for CML stem cell maintenance in vivo establish a new, biologically significant mechanism of cancer recurrence that is independent of oncogene addiction.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Células-Tronco/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box O3/metabolismo , Lisofosfolipídeos/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Recidiva Local de Neoplasia/metabolismo , Diester Fosfórico Hidrolases/genética , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais , beta Catenina/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(38): 23617-23625, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32879008

RESUMO

Low-glucose and -insulin conditions, associated with ketogenic diets, can reduce the activity of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, potentially leading to a range of positive medical and health-related effects. Here, we determined whether mTORC1 signaling is also a target for decanoic acid, a key component of the medium-chain triglyceride (MCT) ketogenic diet. Using a tractable model system, Dictyostelium, we show that decanoic acid can decrease mTORC1 activity, under conditions of constant glucose and in the absence of insulin, measured by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). We determine that this effect of decanoic acid is dependent on a ubiquitin regulatory X domain-containing protein, mediating inhibition of a conserved Dictyostelium AAA ATPase, p97, a homolog of the human transitional endoplasmic reticulum ATPase (VCP/p97) protein. We then demonstrate that decanoic acid decreases mTORC1 activity in the absence of insulin and under high-glucose conditions in ex vivo rat hippocampus and in tuberous sclerosis complex (TSC) patient-derived astrocytes. Our data therefore indicate that dietary decanoic acid may provide a new therapeutic approach to down-regulate mTORC1 signaling.


Assuntos
Ácidos Decanoicos/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Astrócitos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Dictyostelium/efeitos dos fármacos , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Epilepsia , Glucose/metabolismo , Hipocampo/química , Hipocampo/metabolismo , Humanos , Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/farmacologia , Fatores de Iniciação de Peptídeos , Fosforilação , Ratos
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