RESUMO
The integrated stress response (ISR) refers to signaling pathways initiated by stress-activated eIF2α kinases. Distinct eIF2α kinases respond to different stress signals, including amino acid deprivation and mitochondrial stress. Such stress-induced eIF2α phosphorylation attenuates general mRNA translation and, at the same time, stimulates the preferential translation of specific downstream factors to orchestrate an adaptive gene expression program. In recent years, there have been significant new advances in our understanding of ISR during metabolic stress adaptation. Here, I discuss those advances, reviewing among others the ISR activation mechanisms in response to amino acid deprivation and mitochondrial stress. In addition, I review how ISR regulates the amino acid metabolic pathways and how changes in the ISR impact the physiology and pathology of various disease models.
Assuntos
Adaptação Fisiológica , Aminoácidos , Fator de Iniciação 2 em Eucariotos , Estresse Fisiológico , Animais , Humanos , Aminoácidos/deficiência , eIF-2 Quinase/metabolismo , eIF-2 Quinase/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Mitocôndrias/metabolismo , Fosforilação , Biossíntese de Proteínas , Transdução de SinaisRESUMO
GCN2 (general control nonderepressible 2) is a serine/threonine-protein kinase that controls messenger RNA translation in response to amino acid availability and ribosome stalling. Here, we show that GCN2 controls erythrocyte clearance and iron recycling during stress. Our data highlight the importance of liver macrophages as the primary cell type mediating these effects. During different stress conditions, such as hemolysis, amino acid deficiency or hypoxia, GCN2 knockout (GCN2-/-) mice displayed resistance to anemia compared with wild-type (GCN2+/+) mice. GCN2-/- liver macrophages exhibited defective erythrophagocytosis and lysosome maturation. Molecular analysis of GCN2-/- cells demonstrated that the ATF4-NRF2 pathway is a critical downstream mediator of GCN2 in regulating red blood cell clearance and iron recycling.
Assuntos
Aminoácidos , Eritrócitos , Ferro , Fígado , Macrófagos , Proteínas Serina-Treonina Quinases , Fator 4 Ativador da Transcrição/metabolismo , Aminoácidos/deficiência , Aminoácidos/metabolismo , Anemia/metabolismo , Animais , Citofagocitose , Eritrócitos/metabolismo , Deleção de Genes , Hemólise , Hipóxia/metabolismo , Ferro/metabolismo , Fígado/citologia , Lisossomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estresse FisiológicoRESUMO
Oxidative metabolism in mitochondria regulates cellular differentiation and gene expression through intermediary metabolites and reactive oxygen species. Its role in kidney development and pathogenesis is not completely understood. Here we inactivated ubiquinone-binding protein QPC, a subunit of mitochondrial complex III, in two types of kidney progenitor cells to investigate the role of mitochondrial electron transport in kidney homeostasis. Inactivation of QPC in sine oculis-related homeobox 2 (SIX2)-expressing cap mesenchyme progenitors, which give rise to podocytes and all nephron segments except collecting ducts, resulted in perinatal death from severe kidney dysplasia. This was characterized by decreased proliferation of SIX2 progenitors and their failure to differentiate into kidney epithelium. QPC inactivation in cap mesenchyme progenitors induced activating transcription factor 4-mediated nutritional stress responses and was associated with a reduction in kidney tricarboxylic acid cycle metabolites and amino acid levels, which negatively impacted purine and pyrimidine synthesis. In contrast, QPC inactivation in ureteric tree epithelial cells, which give rise to the kidney collecting system, did not inhibit ureteric differentiation, and resulted in the development of functional kidneys that were smaller in size. Thus, our data demonstrate that mitochondrial oxidative metabolism is critical for the formation of cap mesenchyme-derived nephron segments but dispensable for formation of the kidney collecting system. Hence, our studies reveal compartment-specific needs for metabolic reprogramming during kidney development.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons , Rim , Néfrons , Organogênese , Podócitos , Aminoácidos/deficiência , Diferenciação Celular , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Rim/embriologia , Rim/metabolismo , Mesoderma/metabolismo , Néfrons/metabolismo , Organogênese/genética , Podócitos/metabolismo , Gravidez , Ureter/embriologiaRESUMO
Oncogene activation, massive proliferation, and increased nutrient demands often result in nutrient and oxygen deprivation in solid tumors including breast cancer (BC), leading to the induction of oxidative stress and endoplasmic reticulum (ER) stress, and subsequently triggering integrated stress response (ISR). To elucidate the role of long non-coding RNAs (lncRNAs) in the ISR of BC, we performed transcriptome analyses and identified a lncRNA, UBA6-AS1, which was upregulated upon amino acid deprivation and ER stress. UBA6-AS1 was preferentially induced in triple-negative BC (TNBC) cells deprived of arginine or glutamine, two critical amino acids required for cancer cell growth, or treated with ER stress inducers. Mechanistically, UBA6-AS1 was regulated through the GCN2/eIF2α/ATF4 pathway, one of the major routes mediating ISR in amino acid sensing. In addition, both in vitro and in vivo assays indicated that UBA6-AS1 promoted TNBC cell survival when cells encountered metabolic stress, implicating a regulatory role of UBA6-AS1 in response to intratumoral metabolic stress during tumor progression. Moreover, PARP1 expression and activity were positively regulated by the GCN2/UBA6-AS1 axis upon amino acid deprivation. In conclusion, our data suggest that UBA6-AS1 is a novel lncRNA regulating ISR upon metabolic stress induction to promote TNBC cell survival. Furthermore, the GCN2-ATF4 axis is important for UBA6-AS1 induction to enhance PARP1 activity and could serve as a marker for the susceptibility of PARP inhibitors in TNBC.
Assuntos
Aminoácidos/deficiência , Estresse do Retículo Endoplasmático , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Aminoácidos/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Serina-Treonina Quinases/genética , RNA Antissenso , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
The hematopoietic system is highly sensitive to perturbations in the translational machinery, of which an emerging level of regulation lies in the epitranscriptomic modification of transfer RNAs (tRNAs). Here, we interrogate the role of tRNA anticodon modifications in hematopoiesis by using mouse models of conditional inactivation of Elp3, the catalytic subunit of Elongator that modifies wobble uridine in specific tRNAs. Loss of Elp3 causes bone marrow failure by inducing death in committing progenitors and compromises the grafting activity of hematopoietic stem cells. Mechanistically, Elp3 deficiency activates a p53-dependent checkpoint in what resembles a misguided amino acid deprivation response that is accompanied by Atf4 overactivation and increased protein synthesis. While deletion of p53 rescues hematopoiesis, loss of Elp3 prompts the development of p53-mutated leukemia/lymphoma, and inactivation of p53 and Elongator cooperatively promotes tumorigenesis. Specific tRNA-modifying enzymes thus condition differentiation and antitumor fate decisions in hematopoietic stem cells and progenitors.
Assuntos
Hematopoese , Histona Acetiltransferases/metabolismo , RNA de Transferência/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Aminoácidos/deficiência , Animais , Linhagem Celular , Sobrevivência Celular , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/ultraestrutura , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Regulação para CimaRESUMO
In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.
Assuntos
Fator 4 Ativador da Transcrição/genética , Edição de Genes/métodos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mitocôndrias/genética , Proteínas Serina-Treonina Quinases/genética , 3-Hidroxiacil-CoA Desidrogenases/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aminoácidos/deficiência , Aminoácidos/farmacologia , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Meios de Cultura/química , Meios de Cultura/farmacologia , Regulação da Expressão Gênica , Genoma Humano , Glucose/deficiência , Glucose/farmacologia , Células HEK293 , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligomicinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Transdução de Sinais , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
During pregnancy, the appropriate allocation of nutrients between the mother and the fetus is dominated by maternal-fetal interactions, which is primarily governed by the placenta. The syncytiotrophoblast (STB) lining at the outer surface of the placental villi is directly bathed in maternal blood and controls feto-maternal exchange. The STB is the largest multinucleated cell type in the human body, and is formed through syncytialization of the mononucleated cytotrophoblast. However, the physiological advantage of forming such an extensively multinucleated cellular structure remains poorly understood. Here, we discover that the STB uniquely adapts to nutrient stress by inducing the macropinocytosis machinery through repression of mammalian target of rapamycin (mTOR) signaling. In primary human trophoblasts and in trophoblast cell lines, differentiation toward a syncytium triggers macropinocytosis, which is greatly enhanced during amino acid shortage, induced by inhibiting mTOR signaling. Moreover, inhibiting mTOR in pregnant mice markedly stimulates macropinocytosis in the syncytium. Blocking macropinocytosis worsens the phenotypes of fetal growth restriction caused by mTOR-inhibition. Consistently, placentas derived from fetal growth restriction patients display: 1) Repressed mTOR signaling, 2) increased syncytialization, and 3) enhanced macropinocytosis. Together, our findings suggest that the unique ability of STB to undergo macropinocytosis serves as an essential adaptation to the cellular nutrient status, and support fetal survival and growth under nutrient deprivation.
Assuntos
Adaptação Fisiológica , Retardo do Crescimento Fetal/metabolismo , Troca Materno-Fetal/fisiologia , Pinocitose/genética , Proteínas da Gravidez/genética , Serina-Treonina Quinases TOR/genética , Trofoblastos/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoácidos/deficiência , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Vilosidades Coriônicas/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Regulação da Expressão Gênica , Humanos , Camundongos , Gravidez , Proteínas da Gravidez/metabolismo , Cultura Primária de Células , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Trofoblastos/citologiaRESUMO
Development of the mammalian preimplantation embryo is influenced by autocrine/paracrine factors and the availability of nutrients. Deficiencies of these during in vitro culture reduce the success of assisted reproductive technologies. The mechanistic target of rapamycin complex 1 (mTORC1) pathway integrates external and internal signals, including those by amino acids (AAs), to promote normal preimplantation development. For this reason, AAs are often included in embryo culture media. In this study, we examined how withdrawal and addition of AAs to culture media modulate mTORC1 pathway activity compared with its activity in mouse embryos developed in vivo. Phosphorylation of signaling components downstream of mTORC1, namely, p70 ribosomal protein S6 kinase (p70S6K), ribosomal protein S6, and 4E binding protein 1 (4E-BP1), and that of protein kinase B (Akt), which lies upstream of mTORC1, changed significantly across stages of embryos developed in vivo. For freshly isolated blastocysts placed in vitro, the absence of AAs in the culture medium, even for a few hours, decreased mTORC1 signaling, which could only be partially restored by their addition. Long-term culture of early embryos to blastocysts in the absence of AAs decreased mTORC1 signaling to a greater extent and again this could only be partially restored by their inclusion. This failure to fully restore is probably due to decreased phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC2 signaling in culture, as indicated by decreased P-AktS473. mTORC2 lies upstream of mTORC1 and is insensitive to AAs, and its reduced activity probably results from loss of maternal/autocrine factors. These data highlight reduced mTORC1/2 signaling activity correlating with compromised development in vitro and show that the addition of AAs can only partially offset these effects.
Assuntos
Aminoácidos/deficiência , Blastocisto/enzimologia , Meios de Cultura/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Técnicas de Cultura Embrionária , Feminino , Masculino , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Transplantation of in vitro-manipulated cells is widely used in hematology. While transplantation is well recognized to impose severe stress on transplanted cells, the nature of transplant-induced stress remains elusive. Here, we propose that the lack of amino acids in serum is the major cause of transplant-induced stress. Mechanistically, amino acid deficiency decreases protein synthesis and nutrient consummation. However, in cells with overactive AKT and ERK, mTORC1 is not inhibited and protein synthesis remains relatively high. This impaired signaling causes nutrient depletion, cell cycle block, and eventually autophagy and cell death, which can be inhibited by cycloheximide or mTORC1 inhibitors. Thus, mTORC1-mediated amino acid signaling is critical in cell fate determination under transplant-induced stress, and protein synthesis inhibition can improve transplantation efficiency.
Assuntos
Aminoácidos/sangue , Regulação Leucêmica da Expressão Gênica , Leucemia/genética , Leucócitos/metabolismo , Transdução de Sinais/genética , Aminoácidos/deficiência , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Rastreamento de Células , Transplante de Células , Ciclinas/genética , Ciclinas/metabolismo , Cicloeximida/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Xenoenxertos , Humanos , Leucemia/metabolismo , Leucemia/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Células THP-1RESUMO
OBJECTIVE: We generated induced pluripotent stem (iPS) cells from a patient with distal myopathy with rimmed vacuoles (DMRV), in which sialic acids synthesis is reported to be defective. In this study, we examined whether the differentiation to retinal pigment epithelial (RPE) cells and autophagy was affected in the patient derived cells. METHODS: Patient derived iPS cells were established through the transduction of re-programming factors into peripheral mononuclear cells via retrovirus vectors. RPE cells were induced from iPS cells through aggregation culture. Then the autophagy induced by amino acid starvation was estimated by measuring LC3-containing "puncta" structure. RESULTS: A 3D aggregate culture of patient-derived iPS cells resulted in some irregular shapes, and the aggregate contained large vacuoles filled with lipid droplets and cellular components such as damaged mitochondria. RPE cells induced from patient-derived iPS cells showed impaired autophagy flux under amino acid starvation. CONCLUSION: These findings were similar to those of sialidosis patient-derived iPS cells, in which cleavage of terminal sialic acids in oligosaccharide chains is defective. This suggests that the control of both the addition and removal of sialic acids are pivotal for autophagy progression.
Assuntos
Autofagia , Miopatias Distais/patologia , Células Epiteliais/fisiologia , Células-Tronco Pluripotentes/patologia , Pigmentos da Retina , Ácidos Siálicos , Vacúolos/patologia , Adulto , Aminoácidos/deficiência , Diferenciação Celular , Células Cultivadas , Miopatias Distais/etiologia , Miopatias Distais/metabolismo , Feminino , Humanos , Mucolipidoses/patologia , Oligossacarídeos/metabolismo , Células-Tronco Pluripotentes/citologia , Ácidos Siálicos/deficiência , Ácidos Siálicos/metabolismoRESUMO
An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino-acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core-binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino-acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as key sensors of glutamine availability in mammalian cells.
Assuntos
Aminoácidos/deficiência , Biossíntese de Proteínas , RNA de Transferência de Glutamina/metabolismo , Aminoacilação de RNA de Transferência , Animais , Linhagem Celular Tumoral , Glutaminase/antagonistas & inibidores , Glutaminase/metabolismo , Glutamina/metabolismo , Humanos , Camundongos , Peptídeos/metabolismoRESUMO
Non-alcoholic steatohepatitis (NASH) is a severe, advanced form of non-alcoholic fatty liver disease (NAFLD) that is associated with features of metabolic syndrome and characterized by hepatic steatosis, inflammation, and fibrosis. In addition, NASH is associated with endothelial dysfunction within the hepatic vasculature. Treatment with CU06-1004 (previously called Sac-1004) ameliorates endothelial dysfunction by inhibiting hyperpermeability and inflammation. In this study, we investigated the protective effects of CU06-1004 in a choline-deficient L-amino acid (CDAA)-induced mouse model of NASH for 3 or 6 weeks. Specifically, we evaluated the effects of CU06-1004 on lipid accumulation, inflammation, hepatic fibrosis, and liver sinusoidal endothelial cell (LSEC) capillarization through biochemical analysis, immunohistochemistry, and real-time PCR. We found that the administration of CU06-1004 to mice improved liver triglyceride (TG) and serum alanine aminotransferase (ALT) in this CDAA-induced model of NASH for 6 weeks. In groups of NASH induced mice for both 3 and 6 weeks, CU06-1004 significantly reduced the hepatic expression of genes related to lipogenesis, inflammation, and cell adhesion. However, expression of genes related to hepatic fibrosis and vascular endothelial changes were only decreased in animals with mild NASH. These results suggest that the administration of CU06-1004 suppresses hepatic steatosis, inflammation, fibrosis, and LSEC capillarization in a CDAA-induced mouse model of NASH. This suggests that CU06-1004 has therapeutic potential for the treatment of mild NASH.
Assuntos
Dieta/veterinária , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Alanina Transaminase/sangue , Aminoácidos/deficiência , Aminoácidos/metabolismo , Animais , Adesão Celular/genética , Colina/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inflamação/genética , Lipogênese/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cisplatin is a mainstay of cancer chemotherapy. It forms DNA adducts, thereby activating poly(ADP-ribose) polymerases (PARPs) to initiate DNA repair. The PARP substrate NAD+ is synthesized from 5-phosphoribose-1-pyrophosphate (PRPP), and we found that treating cells for 6 h with cisplatin reduced intracellular PRPP availability. The decrease in PRPP was likely from (1) increased PRPP consumption, because cisplatin increased protein PARylation and PARP1 shRNA knock-down returned PRPP towards normal, and (2) decreased intracellular phosphate, which down-regulated PRPP synthetase activity. Depriving cells of a single essential amino acid decreased PRPP synthetase activity with a half-life of ~ 8 h, and combining cisplatin and amino acid deprivation synergistically reduced intracellular PRPP. PRPP is a rate-limiting substrate for purine nucleotide synthesis, and cisplatin inhibited de novo purine synthesis and DNA synthesis, with amino acid deprivation augmenting cisplatin's effects. Amino acid deprivation enhanced cisplatin's cytotoxicity, increasing cellular apoptosis and DNA strand breaks in vitro, and intermittent deprivation of lysine combined with a sub-therapeutic dose of cisplatin inhibited growth of ectopic hepatomas in mice. Augmentation of cisplatin's biochemical and cytotoxic effects by amino acid deprivation suggest that intermittent deprivation of an essential amino acid could allow dose reduction of cisplatin; this could reduce the drug's side effects, and allow its use in cisplatin-resistant tumors.
Assuntos
Aminoácidos/deficiência , Apoptose , Carcinoma Hepatocelular/patologia , Cisplatino/farmacologia , Neoplasias Hepáticas/patologia , Fosforribosil Pirofosfato/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The dysregulation of the metabolic regulator TOR complex I (TORC1) contributes to a wide array of human pathologies. Tuberous sclerosis complex (TSC) is a potent inhibitor of TORC1. Here, we demonstrate that the Rag GTPase acts in both the amino-acid-sensing and growth factor signaling pathways to control TORC1 activity through the regulation of TSC dynamics in HeLa cells and Drosophila. We find that TSC lysosomal-cytosolic exchange increases in response to both amino acid and growth factor restriction. Moreover, the rate of exchange mirrors TSC function, with depletions of the Rag GTPase blocking TSC lysosomal mobility and rescuing TORC1 activity. Finally, we show that the GATOR2 complex controls the phosphorylation of TSC2, which is essential for TSC exchange. Our data support the model that the amino acid and growth factor signaling pathways converge on the Rag GTPase to inhibit TORC1 activity through the regulation of TSC dynamics.
Assuntos
Aminoácidos/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Esclerose Tuberosa/metabolismo , Animais , Drosophila , Feminino , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Lisossomos/metabolismo , Mutação/genética , Ovário/metabolismo , Fosforilação , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells.
Assuntos
Autofagia/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Ativação Transcricional/genética , Aminoácidos/deficiência , Autofagia/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células HCT116 , Células HEK293 , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Sirolimo/farmacologia , Ativação Transcricional/efeitos dos fármacos , Proteína com Valosina/metabolismoRESUMO
Tumours depend on nutrients supplied by the host for their growth and survival. Modifications to the host's diet can change nutrient availability in the tumour microenvironment, which might represent a promising strategy for inhibiting tumour growth. Dietary modifications can limit tumour-specific nutritional requirements, alter certain nutrients that target the metabolic vulnerabilities of the tumour, or enhance the cytotoxicity of anti-cancer drugs. Recent reports have suggested that modification of several nutrients in the diet can alter the efficacy of cancer therapies, and some of the newest developments in this quickly expanding field are reviewed here. The results discussed indicate that the dietary habits and nutritional state of a patient must be taken into account during cancer research and therapy.
Assuntos
Dieta , Neoplasias/dietoterapia , Neoplasias/terapia , Estado Nutricional , Aminoácidos/deficiência , Aminoácidos/metabolismo , Animais , Suplementos Nutricionais , Jejum/fisiologia , Ácidos Graxos/metabolismo , Ácido Fólico/metabolismo , Frutose/deficiência , Frutose/metabolismo , Glucose/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
Mammalian target of rapamycin complex 1 (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC1 via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn activates mTORC1 at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)-phosphatidic acid (PA) pathway, required for mTORC1 signaling through mechanisms that are not fully understood. Here, using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, we show that exogenously supplied PA vesicles deliver mTORC1 to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC1 lysosomal translocation. We observed that precancerous cells with constitutive Rheb activation through loss of tuberous sclerosis complex subunit 2 (TSC2) exploit the PLD-PA pathway and thereby sustain mTORC1 activation at the lysosome in the absence of amino acids. Our findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC1 translocation and activation at the lysosome.
Assuntos
Aminoácidos/deficiência , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Ácidos Fosfatídicos/metabolismo , Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Endocitose , Humanos , Camundongos , Fosfolipase D/metabolismo , Transporte Proteico , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
OBJECTIVES: Surgery triggers a stress response that produces insulin resistance and hyperglycemia. During cardiac surgery, the administration of high-dose insulin along with dextrose titration maintains normoglycemia, but dramatically decreases plasma amino acids (AAs) compared with preoperative fasting levels. Hypoaminoacidemia limits protein synthesis and prevents anabolic responses after surgery. We investigated whether parenteral infusion of AAs during and immediately after cardiac surgery would prevent hypoaminoacidemia in patients who receive high-dose insulin therapy. METHODS: Sixteen patients undergoing coronary artery bypass grafting surgery were randomly allocated to receive AAs with % kcal equivalent to either 20% (nâ¯=â¯8) or 35% (nâ¯=â¯8) of their measured resting energy expenditure (REE). Insulin was infused at a constant rate of 5 mU/(kg × min), whereas dextrose was titrated to maintain normoglycemia during and until 5 h after surgery. Plasma AA concentrations were measured at baseline before and after surgery. RESULTS: Compared with the 20% AA group after surgery, AA concentrations were significantly higher in the 35% AA group for 12 of 20 AAs (P < 0.032), including all branched-chain AAs. In the 20% AA group, total essential AAs decreased by 21% and nonessential AAs decreased by 14% after surgery compared with preoperative fasting levels. In contrast, giving 35% AAs prevented this unfavorable decrease in AAs, and in fact allowed for a 23% and 12% increase in essential and nonessential AAs, respectively. CONCLUSIONS: AA supplementation at 35% REE, but not 20% REE, can effectively prevent hypoaminoacidemia caused by high-dose insulin therapy during cardiac surgery.
Assuntos
Aminoácidos/deficiência , Ponte de Artéria Coronária/efeitos adversos , Suplementos Nutricionais , Insulina/administração & dosagem , Nutrição Parenteral/métodos , Complicações Pós-Operatórias/prevenção & controle , Aminoácidos/sangue , Glicemia/efeitos dos fármacos , Ponte de Artéria Coronária/métodos , Relação Dose-Resposta a Droga , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Hiperglicemia/sangue , Hiperglicemia/prevenção & controle , Resistência à Insulina , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/sangue , Biossíntese de Proteínas/efeitos dos fármacos , Resultado do TratamentoRESUMO
Diet composition impacts metabolic and cardiovascular health with high caloric diets contributing to obesity related disorders. Dietary interventions such as caloric restriction exert beneficial effects in the cardiovascular system, but alteration of which specific nutrient is responsible is less clear. This study investigates the effects of a low protein diet (LPD) on morphology, tissue composition and function of the neonatal and adult mouse heart. Mice were subjected to LPD (8.8% protein) or standard protein diet (SPD, 22% protein) throughout intrauterine and postnatal life. At birth LPD female but not male offspring exhibit reduced body weight whereas heart weight was unchanged in both sexes. Cardiomyocyte cross sectional area was increased in newborn LPD females compared to SPD, whereas proliferation, cellular tissue composition and vascularization were unaffected. Adult female mice on LPD exhibit reduced body weight but normal heart weight compared to SPD controls. Echocardiography revealed normal left ventricular contractility in LPD animals. Histology showed reduced interstitial fibrosis, lower cardiomyocyte volume and elevated numbers of cardiomyocyte and non-myocyte nuclei per tissue area in adult LPD versus SPD myocardium. Furthermore, capillary density was increased in LPD hearts. In conclusion, pre- and postnatal dietary protein restriction in mice causes a potentially beneficial myocardial remodeling.
Assuntos
Envelhecimento/fisiologia , Coração/fisiologia , Aminoácidos/deficiência , Animais , Animais Recém-Nascidos , Peso Corporal , Capilares/fisiologia , Contagem de Células , Proliferação de Células , Tamanho Celular , Dieta com Restrição de Proteínas , Comportamento Alimentar , Feminino , Coração/anatomia & histologia , Ventrículos do Coração , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Neovascularização Fisiológica , Tamanho do Órgão , Gravidez , Caracteres Sexuais , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Various rodent models have been proposed for basic research; however, the pathogenesis of human nonalcoholic steatohepatitis (NASH) is difficult to closely mimic. Lipopolysaccharide (LPS) has been reported to play a pivotal role in fibrosis development during NASH progression via activation of toll-like receptor 4 (TLR4) signaling. This study aimed to clarify the impact of low-dose LPS challenge on NASH pathological progression and to establish a novel murine NASH model. C57BL/6J mice were fed a choline-deficient l-amino-acid-defined (CDAA) diet to induce NASH, and low-dose LPS (0.5 mg/kg) was intraperitoneally injected thrice a week. CDAA-fed mice showed hepatic CD14 overexpression, and low-dose LPS challenge enhanced TLR4/NF-κB signaling activation in the liver of CDAA-fed mice. LPS challenge potentiated CDAA-diet-mediated insulin resistance, hepatic steatosis with upregulated lipogenic genes, and F4/80-positive macrophage infiltration with increased proinflammatory cytokines. It is noteworthy that LPS administration extensively boosted pericellular fibrosis with the activation of hepatic stellate cells in CDAA-fed mice. Exogenous LPS administration exacerbated pericellular fibrosis in CDAA-mediated steatohepatitis in mice. These findings suggest a key role for LPS/TLR4 signaling in NASH progression, and the authors therefore propose this as a suitable model to mimic human NASH.