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
Autophagy induction involves extensive molecular and membrane reorganization. Despite substantial progress, the mechanism underlying autophagy initiation remains poorly understood. Here, we used quantitative photoactivated localization microscopy with single-molecule sensitivity to analyze the nanoscopic distribution of endogenous ULK1, the kinase that triggers autophagy. Under amino acid starvation, ULK1 formed large clusters containing up to 161 molecules at the endoplasmic reticulum. Cross-correlation analysis revealed that ULK1 clusters engaging in autophagosome formation require 30 or more molecules. The ULK1 structures with more than the threshold number contained varying levels of Atg13, Atg14, Atg16, LC3B, GEC1, and WIPI2. We found that ULK1 activity is dispensable for the initial clustering of ULK1, but necessary for the subsequent expansion of the clusters, which involves interaction with Atg14, Atg16, and LC3B and relies on Vps34 activity. This quantitative analysis at the single-molecule level has provided unprecedented insights into the behavior of ULK1 during autophagy initiation.
Assuntos
Autofagia , Aminoácidos/deficiência , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , HumanosRESUMO
Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA-limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Despite auxotrophic for most AA, Plasmodium does not have either a TOR complex nor the GCN2-downstream transcription factors. While Ile starvation has been shown to trigger eIF2α phosphorylation and a hibernation-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on an efficient sensing pathway to respond to AA fluctuations. A phenotypic screen of kinase knockout mutant parasites identified nek4, eIK1 and eIK2-the last two clustering with the eukaryotic eIF2α kinases-as critical for Plasmodium to sense and respond to distinct AA-limiting conditions. Such AA-sensing pathway is temporally regulated at distinct life cycle stages, allowing parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data disclose a set of heterogeneous responses to AA depletion in malaria parasites, mediated by a complex mechanism that is critical for modulating parasite growth and survival.
Assuntos
Aminoácidos , Plasmodium , Aminoácidos/deficiência , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Fosforilação , Fosfotransferases/metabolismo , Plasmodium/enzimologia , Plasmodium/genéticaRESUMO
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
Angiotensin-converting enzyme 2 (ACE2) has been identified as a primary receptor for severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2). Here, we investigated the expression regulation of ACE2 in enterocytes under amino acid deprivation conditions. In this study, we found that ACE2 expression was upregulated upon all or single essential amino acid deprivation in human colonic epithelial CCD841 cells. Furthermore, we found that knockdown of general control nonderepressible 2 (GCN2) reduced intestinal ACE2 mRNA and protein levels in vitro and in vivo. Consistently, we revealed two GCN2 inhibitors, GCN2iB and GCN2-IN-1, downregulated ACE2 protein expression in CCD841 cells. Moreover, we found that increased ACE2 expression in response to leucine deprivation was GCN2 dependent. Through RNA-sequencing analysis, we identified two transcription factors, MAFB and MAFF, positively regulated ACE2 expression under leucine deprivation in CCD841 cells. These findings demonstrate that amino acid deficiency increases ACE2 expression and thereby likely aggravates intestinal SARS-CoV-2 infection.
Assuntos
Aminoácidos , Enzima de Conversão de Angiotensina 2 , COVID-19 , Enterócitos , Proteínas Serina-Treonina Quinases , Aminoácidos/deficiência , Aminoácidos/metabolismo , Enzima de Conversão de Angiotensina 2/biossíntese , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/enzimologia , COVID-19/genética , COVID-19/virologia , Enterócitos/enzimologia , Enterócitos/metabolismo , Humanos , Leucina/farmacologia , Peptidil Dipeptidase A/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , SARS-CoV-2/metabolismoRESUMO
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
Animals have developed various nutrient-sensing mechanisms for survival under fluctuating environmental conditions. Although extensive cell-culture-based analyses have identified diverse mediators of amino acid sensing upstream of mTOR, studies using animal models to examine intestine-initiated amino acid sensing mechanisms under specific physiological conditions are lacking. Here, we developed a Caenorhabditis elegans model to examine the impact of amino acid deficiency on development. We discovered a leucine-derived monomethyl branched-chain fatty acid and its downstream metabolite, glycosphingolipid, which critically mediates the overall amino acid sensing by intestinal and neuronal mTORC1, which in turn regulates postembryonic development at least partly by controlling protein translation and ribosomal biogenesis. Additional data suggest that a similar mechanism may operate in mammals. This study uncovers an amino-acid-sensing mechanism mediated by a lipid biosynthesis pathway.
Assuntos
Aminoácidos/deficiência , Ácidos Graxos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Citoplasma/metabolismo , Glicoesfingolipídeos/metabolismo , Intestinos , Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Modelos Animais , Biossíntese de ProteínasRESUMO
Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.
Assuntos
Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mapas de Interação de Proteínas , Aminoácidos/deficiência , Animais , Proteínas Sanguíneas/farmacologia , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Organogênese/efeitos dos fármacos , Análise de Componente Principal , Mapas de Interação de Proteínas/efeitos dos fármacos , Sirolimo/farmacologiaRESUMO
Single-cell sequencing methods have enabled in-depth analysis of the diversity of cell types and cell states in a wide range of organisms. These tools focus predominantly on sequencing the genomes1, epigenomes2 and transcriptomes3 of single cells. However, despite recent progress in detecting proteins by mass spectrometry with single-cell resolution4, it remains a major challenge to measure translation in individual cells. Here, building on existing protocols5-7, we have substantially increased the sensitivity of these assays to enable ribosome profiling in single cells. Integrated with a machine learning approach, this technology achieves single-codon resolution. We validate this method by demonstrating that limitation for a particular amino acid causes ribosome pausing at a subset of the codons encoding the amino acid. Of note, this pausing is only observed in a sub-population of cells correlating to its cell cycle state. We further expand on this phenomenon in non-limiting conditions and detect pronounced GAA pausing during mitosis. Finally, we demonstrate the applicability of this technique to rare primary enteroendocrine cells. This technology provides a first step towards determining the contribution of the translational process to the remarkable diversity between seemingly identical cells.
Assuntos
Ciclo Celular/genética , Códon/genética , Biossíntese de Proteínas , RNA-Seq/métodos , Ribossomos/metabolismo , Análise de Célula Única , Aminoácidos/deficiência , Aminoácidos/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Aprendizado de Máquina , Masculino , Camundongos , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Biossíntese de Proteínas/efeitos dos fármacos , Reprodutibilidade dos Testes , Ribossomos/efeitos dos fármacosRESUMO
The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the ß-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.
Assuntos
Aminoácidos/deficiência , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/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
Hyaluronan (HA) as a glycosaminoglycan can bind to cell-surface receptors, such as TLR4, to regulate inflammation, tissue injury, repair, and fibrosis. 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, is a drug used for the treatment of biliary spasms. Currently, therapeutic interventions are not available for non-alcoholic steatohepatitis (NASH). In this study, we investigated the effects of 4-MU on NASH using a choline-deficient amino acid (CDAA) diet model. CDAA diet-fed mice showed NASH characteristics, including hepatocyte injury, hepatic steatosis, inflammation, and fibrogenesis. 4-MU treatment significantly reduced hepatic lipid contents in CDAA diet-fed mice. 4-MU reversed CDAA diet-mediated inhibition of Ppara and induction of Srebf1 and Slc27a2. Analysis of serum ALT and AST levels revealed that 4-MU treatment protected against hepatocellular damage induced by CDAA diet feeding. TLR4 regulates low molecular weight-HA-induced chemokine expression in hepatocytes. In CDAA diet-fed, 4-MU-treated mice, the upregulated chemokine/cytokine expression, such as Cxcl1, Cxcl2, and Tnf was attenuated with the decrease of macrophage infiltration into the liver. Moreover, HA inhibition repressed CDAA diet-induced mRNA expression of fibrogenic genes, Notch1, and Hes1 in the liver. In conclusion, 4-MU treatment inhibited liver steatosis and steatohepatitis in a mouse model of NASH, implicating that 4-MU may have therapeutic potential for NASH.
Assuntos
Deficiência de Colina/metabolismo , Ácido Hialurônico/antagonistas & inibidores , Ácido Hialurônico/biossíntese , Himecromona/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Aminoácidos/administração & dosagem , Aminoácidos/deficiência , Animais , Colina/administração & dosagem , Deficiência de Colina/induzido quimicamente , Deficiência de Colina/complicações , Himecromona/farmacologia , Indicadores e Reagentes/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologiaRESUMO
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
Due to the ever-expanding functions attributed to autophagy, there is widespread interest in understanding its contribution to human physiology; however, its specific cellular role as a stress-response mechanism is still poorly defined. To investigate autophagy's role in this regard, we repeatedly subjected cultured mouse myoblasts to two stresses with diverse impacts on autophagic flux: amino acid and serum withdrawal (Hank's balanced salt solution [HBSS]), which robustly induces autophagy, or low-level toxic stress (staurosporine, STS). We found that intermittent STS (int-STS) administration caused cell cycle arrest, development of enlarged and misshapen cells/nuclei, increased senescence-associated heterochromatic foci and senescence-associated ß-galactosidase activity, and prevented myogenic differentiation. These features were not observed in cells intermittently incubated in HBSS (int-HB). While int-STS cells displayed less DNA damage (phosphorylated H2A histone family, member X content) and caspase activity when administered cisplatin, int-HB cells were protected from STS-induced cell death. Interestingly, STS-induced senescence was attenuated in autophagy related 7-deficient cells. Therefore, while repeated nutrient withdrawal did not cause senescence, autophagy was required for senescence caused by toxic stress. These results illustrate the context-dependent effects of different stressors, potentially highlighting autophagy as a distinguishing factor.
Assuntos
Aminoácidos/deficiência , Autofagia , Senescência Celular , Células Musculares/patologia , Músculo Esquelético/patologia , Estresse Fisiológico , Animais , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Forma do Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Camundongos , Células Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Estresse Fisiológico/efeitos dos fármacosRESUMO
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-1Assuntos
Aminoácidos/deficiência , COVID-19/complicações , Deficiências Nutricionais/complicações , Transtornos Mentais/etiologia , Deficiência de Vitamina D/sangue , Transtornos de Ansiedade/sangue , Transtornos de Ansiedade/etiologia , COVID-19/sangue , Deficiências Nutricionais/sangue , Transtorno Depressivo/sangue , Transtorno Depressivo/etiologia , Humanos , Transtornos Mentais/sangueRESUMO
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.