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1.
Nat Commun ; 12(1): 366, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446657

RESUMO

Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet.


Assuntos
Glicina/metabolismo , Neoplasias/dietoterapia , Serina/biossíntese , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Glicina/análise , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Fosfoglicerato Desidrogenase/metabolismo , Serina/análise
2.
Nat Metab ; 3(1): 33-42, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33462515

RESUMO

Mitochondrial diseases (MDs) are a heterogeneous group of disorders resulting from mutations in nuclear or mitochondrial DNA genes encoding mitochondrial proteins1,2. MDs cause pathologies with severe tissue damage and ultimately death3,4. There are no cures for MDs and current treatments are only palliative5-7. Here we show that tetracyclines improve fitness of cultured MD cells and ameliorate disease in a mouse model of Leigh syndrome. To identify small molecules that prevent cellular damage and death under nutrient stress conditions, we conduct a chemical high-throughput screen with cells carrying human MD mutations and discover a series of antibiotics that maintain survival of various MD cells. We subsequently show that a sub-library of tetracycline analogues, including doxycycline, rescues cell death and inflammatory signatures in mutant cells through partial and selective inhibition of mitochondrial translation, resulting in an ATF4-independent mitohormetic response. Doxycycline treatment strongly promotes fitness and survival of Ndufs4-/- mice, a preclinical Leigh syndrome mouse model8. A proteomic analysis of brain tissue reveals that doxycycline treatment largely prevents neuronal death and the accumulation of neuroimmune and inflammatory proteins in Ndufs4-/- mice, indicating a potential causal role for these proteins in the brain pathology. Our findings suggest that tetracyclines deserve further evaluation as potential drugs for the treatment of MDs.


Assuntos
Antibacterianos/uso terapêutico , Doenças Mitocondriais/tratamento farmacológico , Tetraciclinas/uso terapêutico , Fator 4 Ativador da Transcrição/metabolismo , Animais , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Doença de Leigh/tratamento farmacológico , Doença de Leigh/patologia , Expectativa de Vida , Metabolômica , Camundongos , Camundongos Knockout , Doenças Mitocondriais/mortalidade , Doenças Mitocondriais/patologia , Aptidão Física , Análise de Sobrevida
3.
Chem Biol Interact ; 334: 109353, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33309543

RESUMO

Perhexiline is a coronary vasodilator for angina treatment that was first developed in the 1960s. Perhexiline enjoyed worldwide success before reports of severe side effects, such as hepatotoxicity and neurotoxicity, caused its withdrawal from most of the markets. The underlying mechanism of the cytotoxicity of perhexiline, however, is not yet well understood. Here we demonstrated that perhexiline induced cellular damage in primary human hepatocytes, HepaRG cells and HepG2 cells. Analysis of gene and protein expression levels of endoplasmic reticulum (ER) stress markers showed that perhexiline caused ER stress in primary human hepatocytes and HepG2 cells. The splicing of XBP1 mRNA, a hallmark of ER stress, was observed upon perhexiline treatment. Using Gluc-Fluc-HepG2 cell line, we demonstrated that protein secretion was impaired upon perhexiline treatment, suggesting functional deficits in ER. Inhibition of ER stress using ER inhibitor 4-PBA or salubrinal attenuated the cytotoxicity of perhexiline. Directly knocking down ATF4 using siRNA also partially rescued HepG2 cells upon perhexiline exposure. In addition, inhibition of ER stress using either inhibitors or siRNA transfection attenuated perhexiline-induced increase in caspase 3/7 activity, indicating that ER stress contributed to perhexiline-induced apoptosis. Moreover, perhexiline treatment resulted in activation of p38 and JNK signaling pathways, two branches of MAPK cascade. Pre-treating HepG2 cells with p38 inhibitor SB239063 attenuated perhexiline-induced apoptosis and cell death. The inhibitor also prevented the activation of CHOP and ATF4. Overall, our study demonstrated that ER stress is one important mechanism underlying the hepatotoxicity of perhexiline, and p38 signaling pathway contributes to this process. Our finding shed light on the role of both ER stress and p38 signaling pathway in drug-induced liver injury.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Perexilina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo
4.
Nat Commun ; 11(1): 5594, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154371

RESUMO

The underlying pathological mechanisms of glaucomatous trabecular meshwork (TM) damage and elevation of intraocular pressure (IOP) are poorly understood. Here, we report that the chronic endoplasmic reticulum (ER) stress-induced ATF4-CHOP-GADD34 pathway is activated in TM of human and mouse glaucoma. Expression of ATF4 in TM promotes aberrant protein synthesis and ER client protein load, leading to TM dysfunction and cell death. These events lead to IOP elevation and glaucomatous neurodegeneration. ATF4 interacts with CHOP and this interaction is essential for IOP elevation. Notably, genetic depletion or pharmacological inhibition of ATF4-CHOP-GADD34 pathway prevents TM cell death and rescues mouse models of glaucoma by reducing protein synthesis and ER client protein load in TM cells. Importantly, glaucomatous TM cells exhibit significantly increased protein synthesis along with induction of ATF4-CHOP-GADD34 pathway. These studies indicate a pathological role of ATF4-CHOP-GADD34 pathway in glaucoma and provide a possible treatment for glaucoma by targeting this pathway.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Estresse do Retículo Endoplasmático , Glaucoma de Ângulo Aberto/metabolismo , Biossíntese de Proteínas , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Animais , Humor Aquoso/metabolismo , Morte Celular , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Glaucoma de Ângulo Aberto/tratamento farmacológico , Glaucoma de Ângulo Aberto/patologia , Humanos , Camundongos , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/metabolismo , Hipertensão Ocular/patologia , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Transdução de Sinais , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
5.
Nat Commun ; 11(1): 4676, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938922

RESUMO

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.


Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética
6.
Nat Commun ; 11(1): 4677, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938929

RESUMO

The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estresse do Retículo Endoplasmático/genética , Fatores de Iniciação em Eucariotos/genética , Biossíntese de Proteínas , Fatores de Transcrição/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Linhagem Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Mutação , Fases de Leitura Aberta , Interferência de RNA , Degeneração Retiniana/genética , Fatores de Transcrição/metabolismo
7.
Environ Toxicol ; 35(10): 1100-1113, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32506763

RESUMO

Chronic exposure to arsenic remains a worldwide environmental health issue, affecting hundreds of millions of people. Although, arsenic-induced oxidative stress and apoptosis have been determined, the underlying apoptosis mechanism has not been fully elucidated yet. Oxidative stress integrated-ER stress plays an important role in Life-and-Death decision of cells. The current study was to investigate whether NaAsO2 utilizes oxidative stress integrated-ER stress signaling to exert pro-apoptotic activity in L-02 cells. Results showed that death receptor 5 (DR5) was a mediator of NaAsO2 -induced apoptosis by enhancing construction of the death-inducing signaling complex (DISC). NaAsO2 -sensitized DR5 elevation required maintainable transcription and its transcription factor C/EBP homologous protein (CHOP). Further results showed that NaAsO2 increased expression in biomarker of endoplasmic reticulum (ER) stress and activated the protein kinase R-like ER kinase (PERK)-eukaryotic translation initiation 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway. PERK inhibitor and ATF4 siRNA significantly attenuated NaAsO2 -induced CHOP and DR5 expressions. In addition, the antioxidant N-acetyl-l-cysteine (NAC) treatment led to amelioration of NaAsO2 -induced production of reactive oxygen species (ROS) and some ER stress- and apoptosis- related protein levels and cell viability. Taken together, the results indicate that ROS-mediated PERK-eIF2α-ATF4 pathway activated by NaAsO2 is the critical upstream event for subsequent apoptosis induction via regulating CHOP-DR5 signaling in L-02 cells when chronic exposure to arsenic, and support that antioxidants might be potential therapeutic agents for preventing or delaying the onset and progress of arsenic-induced hepatotoxicity.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Arsenitos/toxicidade , Poluentes Ambientais/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Transdução de Sinais
8.
Am J Respir Cell Mol Biol ; 63(4): 478-489, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32551949

RESUMO

Although endoplasmic reticulum (ER) unfolded protein response (UPRER) is well known, mitochondrial unfolded protein response (UPRmt) has not been recognized in alveolar epithelial cells. Furthermore, ER stress and mitochondrial dysfunction are frequently encountered in alveolar epithelial cells from an array of lung disorders. However, these two scenarios have been often regarded as separate mechanisms contributing to the pathogeneses. It is unclear whether there is interplay between these two phenomena or an integrator that couples these two signaling cascades in the stressed alveolar epithelial cells from those pathologies. In this study, we defined UPRmt in alveolar epithelial cells and identified ATF4 (activating transcription factor 4), but not ATF5, as the key regulator of UPRmt. We found that UPRER led to UPRmt and mitochondrial dysfunction in an ATF4-dependent manner. In contrast, mitochondrial stresses did not activate UPRER. We found that alveolar epithelial ATF4 and UPRmt were induced in aged mice with experimental pulmonary fibrosis as well as in patients with idiopathic pulmonary fibrosis. Finally, we found that the inducible expression of ATF4 in mouse alveolar epithelial cells aggravated pulmonary UPRmt, lung inflammation, body weight loss, and death upon bleomycin-induced lung injury. In conclusion, ER stress induces ATF4-dependent UPRmt and mitochondrial dysfunction, indicating a novel mechanism by which ER stress contributes to the pathogeneses of a variety of pulmonary disorders.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Células Epiteliais Alveolares/metabolismo , Mitocôndrias/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Células Epiteliais Alveolares/fisiologia , Animais , Apoptose/fisiologia , Linhagem Celular , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/fisiopatologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Transdução de Sinais/fisiologia
9.
Nat Microbiol ; 5(9): 1144-1157, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32541947

RESUMO

Quiescence is a hallmark of CD4+ T cells latently infected with human immunodeficiency virus 1 (HIV-1). While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. As a key regulator of T-cell quiescence, FOXO1 promotes latency and suppresses productive HIV infection. We report that, in resting T cells, FOXO1 inhibition impaired autophagy and induced endoplasmic reticulum (ER) stress, thereby activating two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and are necessary for HIV reactivation. Indeed, inhibition of protein kinase R-like ER kinase, an ER stress sensor that can mediate the induction of ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppressed HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a link of FOXO1, ER stress and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/farmacologia , HIV-1/efeitos dos fármacos , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Fator 4 Ativador da Transcrição/metabolismo , Linfócitos T CD4-Positivos/virologia , Proteína Forkhead Box O1/genética , Técnicas de Silenciamento de Genes , Infecções por HIV/virologia , Humanos , Células K562
10.
Toxicol Lett ; 331: 178-187, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32569804

RESUMO

Chromium (Cr) (VI) compounds are known to be serious toxic and carcinogenic, but the mechanism is not clear. In our previous study, we found that Cr (VI)-induced ER stress plays an important role in the crosstalk between apoptosis and autophagy, while autophagy was apoptosis-dependent and subsequently prevents apoptosis cell death to keep A549 cells resistant to Cr (VI)-induced toxicity. In this study, we found that Cr (VI) could induce aerobic glycolysis in A549 cells. Both ER stress inhibitor, phenylbutyric acid (4-PBA) and the inhibitor of autophagy, 3-MA, repressed Cr (VI)-induced glycolysis, indicating that both ER stress and autophagy were involved in Cr (VI)-induced glycolysis in A549 cells. Co-treatment of the inhibitor of aerobic glycolysis, 2-DG and Cr (VI) for 24 h increased Cr (VI)-induced cleaved caspase-3, caspase-9 and the number of apoptotic cells, demonstrating that aerobic glycolysis played an important role in attenuating Cr (VI)-induced apoptosis. Furthermore, knockdown of ATF4 by siATF4 significantly decreased Cr (VI)-induced aerobic glycolysis and apoptosis, suggesting that ATF4 was involved in Cr (VI)-induced aerobic glycolysis and its effect of attenuating apoptosis in A549 cells. Taken together, our results demonstrated that autophagy-dependent glycolysis played a role in attenuating Cr (VI)-induced apoptosis. ER stress was involved in facilitating glycolysis, whose induction was mediated by ATF4. These findings open a window for the development of therapeutic interventions to prevent Cr (VI)-induced toxicity.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cromo/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Glicólise/efeitos dos fármacos , Células A549 , Fator 4 Ativador da Transcrição/genética , Apoptose/genética , Autofagia/genética , Estresse do Retículo Endoplasmático/genética , Técnicas de Silenciamento de Genes , Glicólise/genética , Humanos
11.
Nat Commun ; 11(1): 2847, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504036

RESUMO

The browning of white adipose tissue (WAT) has got much attention for its potential beneficial effects on metabolic disorders, however, the nutritional factors and neuronal signals involved remain largely unknown. We sought to investigate whether WAT browning is stimulated by leucine deprivation, and whether the amino acid sensor, general control non-derepressible 2 (GCN2), in amygdalar protein kinase C-δ (PKC-δ) neurons contributes to this regulation. Our results show that leucine deficiency can induce WAT browning, which is unlikely to be caused by food intake, but is largely blocked by PKC-δ neuronal inhibition and amygdalar GCN2 deletion. Furthermore, GCN2 knockdown in amygdalar PKC-δ neurons blocks WAT browning, which is reversed by over-expression of amino acid responsive gene activating transcription factor 4 (ATF4), and is mediated by the activities of amygdalar PKC-δ neurons and the sympathetic nervous system. Our data demonstrate that GCN2/ATF4 can regulate WAT browning in amygdalar PKC-δ neurons under leucine deprivation.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Tecido Adiposo Branco/fisiologia , Tonsila do Cerebelo/fisiologia , Leucina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/inervação , Tonsila do Cerebelo/citologia , Animais , Técnicas de Silenciamento de Genes , Lipólise/fisiologia , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Proteína Quinase C-delta/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/fisiologia , Técnicas Estereotáxicas , Sistema Nervoso Simpático/fisiologia , Termogênese/fisiologia
12.
Mol Cell ; 79(4): 546-560.e7, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32589964

RESUMO

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.


Assuntos
Complexos Multiproteicos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Códon de Iniciação , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Fatores de Iniciação de Peptídeos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Nat Cell Biol ; 22(7): 882-895, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32451439

RESUMO

It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer-microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.


Assuntos
Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Moléculas de Adesão Celular/metabolismo , Reprogramação Celular , Proteínas da Matriz Extracelular/metabolismo , eIF-2 Quinase/metabolismo , Quinases Associadas a rho/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Moléculas de Adesão Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Humanos , Camundongos , Comunicação Parácrina , eIF-2 Quinase/genética , Quinases Associadas a rho/genética
14.
Res Vet Sci ; 130: 237-239, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32224353

RESUMO

Monocrotaline (MCT) belongs to the category of Pyrrolizdine Alkaloids (PAs), which is one of important hepatotoxic alkaloid in Crotalaria Lin. Apoptosis is one mechanism of toxic responses induced by MCT. However, the underlying mechanism of liver apoptosis caused by MCT through Endoplasmic reticulum (ER) stress continues to be incompletely understood. In this study, we describe the role of ER stress in MCT induced hepatotoxicity in rats. 24 male rats were randomly divided into 3 groups: normal saline group, 45 mg/kg MCT group and 90 mg/kg MCT group. After 48 h of saline/MCT administration, the livers were collected for analysis of ER stress-related proteins by Western blotting. The expression of GRP78, p-IRE1α, ATF6 and caspase-12 showed a dose-dependent increase. PERK/eIF2α/ATF4/CHOP pathway is one of the major ER stress pathways which is required for cell survival. Therefore, through analyzing the effects of MCT on this pathway, we found the protein levels of p-PERK, p-eIF2α, ATF4 and CHOP were increase obviously. All these results indicate that MCT induces ER stress in rat liver. The PERK/eIF2α/ATF4/CHOP pathway is involved in the regulation of MCT-induced ER stress in the liver of rat.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fígado/efeitos dos fármacos , Monocrotalina/efeitos adversos , Fator 4 Ativador da Transcrição/metabolismo , Animais , Fator de Iniciação 2 em Eucariotos/metabolismo , Fígado/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(18): 9932-9941, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312819

RESUMO

Cellular starvation is typically a consequence of tissue injury that disrupts the local blood supply but can also occur where cell populations outgrow the local vasculature, as observed in solid tumors. Cells react to nutrient deprivation by adapting their metabolism, or, if starvation is prolonged, it can result in cell death. Cell starvation also triggers adaptive responses, like angiogenesis, that promote tissue reorganization and repair, but other adaptive responses and their mediators are still poorly characterized. To explore this issue, we analyzed secretomes from glucose-deprived cells, which revealed up-regulation of multiple cytokines and chemokines, including IL-6 and IL-8, in response to starvation stress. Starvation-induced cytokines were cell type-dependent, and they were also released from primary epithelial cells. Most cytokines were up-regulated in a manner dependent on NF-κB and the transcription factor of the integrated stress response ATF4, which bound directly to the IL-8 promoter. Furthermore, glutamine deprivation, as well as the antimetabolic drugs 2-deoxyglucose and metformin, also promoted the release of IL-6 and IL-8. Finally, some of the factors released from starved cells induced chemotaxis of B cells, macrophages, and neutrophils, suggesting that nutrient deprivation in the tumor environment can serve as an initiator of tumor inflammation.


Assuntos
Inflamação/genética , Interleucina-6/genética , Interleucina-8/genética , Neoplasias/metabolismo , Estresse Fisiológico/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Antimetabólitos/farmacologia , Morte Celular/efeitos dos fármacos , Desoxiglucose/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Glucose/metabolismo , Glutamina/metabolismo , Células HeLa , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Metformina/farmacologia , NF-kappa B/genética , Neoplasias/genética , Regiões Promotoras Genéticas/genética , Inanição/genética , Inanição/metabolismo , Estresse Fisiológico/imunologia
16.
Phytomedicine ; 69: 153183, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32113150

RESUMO

BACKGROUND: Osteosarcoma (OS) is a significant threat to the lives of children and young adults. Although neoadjuvant chemotherapy is the first choice of treatment for OS, it is limited by serious side-effects and cancer metastasis. ß-Elemonic acid (ß-EA), an active component extracted from Boswellia carterii Birdw., has been reported to exhibit potential anti-inflammatory and anticancer activities. However, the anti-tumor effects and underlying mechanisms on OS as well as pharmacokinetic characteristics of ß-EA remain unknown. PURPOSE: This study was aimed to investigating the anti-tumor effects of ß-EA on human OS, the underlying mechanisms, and the pharmacokinetic and tissue distribution characteristics. STUDY DESIGN AND METHODS: Cell viability and colony formation assays were performed to determine the effect of ß-EA cell on cell proliferation. Apoptosis rates, mitochondrial membrane potential and cell cycle features were analyzed by flow cytometry. qRT-PCR, Western blot, immunofluorescence and immunohistochemical assays were conducted to evaluate the expression levels of genes or proteins related to the pathways affected by ß-EA in vitro and in vivo. Cell migration and invasion were evaluated in wound healing and Transwell chamber assays. The effects and pharmacokinetic characteristics of ß-EA in vivo were evaluated by analyzing tumor suppression, pharmacokinetics and tissue distribution. RESULTS: Explorations indicated that endoplasmic reticulum (ER) stress conditions provoked by ß-EA activated the PERK/eIF2α/ATF4 branch of the unfolded protein reaction (UPR), stimulating C/EBP homologous protein (CHOP)-regulated apoptosis and inducing Ca2+ leakage leading to caspase-dependent apoptosis. Furthermore, ß-EA induced G0/G1 cell cycle arrest and inhibited metastasis of HOS and 143B cells by attenuating Wnt/ß-catenin signaling effects, which included decreased levels of p-Akt(Ser473), p-Gsk3ß (Ser9), Wnt/ß-catenin target genes (c-Myc and CyclinD1) along with a decline in nuclear ß-catenin accumulation. The fast absorption, short elimination half-life, and linear pharmacokinetic characteristics of ß-EA were also revealed. The distribution of ß-EA was detected in the tumor and bone tissues. CONCLUSIONS: Overall, both in vitro and in vivo investigations showed the potential of ß-EA for the treatment of human OS. The pharmacokinetic profile and considerable distribution in the tumor and bone tissues warrant further preclinical or even clinical studies.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Fenantrenos/farmacologia , Fator 4 Ativador da Transcrição/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacocinética , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Distribuição Tecidual , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Triterpenos , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo , eIF-2 Quinase/metabolismo
18.
Anticancer Res ; 40(3): 1387-1394, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32132035

RESUMO

BACKGROUND/AIM: Cancer cells are frequently exposed to microenvironmental stresses, including amino acid deprivation and hypoxia, which are often targeted for cancer therapy. Here, we examined the effect of hypoxia in cysteine-deprived breast cancer cells and the mechanism to counteract the hypoxia effect. MATERIALS AND METHODS: Cell death was determined by annexin V-FITC and propidium iodide staining. Expression of mRNAs and proteins was determined by reverse transcription polymerase chain reaction and western blot analysis, respectively. RESULTS: Cysteine deprivation or sulfasalazine, a potent inhibitor of cysteine/glutamate transporter, induced cell death by activating transcription factor 4 (ATF4) up-regulation. Hypoxia significantly suppressed cell death and ATF4 up-regulation induced by cysteine deprived conditions. In addition, tumor necrosis factor-related apoptosis-inducing ligand reversed the effect of hypoxia on cysteine deprived conditions. CONCLUSION: Prevention of hypoxia may be a means for augmenting the effect of amino acid deprivation as a strategy for cancer therapy.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Neoplasias da Mama/metabolismo , Cisteína/deficiência , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Morte Celular/fisiologia , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Cisteína/antagonistas & inibidores , Cisteína/metabolismo , Feminino , Humanos , Sulfassalazina/farmacologia , Transfecção , Regulação para Cima
19.
Nature ; 579(7799): 427-432, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132707

RESUMO

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.


Assuntos
Citosol/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Estresse Fisiológico , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/biossíntese , Fator 4 Ativador da Transcrição/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Citosol/enzimologia , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/química , Chaperonas Moleculares/metabolismo , Fosforilação , Ligação Proteica
20.
Respir Med ; 161: 105852, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32056726

RESUMO

OBJECTIVE: The aim of the study was to investigate the mechanism and effect of FBXL10 in myocardial ischemia reperfusion injury in vivo and in vitro. METHODS: The myocardial ischemia reperfusion (I/R) model was established by 30 min of coronary occlusion followed by 2 h of reperfusion in rats. Western blot and TUNEL assay were used to measure the apoptosis during I/R. The expression levels of endoplasmic reticulum related proteins in myocardial tissues and H9c2 cells were detected by immunohistochemistry staining and immunofluorescence staining. Flow cytometry and CCK-8 were used to detect the apoptosis and viability of H9c2 cells. RESULTS: The results revealed that FBXL10 significantly reduced myocardial infarction, improved the pathological morphology of myocardium, markedly reduced inflammatory response in the myocardial ischemia reperfusion rats. Moreover the expressions of endoplasmic reticulum stress key proteins were caused by I/R were suppressed significantly by FBXL10 treatment, including CHOP, GRP78, ATF4 and p-PERK. Additionally FBXL10 inhibited the expression of endoplasmic reticulum stress key proteins in H/R H9c2 cells. Furthermore, FBXL10 reduced the levels of apoptotic cells and inflammatory response compared with I/R and H/R group. CONCLUSION: Taken together, we found that FBXL10 could attenuate I/R injury through inhibiting endoplasmic reticulum stress (ERs).


Assuntos
Estresse do Retículo Endoplasmático/genética , Proteínas F-Box/fisiologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/genética , Células Cultivadas , Expressão Gênica/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Traumatismo por Reperfusão Miocárdica/patologia , Ratos , Ratos Sprague-Dawley , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
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