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1.
Nat Commun ; 11(1): 5661, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168829

RESUMO

Sarcopenia is characterized by decreased skeletal muscle mass and function with age. Aged muscles have altered lipid compositions; however, the role and regulation of lipids are unknown. Here we report that FABP3 is upregulated in aged skeletal muscles, disrupting homeostasis via lipid remodeling. Lipidomic analyses reveal that FABP3 overexpression in young muscles alters the membrane lipid composition to that of aged muscle by decreasing polyunsaturated phospholipid acyl chains, while increasing sphingomyelin and lysophosphatidylcholine. FABP3-dependent membrane lipid remodeling causes ER stress via the PERK-eIF2α pathway and inhibits protein synthesis, limiting muscle recovery after immobilization. FABP3 knockdown induces a young-like lipid composition in aged muscles, reduces ER stress, and improves protein synthesis and muscle recovery. Further, FABP3 reduces membrane fluidity and knockdown increases fluidity in vitro, potentially causing ER stress. Therefore, FABP3 drives membrane lipid composition-mediated ER stress to regulate muscle homeostasis during aging and is a valuable target for sarcopenia.


Assuntos
Envelhecimento/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Proteína 3 Ligante de Ácido Graxo/metabolismo , Lipídeos de Membrana/metabolismo , Músculo Esquelético/metabolismo , Animais , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteína 3 Ligante de Ácido Graxo/genética , Feminino , Técnicas de Silenciamento de Genes , Lipidômica , Fluidez de Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/patologia , Mioblastos/patologia , Mioblastos/fisiologia , Fosfolipídeos/metabolismo , Proteínas Serina-Treonina Quinases , Sarcopenia , Regulação para Cima
2.
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
3.
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
4.
Mol Cell ; 79(4): 588-602.e6, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32615089

RESUMO

The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3' UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2α, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.


Assuntos
Pegada de DNA/métodos , Genoma Fúngico , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/metabolismo , Regiões 3' não Traduzidas , Anisomicina/farmacologia , Códon de Terminação , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Mutação , Fosforilação , Estabilidade de RNA , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética
5.
Nat Microbiol ; 5(11): 1361-1373, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32690955

RESUMO

Eukaryotic cells, when exposed to environmental or internal stress, activate the integrated stress response (ISR) to restore homeostasis and promote cell survival. Specific stress stimuli prompt dedicated stress kinases to phosphorylate eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 (p-eIF2) in turn sequesters the eIF2-specific guanine exchange factor eIF2B to block eIF2 recycling, thereby halting translation initiation and reducing global protein synthesis. To circumvent stress-induced translational shutdown, viruses encode ISR antagonists. Those identified so far prevent or reverse eIF2 phosphorylation. We now describe two viral proteins-one from a coronavirus and the other from a picornavirus-that have independently acquired the ability to counteract the ISR at its very core by acting as a competitive inhibitor of p-eIF2-eIF2B interaction. This allows continued formation of the eIF2-GTP-Met-tRNAi ternary complex and unabated global translation at high p-eIF2 levels that would otherwise cause translational arrest. We conclude that eIF2 and p-eIF2 differ in their interaction with eIF2B to such effect that p-eIF2-eIF2B association can be selectively inhibited.


Assuntos
Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Estresse Fisiológico/fisiologia , Proteínas Virais/metabolismo , Animais , Sítios de Ligação , Chlorocebus aethiops , Células Eucarióticas/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Fosforilação , Picornaviridae/metabolismo , Ligação Proteica , Células Vero
6.
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
7.
PLoS One ; 15(5): e0231894, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365111

RESUMO

Stress granules (SG) are cytoplasmic RNA granules that form during various types of stress known to inhibit general translation, including oxidative stress, hypoxia, endoplasmic reticulum stress (ER), ionizing radiations or viral infection. Induction of these SG promotes cell survival in part through sequestration of proapoptotic molecules, resulting in the inactivation of cell death pathways. SG also form in cancer cells, but studies investigating their formation upon treatment with chemotherapeutics are very limited. Here we identified Lapatinib (Tykerb / Tyverb®), a tyrosine kinase inhibitor used for the treatment of breast cancers as a new inducer of SG in breast cancer cells. Lapatinib-induced SG formation correlates with the inhibition of general translation initiation which involves the phosphorylation of the translation initiation factor eIF2α through the kinase PERK. Disrupting PERK-SG formation by PERK depletion experiments sensitizes resistant breast cancer cells to Lapatinib. This study further supports the assumption that treatment with anticancer drugs activates the SG pathway, which may constitute an intrinsic stress response used by cancer cells to resist treatment.


Assuntos
Grânulos Citoplasmáticos/efeitos dos fármacos , Lapatinib/uso terapêutico , Neoplasias/tratamento farmacológico , Biossíntese de Proteínas/efeitos dos fármacos , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Lapatinib/farmacologia , Células MCF-7 , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , eIF-2 Quinase/metabolismo
8.
Nat Commun ; 11(1): 2498, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427827

RESUMO

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5' Untranslated Regions (5'UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and "fate-switching" toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-cell-like phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.


Assuntos
Regiões 5' não Traduzidas/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Biossíntese de Proteínas/genética , Isoformas de RNA/genética , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Proteína Homeobox Nanog/genética , Proteína Nodal/genética , Fosforilação/efeitos dos fármacos , Fatores de Transcrição da Família Snail/genética
9.
Proc Natl Acad Sci U S A ; 117(20): 10935-10945, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32355000

RESUMO

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.


Assuntos
Relógios Circadianos/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Ritmo Circadiano , Fator de Iniciação 2 em Eucariotos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , eIF-2 Quinase/metabolismo
10.
Nucleic Acids Res ; 48(11): 6223-6233, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374873

RESUMO

As cells encounter adverse environmental conditions, such as hypoxia, oxidative stress or nutrient deprivation, they trigger stress response pathways to protect themselves until transient stresses have passed. Inhibition of translation is a key component of such cellular stress responses and mounting evidence has revealed the importance of a class of tRNA-derived small RNAs called tiRNAs in this process. The most potent of these small RNAs are those with the capability of assembling into tetrameric G-quadruplex (G4) structures. However, the mechanism by which these small RNAs inhibit translation has yet to be elucidated. Here we show that eIF4G, the major scaffolding protein in the translation initiation complex, directly binds G4s and this activity is required for tiRNA-mediated translation repression. Targeting of eIF4G results in an impairment of 40S ribosome scanning on mRNAs leading to the formation of eIF2α-independent stress granules. Our data reveals the mechanism by which tiRNAs inhibit translation and demonstrates novel activity for eIF4G in the regulation of translation.


Assuntos
Fator de Iniciação 4G em Eucariotos/metabolismo , Quadruplex G , Biossíntese de Proteínas , RNA de Transferência/química , RNA de Transferência/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/química , Fator de Iniciação 4F em Eucariotos/metabolismo , Humanos , Iniciação Traducional da Cadeia Peptídica , Fosfoproteínas/metabolismo , Domínios Proteicos , RNA Mensageiro/metabolismo , RNA de Transferência/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
11.
Clin Sci (Lond) ; 134(10): 1127-1141, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32400851

RESUMO

Vitiligo is a depigmentation disorder that develops as a result of the progressive disappearance of epidermal melanocytes. The elevated level of amino acid metabolite homocysteine (Hcy) has been identified as circulating marker of oxidative stress and known as a risk factor for vitiligo. However, the mechanism underlying Hcy-regulated melanocytic destruction is currently unknown. The present study aims to elucidate the effect of Hcy on melanocytic destruction and its involvement in the pathogenesis of vitiligo. Our results showed that Hcy level was significantly elevated in the serum of progressive vitiligo patients. Notably, Hcy induced cell apoptosis in melanocytes via activating reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-C/EBP homologous protein (CHOP) pathway. More importantly, folic acid, functioning in the transformation of Hcy, could lower the intracellular Hcy level and further reverse the apoptotic effect of Hcy on melanocytes. Additionally, Hcy disrupted melanogenesis whereas folic acid supplementation could reverse the melanogenesis defect induced by Hcy in melanocytes. Taken together, Hcy is highly increased in vitiligo patients at progressive stage, and our in vitro studies revealed that folic acid could protect melanocytes from Hcy-induced apoptosis and melanin synthesis inhibition, indicating folic acid as a potential benefit agent for patients with progressive vitiligo.


Assuntos
Apoptose , Fator de Iniciação 2 em Eucariotos/metabolismo , Homocisteína/metabolismo , Melanócitos/metabolismo , Melanócitos/patologia , Fator de Transcrição CHOP/metabolismo , Vitiligo/metabolismo , eIF-2 Quinase/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Ácido Fólico/farmacologia , Homocisteína/sangue , Humanos , Masculino , Melaninas/biossíntese , Melanócitos/efeitos dos fármacos , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Vitiligo/sangue
12.
Chemosphere ; 254: 126785, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32334250

RESUMO

Fetal overexposure to active glucocorticoid (GC) is the major cause for fetal growth restriction (FGR). This study investigated the influences of cadmium (Cd) exposure on active GC and its mechanism in placental trophoblasts. Pregnant mice were exposed to CdCl2 (4.5 mg/kg, i.p.). Human JEG-3 cells were treated with CdCl2 (0-20 µM). Prenatal Cd exposure significantly increased active GC level in amniotic fluid and placenta. Similarly, Cd treatment also elevated active GC level in medium. Expectedly, the expression of 11ß-HSD2 protein was markedly downregulated in Cd-exposed placental trophoblasts. We further found that Cd activated the PERK/p-eIF2α signaling pathway in placental trophoblasts. Mechanistically, PERK siRNA pretreatment completely blocked PERK/p-eIF2α signaling, and thereby restoring Cd-downregulated 11ß-HSD2 protein expression in human placental trophoblasts. We further found that N-acetylcysteine, a well-known antioxidant, obviously reversed Cd-downregulated 11ß-HSD2 protein expression by inhibiting p-PERK/p-eIF2α signaling in placental trophoblasts. Overall, our data suggest that Cd activates the PERK/p-eIF2α signaling, down-regulates the protein expression of 11ß-HSD2, and thereby elevating active GC level in placental trophoblast.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Animais , Cádmio/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Retardo do Crescimento Fetal , Glucocorticoides/metabolismo , Humanos , Camundongos , Placenta/metabolismo , Gravidez , Trofoblastos/fisiologia
13.
Clin Sci (Lond) ; 134(7): 907-920, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32236445

RESUMO

BACKGROUND: Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. AIM: To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. METHODS: Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. RESULTS: Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1ß, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. CONCLUSIONS: Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis.


Assuntos
Albendazol/farmacologia , Proliferação de Células/efeitos dos fármacos , Fármacos Dermatológicos/farmacologia , Queratinócitos/efeitos dos fármacos , Psoríase/tratamento farmacológico , Pele/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Imiquimode , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinas/genética , Queratinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Psoríase/induzido quimicamente , Psoríase/metabolismo , Psoríase/patologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Fosfatases cdc25/metabolismo , eIF-2 Quinase/metabolismo
14.
Science ; 368(6489)2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32327570

RESUMO

Protein quality control is essential for the proper function of cells and the organisms that they make up. The resulting loss of proteostasis, the processes by which the health of the cell's proteins is monitored and maintained at homeostasis, is associated with a wide range of age-related human diseases. Here, we highlight how the integrated stress response (ISR), a central signaling network that responds to proteostasis defects by tuning protein synthesis rates, impedes the formation of long-term memory. In addition, we address how dysregulated ISR signaling contributes to the pathogenesis of complex diseases, including cognitive disorders, neurodegeneration, cancer, diabetes, and metabolic disorders. The development of tools through which the ISR can be modulated promises to uncover new avenues to diminish pathologies resulting from it for clinical benefit.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Proteostase , Estresse Fisiológico , Fatores de Complexo Ternário/metabolismo , Acetamidas/química , Acetamidas/farmacologia , Animais , Cicloexilaminas/química , Cicloexilaminas/farmacologia , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Humanos , Imunidade , Doenças Metabólicas/metabolismo , Camundongos , Neoplasias/metabolismo , Fosfotransferases/metabolismo
15.
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
16.
Nature ; 579(7799): 433-437, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132706

RESUMO

Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies1-3. Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells4,5. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress6. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown1,2,7. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.


Assuntos
Citosol/metabolismo , Citosol/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Genoma Humano/genética , Humanos , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Fosforilação , Ligação Proteica , Estresse Fisiológico , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo
17.
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 , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo , eIF-2 Quinase/metabolismo
18.
Stroke ; 51(5): 1570-1577, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32212900

RESUMO

Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perkf/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results- After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions- Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.


Assuntos
Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Neurônios/metabolismo , Neuroproteção/genética , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/genética , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Cinamatos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/efeitos dos fármacos , Infarto da Artéria Cerebral Média/fisiopatologia , Camundongos , Camundongos Knockout , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
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.
Mol Cells ; 43(3): 264-275, 2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32150794

RESUMO

Reactive oxygen species (ROS) play a significant role in intracellular signaling and regulation, particularly when they are maintained at physiologic levels. However, excess ROS can cause cell damage and induce cell death. We recently reported that eIF2α phosphorylation protects hepatocytes from oxidative stress and liver fibrosis induced by fructose metabolism. Here, we found that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the epidermal growth factor receptor (EGFR) and altered EGFR-mediated signaling pathways. EGFR-mediated signaling pathways are important for cell proliferation, differentiation, and survival in many tissues and cell types. Therefore, we studied whether the reduced amount of EGFR is responsible for the eIF2α phosphorylationdeficient hepatocytes' vulnerability to oxidative stress. ROS such as hydrogen peroxide and superoxides induce both EGFR tyrosine phosphorylation and eIF2α phosphorylation. eIF2α phosphorylation-deficient primary hepatocytes, or EGFR knockdown cells, have decreased ROS scavenging ability compared to normal cells. Therefore, these cells are particularly susceptible to oxidative stress. However, overexpression of EGFR in these eIF2α phosphorylationdeficient primary hepatocytes increased ROS scavenging ability and alleviated ROS-mediated cell death. Therefore, we hypothesize that the reduced EGFR level in eIF2α phosphorylation-deficient hepatocytes is one of critical factors responsible for their susceptibility to oxidative stress.


Assuntos
Receptores ErbB/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Hepatócitos/metabolismo , Estresse Oxidativo/fisiologia , Animais , Linhagem Celular , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
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