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1.
Adv Pharmacol ; 89: 79-99, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32616215

RESUMO

Major depressive disorder is a prevalent and serious form of mental illness. While traditional antidepressants ameliorate some of the symptoms associated with depression, the onset of action typically takes several weeks leaving severely depressed individuals vulnerable to self-injurious behavior and possibly suicide. There has been a major unmet need for the development of pharmacological therapies that can quickly alleviate symptoms associated with depression. Clinical data shows that a single sub-psychomimetic dose of ketamine, a noncompetitive glutamatergic N-methyl-d-aspartate (NMDA) receptor antagonist, has rapid antidepressant responses in patients with treatment-resistant major depressive disorder. We have studied key signaling pathways and synaptic mechanisms underlying the rapid antidepressant action of ketamine. Our studies show ketamine blocks synaptic NMDA receptors involved in spontaneous synaptic transmission, which deactivates calcium/calmodulin-dependent kinase eukaryotic elongation factor 2 kinase (eEF2K), resulting in dephosphorylation of eukaryotic elongation factor 2 (eEF2), and the subsequent desuppression of brain-derived neurotrophic factor (BDNF) protein synthesis in the hippocampus. This signaling pathway then potentiates synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor responses that results in a novel form of synaptic potentiation which corresponds with antidepressant efficacy. In this chapter, we focus on our studies examining ketamine's action and the instructive role of eEF2K in rapid antidepressant action. Our recent studies highlight eEF2K as a major molecular substrate mediating synaptic plasticity and the rapid antidepressant effects of ketamine.


Assuntos
Antidepressivos/farmacologia , Quinase do Fator 2 de Elongação/metabolismo , Ketamina/farmacologia , Animais , Depressão/tratamento farmacológico , Depressão/fisiopatologia , Humanos , Ketamina/uso terapêutico , Plasticidade Neuronal/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo
2.
Biochim Biophys Acta Mol Cell Res ; 1867(8): 118732, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32360667

RESUMO

Nitric oxide is an important neuromodulator in the CNS, and its production within neurons is modulated by NMDA receptors and requires a fine-tuned availability of L-arginine. We have previously shown that globally inhibiting protein synthesis mobilizes intracellular L-arginine "pools" in retinal neurons, which concomitantly enhances neuronal nitric oxide synthase-mediated nitric oxide production. Activation of NMDA receptors also induces local inhibition of protein synthesis and L-arginine intracellular accumulation through calcium influx and stimulation of eucariotic elongation factor type 2 kinase. We hypothesized that protein synthesis inhibition might also increase intracellular L-arginine availability to induce nitric oxide-dependent activation of downstream signaling pathways. Here we show that nitric oxide produced by inhibiting protein synthesis (using cycloheximide or anisomycin) is readily coupled to AKT activation in a soluble guanylyl cyclase and cGKII-dependent manner. Knockdown of cGKII prevents cycloheximide or anisomycin-induced AKT activation and its nuclear accumulation. Moreover, in retinas from cGKII knockout mice, cycloheximide was unable to enhance AKT phosphorylation. Indeed, cycloheximide also produces an increase of ERK phosphorylation which is abrogated by a nitric oxide synthase inhibitor. In summary, we show that inhibition of protein synthesis is a previously unanticipated driving force for nitric oxide generation and activation of downstream signaling pathways including AKT and ERK in cultured retinal cells. These results may be important for the regulation of synaptic signaling and neuronal development by NMDA receptors as well as for solving conflicting data observed when using protein synthesis inhibitors for studying neuronal survival during development as well in behavior and memory studies.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Óxido Nítrico/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Arginina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Galinhas , Proteína Quinase Dependente de GMP Cíclico Tipo II/genética , Quinase do Fator 2 de Elongação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Nitritos , Fosforilação
3.
Pharm Res ; 37(3): 63, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32133571

RESUMO

PURPOSE: To evalauted natural polymeric biomaterials including hyaluronic acid (HA) and its copolymeric form HA:Suc nanoparticles (NPs) as drug carrier systems for delivery of hydrophobic small molecule kinase EF2-kinase inhibitor in breast and pancreatic cancer cells. METHODS: In vitro cellular uptake studies of Rhodamine 6G labaled HA:Suc nanoparticles were evaluated by using flow cytometry analysis and fluorescent microscopy in breast (MDA-MB-231 and MDA-MB-436) and pancreatic cancer cells (PANC-1 and MiaPaca-2). Besides, in vitro release study of compound A (an EF2-kinase inhibitor) as a model hydrophobic drug was performed in the cancer cells. RESULTS: These biological evaluation studies indicated that HA and HA:Suc NPs provided a highly effective delivery of compound A were into breast and pancreatic cancer cells, leading to significant inhibition of cell proliferation and colony formation of breast and pancreatic cancer cells. CONCLUSION: HA-sucrose NPs incorporating an EF2-Kinase inhibitor demonstrate significant biologic activity in breast and pancreatic cancer cells. This is the first study that shows natural polymeric drug carriers succesfully deliver a hydrofobic cancer drug into cancer cells. Graphical Abstract Nanoparticles based on HA:Suc are effective in delivering hydrofobic cancer drugs in breast and pancreatic cancers.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinase do Fator 2 de Elongação/antagonistas & inibidores , Ácido Hialurônico/química , Nanogéis/química , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Quinase do Fator 2 de Elongação/metabolismo , Feminino , Humanos , Neoplasias Pancreáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia
4.
Nat Prod Res ; 34(20): 2880-2886, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30990084

RESUMO

Two new sorbicillinoids, 13-hydroxy-dihydrotrichodermolide (1) and 10,11,27,28-tetrahydrotrisorbicillinone C (2), were isolated from the sponge-derived fungus Penicillium chrysogenum 581F1. Their structures were determined on the basis of spectroscopic analysis. Compounds 1 and 2 displayed high affinities to target proteins GLP-1R (diabetes) and eEF2K (cancer) with Kd values of 0.0285 µM, 0.0162 µM for GLP-1R and 0.118 µM, 0.0746 µM for eEF2K, respectively.


Assuntos
Cicloexanonas/química , Quinase do Fator 2 de Elongação/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Penicillium chrysogenum/química , Animais , Cicloexanonas/isolamento & purificação , Cicloexanonas/metabolismo , Humanos , Estrutura Molecular , Poríferos/microbiologia
5.
Int J Cancer ; 146(5): 1383-1395, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31286509

RESUMO

Hepatocellular carcinoma (HCC) is an aggressive malignancy with increasing mortality in China. Angiogenesis is crucial for tumor formation, development and metastasis in HCC. Previous studies indicated that high expression levels of elongation factor 2 kinase (eEF2K), a protein kinase that negatively regulates the elongation stage of translation, were associated with poor prognosis of HCC. Here, we show that pharmacological inhibition or knockdown of eEF2K in highly metastatic liver cancer cells inhibits their colony forming and migratory capacities, as well as reducing their invasiveness. Importantly, knocking down eEF2K by lentiviral directed shRNA prevented tumor growth and angiogenesis of HCC in mice. Silencing of eEF2K in endothelial cells (HUVECs) led to a reduction in vascularization, evidenced by a decrease in capillary-like structures in the matrigel. Notably, knocking down eEF2K reduced the expression of angiogenesis-related growth factors in liver cancer cells and the expression of growth factor receptors on HUVECs, and thus restricted signaling crosstalk that promotes angiogenesis between HCC cells and endothelial cells. We also showed that silencing of eEF2K effectively reduced protein levels of SP1/KLF5 transcription factors and hence decreased the levels of bound SP1/KLF5 to the VEGF promoter, resulted in a decrease in VEGF mRNA expression. Knocking down eEF2K also led to a striking decrease in the phosphorylation of PI3K/Akt and STAT3, indicating inactivation of these tumorigenic pathways. Taken together, our data suggest that eEF2K contributes to angiogenesis and tumor progression in HCC via SP1/KLF5-mediated VEGF expression, as well as the subsequent stimulation of PI3K/Akt and STAT3 signaling.


Assuntos
Carcinoma Hepatocelular/irrigação sanguínea , Quinase do Fator 2 de Elongação/metabolismo , Neoplasias Hepáticas/irrigação sanguínea , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Hep G2 , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Transdução de Sinais
6.
Neurotox Res ; 37(2): 366-379, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31292883

RESUMO

Attractive due to an alleged high biocompatibility, silica nanoparticles have been widely used in the field of nanomedicine; however, their proven capacity to induce the synthesis and release of pro-inflammatory cytokines in several cellular models has raised concern about their safety. Glutamate, the main excitatory amino acid transmitter triggers a wide variety of signal transduction cascades that regulate protein synthesis at transcriptional and translational levels. A stimulus-dependent dynamic change in the protein repertoire in neurons and glia cells is the molecular framework of higher brain functions. Within the cerebellum, Bergmann glia cells are the most abundant non-neuronal cells and span the entire molecular layer of the cerebellar cortex, wrapping the synapses in this structure. Taking into consideration the functional role of Bergmann glia in terms of the recycling of glutamate, lactate supply to neurons, and prevention of neurotoxic insults, we decided to investigate the possibility that silica nanoparticles affect Bergmann glia and by these means alter the major excitatory neurotransmitter system in the brain. To this end, we exposed cultured chick cerebellar Bergmann glia cells to silica nanoparticles and measured [35S]-methionine incorporation into newly synthesized polypeptides. Our results demonstrate that exposure of the cultured cells to silica nanoparticles exerts a time- and dose-dependent modulation of protein synthesis. Furthermore, altered patterns of eukaryotic initiation factor 2 alpha and eukaryotic elongation factor 2 phosphorylation were present upon nanoparticle exposure. These results demonstrate that glia cells respond to the presence of this nanomaterial modifying their proteome, presumably in an effort to overcome any plausible neurotoxic effect.


Assuntos
Nanopartículas/efeitos adversos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Dióxido de Silício/efeitos adversos , Animais , Embrião de Galinha , Relação Dose-Resposta a Droga , Quinase do Fator 2 de Elongação/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Metionina/metabolismo , Fosforilação , Cultura Primária de Células , Radioisótopos de Enxofre/metabolismo , Fatores de Tempo
7.
Vet Parasitol ; 276: 108991, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31770701

RESUMO

Eimeria tenella, an obligate intracellular parasite, can actively invade the cecal epithelial cells of chickens and cause severe enteric disease. Eukaryotic elongation factor 2 (eEF2) plays a major role in protein synthesis and cell survival. This study aims to explore the exact mechanisms underlying diclazuril inhibition in second-generation merozoites of E. tenella. The eEF2 cDNA of the second-generation merozoites of E. tenella (EtEF2) was cloned by reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Diclazuril-induced expression profiles of EtEF2 were also analyzed. The cloned full-length cDNA (2893 bp) of the EtEF2 nucleotide sequence encompassed a 2499 bp open reading frame (ORF) that encoded a polypeptide of 832 residues with an estimated molecular mass of 93.12 kDa and a theoretical isoelectric point of 5.99. The EtEF2 nucleotide sequence was submitted to the GenBank database with the accession number KF188423. The EtEF2 protein sequence shared 99 % homology with the eEF2 sequence of Toxoplasma gondii (GenBank XP_002367778.1). The GTPase activity domain and ADP-ribosylation domain were conserved signature sequences of the eEF2 gene family. The changes in the transcriptional and translational levels of EtEF2 were detected through quantitative real-time PCR and Western blot analyses. The mRNA expression level of EtEF2 was 2.706 fold increases and the protein level of EtEF2 was increased 67.31 % under diclazuril treatment. In addition, the localization of EtEF2 was investigated through immunofluorescence assay. Experimental results demonstrated that EtEF2 was distributed primarily in the cytoplasm of second-generation merozoites, and its fluorescence intensity was enhanced after diclazuril treatment. These findings indicated that EtEF2 may have an important role in understanding the signaling mechanism underlying the anticoccidial action of diclazuril and could be a promising target for novel drug exploration.


Assuntos
Galinhas/parasitologia , Coccidiose/veterinária , Coccidiostáticos/farmacologia , Eimeria tenella/efeitos dos fármacos , Quinase do Fator 2 de Elongação/metabolismo , Doenças das Aves Domésticas/tratamento farmacológico , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Coccidiose/tratamento farmacológico , Coccidiose/parasitologia , Eimeria tenella/genética , Quinase do Fator 2 de Elongação/genética , Feminino , Imunofluorescência , Masculino , Merozoítos/efeitos dos fármacos , Merozoítos/genética , Camundongos , Camundongos Endogâmicos BALB C , Nitrilos/farmacologia , Filogenia , Doenças das Aves Domésticas/parasitologia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Triazinas/farmacologia
8.
Protein Sci ; 28(12): 2089-2098, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31626716

RESUMO

Eukaryotic elongation factor 2 kinase (eEF-2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF-2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM-regulated kinases. We had previously identified a minimal construct of eEF-2K (TR) that is activated similarly to the wild-type enzyme by CaM in vitro and retains its ability to phosphorylate eEF-2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1-methyls of TR and Ile δ1- and Met ε-methyls of CaM, as probes of their mutual interaction and the influence of Ca2+ thereon. We find that in the absence of Ca2+ , CaM exclusively utilizes its C-terminal lobe (CaMC ) to engage the N-terminal CaM-binding domain (CBD) of TR in a high-affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca2+ -loaded N-terminal lobe of CaM (CaMN ) at increased Ca2+ levels serves to enhance the affinity further. These latter interactions under Ca2+ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca2+ -free state. Our data are consistent with a scenario in which Ca2+ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM-bound species without significantly modifying the conformation of TR within the final, active complex.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Cálcio/química , Calmodulina/química , Quinase do Fator 2 de Elongação/química
9.
Proc Natl Acad Sci U S A ; 116(45): 22583-22590, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31636182

RESUMO

Gene expression is rapidly remodeled by infection and inflammation in part via transcription factor NF-κB activation and regulated protein synthesis. While protein synthesis is largely controlled by mRNA translation initiation, whether cellular translation elongation factors are responsive to inflammation and infection remains poorly understood. Here, we reveal a surprising mechanism whereby NF-κB restricts phosphorylation of the critical translation elongation factor eEF2, which catalyzes the protein synthesis translocation step. Upon exposure to NF-κB-activating stimuli, including TNFα, human cytomegalovirus infection, or double-stranded DNA, eEF2 phosphorylation on Thr56, which slows elongation to limit protein synthesis, and the overall abundance of eEF2 kinase (eEF2K) are reduced. Significantly, this reflected a p65 NF-κB subunit-dependent reduction in eEF2K pre-mRNA, indicating that NF-κB activation represses eEF2K transcription to decrease eEF2K protein levels. Finally, we demonstrate that reducing eEF2K abundance regulates protein synthesis in response to a bacterial toxin that inactivates eEF2. This establishes that NF-κB activation by diverse physiological effectors controls eEF2 activity via a transcriptional repression mechanism that reduces eEF2K polypeptide abundance to preclude eEF2 phosphorylation, thereby stimulating translation elongation and protein synthesis. Moreover, it illustrates how nuclear transcription regulation shapes translation elongation factor activity and exposes how eEF2 is integrated into innate immune response networks orchestrated by NF-κB.


Assuntos
DNA/metabolismo , Quinase do Fator 2 de Elongação/genética , Inflamação/metabolismo , Biossíntese de Proteínas , Fator de Transcrição RelA/metabolismo , Motivos de Aminoácidos , DNA/genética , Quinase do Fator 2 de Elongação/química , Quinase do Fator 2 de Elongação/metabolismo , Humanos , Inflamação/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação , Fator de Transcrição RelA/genética
10.
Pathol Res Pract ; 215(11): 152636, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31558304

RESUMO

BACKGROUND: Gastric cancer (GC) is the fourth most prevalent malignant tumor and the second leading cause of cancer-related death around the world. Aberrant proliferation and metastasis are the mainspring of death in patients with GC. However, the specific mechanism of gastric cancer is far from being fully elucidated. Accumulating evidence revealed that miRNA played a significant role in the tumorigenesis and development. METHODS: The level of miR-183-5p was detected in 102 GC patients by using qRT-PCR. The prognostic value of miR-183-5p in GC was evaluated. Cell function assays (CCK-8 and transwell assays) were conducted to assess the role of miR-183-5p in proliferation and metastasis in GC. Dual luciferase report assay and western blot were performed to validate this potential target regulated by miR-183-5p in GC. RESULTS: miR-183-5p was down-regulated in GC tissues and cell lines. Remarkable pertinence was obtained between miR-183-5p level and TNM stage, tumor size, invasion depth, and lymph node metastasis. TNM stage, differentiation and miR-183-5p level were independent causes impacting on the overall survival in GC in multivariate analysis. GC individuals with high miR-183-5p level would experience a relatively better survival prognosis. Upregulation of miR-183-5p restrained GC cell proliferation and migration. EEF2 may be a potential target gene regulated by miR-183-5p in GC. CONCLUSION: miR-183-5p acts as a potential prognostic biomarker in gastric cancer and regulates cell functions by modulating EEF2.


Assuntos
Biomarcadores Tumorais/genética , Quinase do Fator 2 de Elongação/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/metabolismo , Neoplasias Gástricas/patologia , Adulto , Idoso , Biomarcadores Tumorais/análise , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Prognóstico , Neoplasias Gástricas/genética
11.
J Alzheimers Dis ; 70(3): 925-936, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31306126

RESUMO

General anesthesia increases the risk for cognitive impairment and Alzheimer's disease (AD) in vulnerable individuals such as the elderly. We previously reported that prior administration of insulin through intranasal delivery can prevent the anesthesia-induced cognitive impairment and biochemical changes in the brain. However, little is known about the underlying molecular mechanisms. Here, we report that general anesthesia resulted in downregulation of mammalian/mechanistic target of rapamycin (mTOR) and eukaryotic elongation factor 2 (eEF2) in the brain along with reduction of presynaptic proteins and brain-derived neurotrophic factor and cognitive impairment in aged mice. Prior administration of intranasal insulin prevented these anesthesia-induced changes. These results suggest the involvement of the mTOR-eEF2 signaling pathway in the anesthesia-induced brain changes and cognitive impairment and in the prevention of these changes with insulin. Correlation analyses and the use of eEF2 kinase inhibitor further support our conclusions. These studies shed light on the molecular mechanism by which anesthesia and insulin could act on synaptic proteins and cognitive function.


Assuntos
Doença de Alzheimer , Encéfalo/metabolismo , Disfunção Cognitiva , Quinase do Fator 2 de Elongação , Insulina/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Administração Intranasal , Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Anestesia Geral/efeitos adversos , Animais , Cognição/efeitos dos fármacos , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/prevenção & controle , Quinase do Fator 2 de Elongação/antagonistas & inibidores , Quinase do Fator 2 de Elongação/metabolismo , Hipoglicemiantes/farmacologia , Camundongos , Transdução de Sinais/efeitos dos fármacos
12.
Physiol Rep ; 7(14): e14158, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31353827

RESUMO

Prior work established that exercise alleviates muscle function loss in a clinically relevant rodent model mimicking the clinical sequelae of severely burned patients. On the basis of these data, we posit that pharmacologic treatment with insulin combined with exercise further mitigates loss of muscle function following severe burn with immobilization. Twenty-four Sprague-Dawley rats were assessed and trained to complete a climbing exercise. All rats followed a standardized protocol to mimic severe burn patients (40% total body surface area scald burn); all rats were immediately placed into a hindlimb unloading apparatus to simulate bedrest. The rats were then randomly assigned to four treatment groups: saline vehicle injection without exercise (VEH/NEX), insulin (5 U/kg) injection without exercise (INS/NEX), saline vehicle with daily exercise (VEH/EX), and insulin with daily exercise (INS/EX). The animals were assessed for 14 days following injury. The groups were compared for multiple variables. Isometric tetanic (Po) and twitch (Pt) forces were significantly elevated in the plantaris and soleus muscles of the INS/EX rats (P < 0.05). Genomic analysis revealed mechanistic causes with specific candidate changes. Molecular analysis of INS/EX rats revealed Akt phosphorylated by PDPK1 was increased with this treatment, and it further activated downstream signals mTOR, eEF2, and GSK3-ß (P < 0.05). Furthermore, muscle RING-finger protein-1 (MuRF-1), an E3 ubiquitin ligase, was reduced in the INS/EX group (P < 0.05). Insulin and resistance exercise have a positive combined effect on the muscle function recovery in this clinically relevant rodent model of severe burn. Both treatments altered signaling pathways of increasing protein synthesis and decreasing protein degradation.


Assuntos
Queimaduras/terapia , Insulina/uso terapêutico , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/métodos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Animais , Queimaduras/tratamento farmacológico , Quinase do Fator 2 de Elongação/genética , Quinase do Fator 2 de Elongação/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Elevação dos Membros Posteriores/métodos , Insulina/administração & dosagem , Masculino , Contração Muscular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiopatologia , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
13.
BMC Cancer ; 19(1): 649, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266475

RESUMO

BACKGROUND: Prognostication of patients with colorectal cancer (CRC) currently relies on tumor-node-metastasis (TNM) staging but clinical outcomes of patients of the same histoclinical stage are heterogeneous. It is therefore imperative to devise novel molecular tests to stratify CRC patients. Our previous work demonstrated that eukaryotic elongation factor-2 kinase (EEF2K) is a tumor suppressor in CRC. Herein, we investigated EEF2K expression in CRC and determined its relationship with clinicopathological parameters. METHODS: Quantitative RT-PCR and Westerns blots were used to examine EEF2K expression in primary tumor and the adjacent non-tumor tissues of CRC patients (n = 20). Kaplan-Meier curves and Cox regression analysis were used to assess the association between clinical outcomes of CRC patients and EEF2K protein expression determined by immunohistochemistry on tissue microarray (n = 151). RESULTS: EEF2K was significantly downregulated at both mRNA and protein levels in tumors of CRC patients. Univariate Cox regression analysis revealed that CRC patients with high tumor grade, advanced TNM staging and low EEF2K expression were associated with worse overall survival. Multivariate analysis further demonstrated that low EEF2K expression was an independent factor for predicting poorer overall survival in CRC patients (p = 0.014; Hazard ratio = 2.951; 95% confidence interval: 1.240-7.024). The 5-year survival rate was 82.8% in the EEF2K-high-expression group versus 63.9% in the EEF2K-low-expression group (p = 0.0118). The association of overall survival with EEF2K expression in CRC patients was verified in The Cancer Genome Atlas (TCGA) cohort. CONCLUSIONS: EEF2K is downregulated in CRC and its expression can be employed as a prognostic marker for CRC patients independent of TNM staging.


Assuntos
Neoplasias do Colo/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Neoplasias Retais/metabolismo , Idoso , Colo/metabolismo , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Regulação para Baixo , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/metabolismo , Neoplasias Retais/mortalidade , Neoplasias Retais/patologia , Reto/metabolismo , Análise de Regressão , Taxa de Sobrevida , Resultado do Tratamento , Proteínas Supressoras de Tumor
14.
Eur J Pharmacol ; 857: 172470, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31226250

RESUMO

Eukaryotic elongation factor 2 kinase (eEF-2K) is known as calcium/calmodulin-dependent protein kinase III and identified as a calcium/calmodulin (Ca2+/CaM)-dependent protein kinase (CaM-PK) that phosphorylates its only substrate eukaryotic elongation factor-2 (eEF-2) and blocks the ability of eEF-2 to bind the ribosome and translation elongation and inhibits global protein synthesis. The activators of eEF-2K include allosteric activator Ca/CaM, Ca/CaM-independent activator cAMP-dependent protein kinase (PKA) and H+. On the other hand, eEF-2K is inactivated by the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. However, the role of eEF-2K in cancer is not well understood. To provide opinion for the diagnosis and treatment of cancer, we summarized the role of eEF-2K in cancer. Based on the fundamental research on eEF-2K, scientists further investigated the role of eEF-2K in cancer and have reported its different effects in many kinds of cancer. eEF-2K involves in many signal pathways, including proliferation, apoptosis, autophagy, invasion and glycolysis, and promotes the development of cancer as an oncogene. Inhibition of eEF-2K by eEF-2K siRNA and little molecular inhibitors resulted in the suppression of proliferation, autophagy, invasion and glycolysis, and accelerate apoptosis to play an antitumor role. In this review, we summarize the regulation and role of eEF-2K in cancer as an oncogene and the exploitation of the inhibitor of eEF-2K. Combined treatment of eEF-2K inhibitor and chemotherapeutics should be a potential tool in cancer therapy.


Assuntos
Quinase do Fator 2 de Elongação/metabolismo , Neoplasias/enzimologia , Animais , Ativação Enzimática , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia
15.
J Mol Biol ; 431(15): 2700-2717, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31108082

RESUMO

Eukaryotic elongation factor 2 kinase (eEF-2K), an atypical calmodulin-activated protein kinase, regulates translational elongation by phosphorylating its substrate, eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome. The activation and activity of eEF-2K are critical for survival under energy-deprived conditions and is implicated in a variety of essential physiological processes. Previous biochemical experiments have indicated that the binding site for the substrate eEF-2 is located in the C-terminal domain of eEF-2K, a region predicted to harbor several α-helical repeats. Here, using NMR methodology, we have determined the solution structure of a C-terminal fragment of eEF-2K, eEF-2K562-725 that encodes two α-helical repeats. The structure of eEF-2K562-725 shows signatures characteristic of TPR domains and of their SEL1-like sub-family. Furthermore, using the analyses of NMR spectral perturbations and ITC measurements, we have localized the eEF-2 binding site on eEF-2K562-725. We find that eEF-2K562-725 engages eEF-2 with an affinity comparable to that of the full-length enzyme. Furthermore, eEF-2K562-725 is able to inhibit the phosphorylation of eEF-2 by full-length eEF-2K in trans. Our present studies establish that eEF-2K562-725 encodes the major elements necessary to enable the eEF-2K/eEF-2 interactions.


Assuntos
Quinase do Fator 2 de Elongação/química , Sequência de Aminoácidos , Quinase do Fator 2 de Elongação/metabolismo , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Especificidade por Substrato
16.
J Biol Chem ; 294(18): 7169-7176, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30890561

RESUMO

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation stage of mRNA translation and is activated under different stress conditions to slow down protein synthesis. One effect of eEF2K is to alter the repertoire of expressed proteins, perhaps to aid survival of stressed cells. Here, we applied pulsed stable isotope labeling with amino acids in cell culture (SILAC) to study changes in the synthesis of specific proteins in human lung adenocarcinoma (A549) cells in which eEF2K had been depleted by an inducible shRNA. We discovered that levels of heat-shock protein 90 (HSP90) are increased in eEF2K-depleted human cells as well as in eEF2K-knockout (eEF2K-/-) mouse embryonic fibroblasts (MEFs). This rise in HSP90 coincided with an increase in the fraction of HSP90 mRNAs associated with translationally active polysomes, irrespective of unchanged total HSP90 levels. These results indicate that blocking eEF2K function can enhance expression of HSP90 chaperones. In eEF2K-/- mouse embryonic fibroblasts (MEFs), inhibition of HSP90 by its specific inhibitor AUY922 promoted the accumulation of ubiquitinated proteins. Notably, HSP90 inhibition promoted apoptosis of eEF2K-/- MEFs under proteostatic stress induced by the proteasome inhibitor MG132. Up-regulation of HSP90 likely protects cells from protein folding stress, arising, for example, from faster rates of polypeptide synthesis due to the lack of eEF2K. Our findings indicate that eEF2K and HSPs closely cooperate to maintain proper proteostasis and suggest that concomitant inhibition of HSP90 and eEF2K could be a strategy to decrease cancer cell survival.


Assuntos
Quinase do Fator 2 de Elongação/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Estresse Oxidativo , Células A549 , Animais , Morte Celular , Células Cultivadas , Quinase do Fator 2 de Elongação/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Marcação por Isótopo , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Ubiquitinação
17.
Acta Pharmacol Sin ; 40(9): 1237-1244, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30914761

RESUMO

Eukaryotic elongation factor-2 kinase (eEF-2K), a negative regulator of protein synthesis, has been shown to play an important role in modulating autophagy and apoptosis in tumor cells under various stresses. In this study, we investigated the regulatory role of eEF-2K in pyroptosis (a new form of programmed necrosis) in doxorubicin-treated human melanoma cells. We found that doxorubicin (0.5-5 µmol/L) induced pyroptosis in melanoma cell lines SK-MEL-5, SK-MEL-28, and A-375 with high expression of DFNA5, but not in human breast cancer cell line MCF-7 with little expression of DFNA5. On the other hand, doxorubicin treatment activated autophagy in the melanoma cells; inhibition of autophagy by transfecting the cells with siRNA targeting Beclin1 or by pretreatment with chloroquine (20 µmol/L) significantly augmented pyroptosis, thus sensitizing the melanoma cells to doxorubicin. We further demonstrated that doxorubicin treatment activated eEF-2K in the melanoma cells, and silencing of eEF-2K blunted autophagic responses, but promoted doxorubicin-induced pyroptotic cell death. Taken together, the above results demonstrate that eEF-2K dictates the cross-talk between pyroptosis and autophagy in doxorubicin-treated human melanoma cells; suppression of eEF-2K results in inhibiting autophagy and augmenting pyroptosis, thus modulating the sensitivity of melanoma cells to doxorubicin, suggesting that targeting eEF-2K may reinforce the antitumor efficacy of doxorubicin, offering a new insight into tumor chemotherapy.


Assuntos
Antineoplásicos/farmacologia , Autofagia/fisiologia , Doxorrubicina/farmacologia , Quinase do Fator 2 de Elongação/metabolismo , Melanoma/metabolismo , Piroptose/fisiologia , Autofagia/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Melanoma/tratamento farmacológico , Proteínas Associadas aos Microtúbulos/metabolismo , Piroptose/efeitos dos fármacos , Receptores Estrogênicos/metabolismo
18.
Curr Biol ; 29(5): 737-749.e5, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30773367

RESUMO

Maintaining accuracy during protein synthesis is crucial to avoid producing misfolded and/or non-functional proteins. The target of rapamycin complex 1 (TORC1) pathway and the activity of the protein synthesis machinery are known to negatively regulate lifespan in many organisms, although the precise mechanisms involved remain unclear. Mammalian TORC1 signaling accelerates the elongation stage of protein synthesis by inactivating eukaryotic elongation factor 2 kinase (eEF2K), which, when active, phosphorylates and inhibits eEF2, which mediates the movement of ribosomes along mRNAs, thereby slowing down the rate of elongation. We show that eEF2K enhances the accuracy of protein synthesis under a range of conditions and in several cell types. For example, our data reveal it links mammalian (m)TORC1 signaling to the accuracy of translation. Activation of eEF2K decreases misreading or termination readthrough errors during elongation, whereas knocking down or knocking out eEF2K increases their frequency. eEF2K also promotes the correct recognition of start codons in mRNAs. Reduced translational fidelity is known to correlate with shorter lifespan. Consistent with this, deletion of the eEF2K ortholog or other factors implicated in translation fidelity in Caenorhabditis elegans decreases lifespan, and eEF2K is required for lifespan extension induced by nutrient restriction. Our data uncover a novel mechanism linking nutrient supply, mTORC1 signaling, and the elongation stage of protein synthesis, which enhances the accuracy of protein synthesis. Our data also indicate that modulating translation elongation and its fidelity affects lifespan.


Assuntos
Caenorhabditis elegans/fisiologia , Quinase do Fator 2 de Elongação/genética , Longevidade/genética , Biossíntese de Proteínas/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Fatores de Transcrição E2F , Quinase do Fator 2 de Elongação/metabolismo
19.
Apoptosis ; 24(3-4): 359-368, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30737648

RESUMO

During the development of cardiac hypertrophy, glucose deprivation (GD) associated with coronary microvascular rarefaction is caused, leading to cardiomyocyte death. Phosphorylation (inactivation) of eukaryotic elongation factor 2 (eEF2) by eEF2 kinase (eEF2K) inhibits protein translation, a highly energy consuming process, which plays protective roles against nutrient deprivation-induced cell death. We previously showed that eEF2 phosphorylation was increased in isolated heart from several cardiac hypertrophy models. In this study, we investigated whether eEF2K/eEF2 mediates the inhibition of cardiomyocyte death under GD condition. In H9c2 rat cardiomyoblasts cultured with serum-free medium, GD significantly augmented eEF2 phosphorylation and signals related to autophagy [increase of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I ratio] and apoptosis (cleavage of caspase-3) as determined by Western blotting. GD induced cell death, which was augmented by eEF2K gene knockdown using a small interfering RNA. eEF2K gene knockdown significantly augmented GD-induced cleavage of caspase-3 and apoptotic nuclear condensation as determined by 4', 6-diamidino-2-phenylindole staining. In contrast, eEF2K gene knockdown significantly inhibited GD-induced increase of LC3-II to LC3-I ratio and autophagosome formation as determined by an immunofluorescence staining. An inhibitor of autophagy, 3-methyladenine or bafilomycin A1 significantly augmented GD-induced cleavage of caspase-3. Further, eEF2K gene knockdown significantly inhibited GD-induced phosphorylation of adenosine monophosphate-activated protein kinase (AMPK)α and its downstream substrate, unc-51 like autophagy activating kinase (ULK)1. An inhibitor of AMPK, dorsomorphin significantly inhibited GD-induced increase of LC3-II to LC3-I ratio. In conclusion, we for the first time revealed that eEF2K/eEF2 axis under GD condition mediates the inhibition of apoptotic H9c2 cell death at least in part via promotion of autophagy through AMPKα/ULK1 signaling pathway.


Assuntos
Morte Celular/fisiologia , Quinase do Fator 2 de Elongação/metabolismo , Glucose/metabolismo , Mioblastos Cardíacos/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/fisiologia , Autofagossomos/metabolismo , Autofagia/fisiologia , Caspase 3/metabolismo , Linhagem Celular , Mioblastos Cardíacos/fisiologia , Fosforilação/fisiologia , Ratos , Transdução de Sinais/fisiologia
20.
Neuropharmacology ; 149: 35-44, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30731135

RESUMO

Ketamine is a fast acting experimental antidepressant with significant therapeutic potential for emotional disorders such as major depressive disorder and alcohol use disorders. Of particular interest is binge alcohol use, which during intermittent withdrawal from drinking involves depressive-like symptoms reminiscent of major depressive disorder. Binge drinking has been successfully modeled in mice with the Drinking in the Dark (DID) paradigm, which involves daily access to 20% ethanol, for a limited duration and selectively during the dark phase of the circadian light cycle. Here we demonstrate that DID exposure reduces the cell surface expression of NMDA- and AMPA-type glutamate receptors in the prelimbic cortex (PLC) of female but not male mice, along with reduced activity of the mammalian target of rapamycin (mTOR) signaling pathway. Pretreatment with an acute subanesthetic dose of ketamine suppresses binge-like ethanol consumption in female but not male mice. Lastly, DID-exposure reduces spontaneous glutamatergic synaptic transmission in the PLC of both sexes, but synaptic transmission is rescued by ketamine selectively in female mice. Thus, ketamine may have therapeutic potential as an ethanol binge suppressing agent selectively in female subjects.


Assuntos
Bebedeira/metabolismo , Bebedeira/terapia , Ácido Glutâmico/metabolismo , Ketamina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Córtex Pré-Frontal , Receptores de Glutamato/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Serina-Treonina Quinases TOR/metabolismo
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