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1.
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
2.
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
3.
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
4.
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
5.
Biomed Res Int ; 2019: 6302950, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31317034

RESUMO

This study aimed to investigate the effects of isoleucine (Ile) on the synthesis and secretion of digestive enzymes and cellular signalling in the pancreatic tissue of dairy goats. The pancreatic tissues were incubated in buffer containing 0, 0.40, 0.80, and 1.60 mM Ile. High levels of Ile significantly increased the buffer release and total concentration of ɑ-amylase in the tissues (P < 0.001). The total trypsin and chymotrypsin concentrations in each of the Ile groups were significantly higher than those in the control group (P < 0.05); however, lipase was not affected. High levels of Ile significantly increased ɑ-amylase mRNA expression (P < 0.001) but had no effect on the mRNA expression of trypsin, chymotrypsin, or lipase. Ile did not affect S6K1 phosphorylation levels. High levels of Ile significantly increased the expression of the γ isoform of 4EBP1 (P < 0.001), which indicated that the phosphorylation of 4EBP1 was significantly increased. The phosphorylation level of eEF2 gradually decreased with the addition of Ile (P < 0.001). These results suggested that high doses of Ile can regulate the excretion of enzymes, especially ɑ-amylase, in the pancreatic tissues of dairy goats by modulating mTOR signalling, and this regulation is independent of the mTOR-S6K1 pathway.


Assuntos
Cabras/metabolismo , Isoleucina/metabolismo , Pâncreas/enzimologia , alfa-Amilases/biossíntese , Animais , Quimotripsina/biossíntese , Quimotripsina/metabolismo , Quinase do Fator 2 de Elongação/genética , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica/genética , Lipase/biossíntese , Lipase/metabolismo , Pâncreas/metabolismo , Fosforilação , RNA Mensageiro/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Tripsina/biossíntese , Tripsina/metabolismo , alfa-Amilases/metabolismo
6.
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
7.
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
8.
Arthritis Res Ther ; 21(1): 97, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987676

RESUMO

BACKGROUND: Intervertebral disc degeneration (IDD) is a major contributor to back, neck, and radicular pain, and the treatment of IDD is costly and relatively ineffective. Dysregulation of microRNAs (miRNAs) has been reported to be involved in IDD. The purpose of our study is to illustrate the potential that miR-143-5p targeting eEF2 gene mediates IDD. METHODS: Following the establishment of the IDD rat models, expression of miR-143-5p, eEF2, Bcl-2, Bax, AMPK, mTOR, cyclinD, COL2, ACAN, and DCN was detected. The NP cells isolated from degenerative intervertebral disc (IVD) were introduced with a series of mimic, inhibitor, or AICAR to explore the functional role of miR-143-5p in IDD and to characterize the relationship between miR-143-5p and eEF2. Cell viability, cell cycle, apoptosis, and senescence were also evaluated. RESULTS: A reduction in eEF2, an increase in miR-143-5p, and activation of the AMPK signaling pathway were observed in degenerative IVD. Moreover, increased senescent NP cells were observed in degenerative IVD. eEF2 was confirmed as a target gene of miR-143-5p. miR-143-5p was found to activate the AMPK signaling pathway. The restoration of miR-143-5p or the activation of AMPK signaling pathway decreased COL2, ACAN, and DCN expression, coupled with the inhibition of NP cell proliferation and differentiation, and promotion of NP apoptosis and senescence. On the contrary, the inhibition of miR-143-5p led to the reversed results. CONCLUSION: The results demonstrated that the inhibition of miR-143-5p may act as a suppressor for the progression of IDD.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Quinase do Fator 2 de Elongação/biossíntese , Degeneração do Disco Intervertebral/metabolismo , MicroRNAs/biossíntese , Transdução de Sinais/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Células Cultivadas , Quinase do Fator 2 de Elongação/antagonistas & inibidores , Quinase do Fator 2 de Elongação/genética , Degeneração do Disco Intervertebral/genética , Masculino , MicroRNAs/genética , Ratos , Ratos Endogâmicos Lew
9.
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
10.
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
11.
Genes Brain Behav ; 18(5): e12563, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30838762

RESUMO

A common feature of several psychiatric disorders is the attentional impairment. eEF2K -/- , IL1RAPL1 -/- and SHANK3Δ11 -/- mice were used as animal models consistently linked to changes in synaptic plasticity, learning and memory. All knockout (KO) mice and their corresponding littermates were submitted to the novel object recognition (NOR) and visual object recognition (VOR) tasks. In the NOR, eEF2K-/- mice exhibited a normal performance in terms of mean discrimination index, while SHANK3Δ11-/- and IL1RAPL1 -/- mice were impaired when a delay of 2 and 24 hours was introduced. Surprisingly, when submitted to VOR, where the two objects were replaced with two shapes delivered from two iPods, all the mutant mice performed worse than those in the NOR. In VOR, the application of motion to different shapes, to increase attention, improved performance in eEF2K -/- and IL1RAPL1 -/- but not in SHANK3Δ11 -/- mice. In SHANK3Δ11 -/- mice, attentional deficit was also present even if different motions were applied to the same shapes or when these mice were repeatedly exposed for 5 days to the context. Behavioral analysis showed that eEF2K-/- and IL1RAPL1 -/- mice had a good flexibility tested in the T-maze. eEF2K-/- showed normal self-grooming. On the basis of previous literature data indicating that SHANK3Δ11 -/- showed impaired flexibility and reduced sociability, we identified in this genotype the most exhaustive model showing all the core symptoms of autism spectrum disorder including a heavy visual attention deficit. These findings show the importance of VOR to identify mouse models of autism.


Assuntos
Atenção , Quinase do Fator 2 de Elongação/genética , Proteína Acessória do Receptor de Interleucina-1/genética , Proteínas do Tecido Nervoso/genética , Percepção Visual/genética , Animais , Discriminação Psicológica , Deleção de Genes , Asseio Animal , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Comportamento Social
12.
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
13.
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
14.
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
15.
J Clin Invest ; 129(2): 820-833, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30667373

RESUMO

Molecular signaling mechanisms underlying Alzheimer's disease (AD) remain unclear. Maintenance of memory and synaptic plasticity depend on de novo protein synthesis, dysregulation of which is implicated in AD. Recent studies showed AD-associated hyperphosphorylation of mRNA translation factor eukaryotic elongation factor 2 (eEF2), which results in inhibition of protein synthesis. We tested to determine whether suppression of eEF2 phosphorylation could improve protein synthesis capacity and AD-associated cognitive and synaptic impairments. Genetic reduction of the eEF2 kinase (eEF2K) in 2 AD mouse models suppressed AD-associated eEF2 hyperphosphorylation and improved memory deficits and hippocampal long-term potentiation (LTP) impairments without altering brain amyloid ß (Aß) pathology. Furthermore, eEF2K reduction alleviated AD-associated defects in dendritic spine morphology, postsynaptic density formation, de novo protein synthesis, and dendritic polyribosome assembly. Our results link eEF2K/eEF2 signaling dysregulation to AD pathophysiology and therefore offer a feasible therapeutic target.


Assuntos
Doença de Alzheimer , Espinhas Dendríticas , Quinase do Fator 2 de Elongação , Potenciação de Longa Duração , Densidade Pós-Sináptica , Transdução de Sinais/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Espinhas Dendríticas/genética , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Modelos Animais de Doenças , Quinase do Fator 2 de Elongação/genética , Quinase do Fator 2 de Elongação/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação/genética , Densidade Pós-Sináptica/genética , Densidade Pós-Sináptica/metabolismo , Densidade Pós-Sináptica/patologia
16.
J Vet Med Sci ; 81(1): 35-41, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30429409

RESUMO

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) inhibits protein translation through the phosphorylation of its specific substrate, eEF2. We previously demonstrated that eEF2K expression increases in superior mesenteric artery from spontaneously hypertensive rats (SHR) and that eEF2K mediates development of hypertension in SHR. In addition, we recently revealed that A484954, a selective eEF2K inhibitor induced relaxation via opening smooth muscle inward rectifier K+ (Kir) channel in rat isolated superior mesenteric artery. Here, we further examined the effects of A484954 on contractility and blood pressure (BP) in rats. Isometric contraction of rat isolated superior mesenteric artery was measured. BP was measured by a carotid cannulation method. A484954 (10 µM) inhibited noradrenaline (NA)-induced contraction in a biphasic manner (magnitude of inhibition higher at high dose NA). A484954 also inhibited an α1-receptor agonist, phenylephrine-induced contraction, while it was not biphasic. Specifically, a ß-receptor antagonist, propranolol (1 µM) prevented the A484954-mediated inhibition of NA (high-dose)-induced contraction. A484954 (10 µM) potentiated a ß-receptor agonist, isoproterenol-induced relaxation, which was completely prevented by BaCl2 (1 mM), a Kir channel blocker. In vivo, A484954 (122 µg/kg) inhibited NA-induced increase of BP in rats. Another eEF2K inhibitor, NH125 (22 µg/kg) also inhibited the NA-induced BP increase in rats. In summary, it was concluded that A484954 lowers NA-induced BP rise perhaps through activation of ß2-receptor-Kir channel and subsequent vasorelaxation via inhibiting eEF2K activity.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Ciclopropanos/farmacologia , Quinase do Fator 2 de Elongação/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirrolidinas/farmacologia , Animais , Contração Isométrica/efeitos dos fármacos , Masculino , Norepinefrina/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos Endogâmicos SHR , Ratos Wistar , Vasodilatação/efeitos dos fármacos
17.
Biol Chem ; 400(4): 501-512, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30218597

RESUMO

The functionality of eukaryotic translation elongation factor 2 (eEF2) is modulated by phosphorylation, eEF2 is simultaneously the molecular target of ADP-ribosylating toxins. We analyzed the interplay between phosphorylation and diphthamide-dependent ADP-ribosylation. Phosphorylation does not require diphthamide, eEF2 without it still becomes phosphorylated. ADP-ribosylation not only modifies the H715 diphthamide but also inhibits phosphorylation of S595 located in proximity to H715, and stimulates phosphorylation of T56. S595 can be phosphorylated by CDK2 and CDK1 which affects EEF2K-mediated T56-phosphorylation. Thus, ADP-ribosylation and S595-phosphorylation by kinases occur within the same vicinity and both trigger T56-phosphorylation. Diphthamide is surface-accessible permitting access to ADP-ribosylating enzymes, the adjacent S595 side chain extends into the interior. This orientation is incompatible with phosphorylation, neither allowing kinase access nor phosphate attachment. S595 phosphorylation must therefore be accompanied by structural alterations affecting the interface to ADP-ribosylating toxins. In agreement with that, replacement of S595 with Ala, Glu or Asp prevents ADP-ribosylation. Phosphorylation (starvation) as well as ADP-ribosylation (toxins) inhibit protein synthesis, both affect the S595/H715 region of eEF2, both trigger T57-phosphorylation eliciting similar transcriptional responses. Phosphorylation is short lived while ADP-ribosylation is stable. Thus, phosphorylation of the S595/H715 'modifier region' triggers transient interruption of translation while ADP-ribosylation arrests irreversibly.


Assuntos
ADP-Ribosilação , Quinase do Fator 2 de Elongação/metabolismo , Quinase do Fator 2 de Elongação/genética , Humanos , Células MCF-7 , Modelos Moleculares , Fosforilação
18.
Med Sci Sports Exerc ; 51(1): 75-83, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30157103

RESUMO

BACKGROUND: A rapid digestibility and high leucine content are considered important for maximal stimulation of muscle protein synthesis. Consequently, with these properties, native whey may hold greater anabolic potential than milk, when supplemented in combination with strength training. Our aim was to compare the effects of supplementation with milk or native whey, during a 12-wk strength training period, on gains in muscle mass and strength in young adults. METHODS: In this double-blinded, randomized, controlled study a total of 40 untrained young men and women received two daily servings of either milk or native whey containing 20 g of protein, during a 12-wk strength training intervention. Muscle strength, lean mass, thigh muscle cross-sectional area, m. vastus lateralis thickness and muscle fiber cross-sectional area were assessed before and after the training period. In addition, the acute phosphorylation of the anabolic kinases p70S6K, 4E-BP1 and eEF-2 in response to a standardized workout and supplementation was investigated before and after the 12-wk training period. RESULTS: Muscle mass and strength increased, by all measures applied (5%-16%, P < 0.001), with no differences between groups (P > 0.25). p70S6K phosphorylation increased (~1000%, P < 0.02) 2 h after exercise in the untrained and trained state, but no differences in anabolic signaling were observed between supplements (P > 0.40). No correlation between these acute measures and changes in muscle mass or strength were observed. CONCLUSION: Supplementation with milk or native whey during a 12-wk strength training period did not differentially affect muscle mass and strength in young untrained individuals.


Assuntos
Suplementos Nutricionais , Proteínas do Leite/administração & dosagem , Força Muscular/fisiologia , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/fisiologia , Substâncias para Melhoria do Desempenho/administração & dosagem , Treinamento de Resistência , Proteínas do Soro do Leite/administração & dosagem , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Aminoácidos/sangue , Animais , Glicemia/metabolismo , Creatina Quinase/sangue , Método Duplo-Cego , Quinase do Fator 2 de Elongação/metabolismo , Feminino , Humanos , Insulina/sangue , Masculino , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Ureia/sangue , Adulto Jovem
19.
Cell Cycle ; 17(23): 2637-2643, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30507330

RESUMO

The transcription factor p73 is a member of the p53 family, of which the transactivation domain containing isoform (TAp73) plays key roles in brain development and neuronal stem cells. TAp73 also facilitates homoeostasis and prevents oxidative damage in vivo by inducing the expression of its target genes. Recently, we found that in addition to its role in regulation of transcription, TAp73 also affects mRNA translation. In cultured cells, acute TAp73 depletion activates eEF2K, which phosphorylates eEF2 reducing mRNA translation elongation. As a consequence, there is a reduction in global proteins synthesis rates and reprogramming of the translatome, leading to a selective decrease in the translation of rRNA processing factors. Given the dramatic effects of Tap73 depletion in vitro it was important to determine whether similar effects were observed in vivo. Here, we report the surprising finding that in brains of TAp73 KO mice there is a reduced level of eEF2K, which allows protein synthesis rates to be maintained suggesting a compensation model. These data provide new insights to the role of TAp73 in translation regulation and the eEF2K pathway in the brain.


Assuntos
Encéfalo/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Proteína Tumoral p73/genética , Animais , Regulação para Baixo , Camundongos , Camundongos Knockout , Fosforilação , Biossíntese de Proteínas , Proteína Tumoral p73/deficiência , Proteína Tumoral p73/metabolismo
20.
Cell Physiol Biochem ; 51(5): 2136-2147, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30522114

RESUMO

BACKGROUND/AIMS: Long non-coding RNAs (lncRNAs) play vital roles in carcinogenesis as oncogenes or tumor suppressor genes. This study explored the biological function of lncRNA gastric adenocarcinoma predictive long intergenic non-coding RNA (GAPLINC) in human non-small cell lung cancer (NSCLC). METHODS: GAPLINC expression in NSCLC specimens and cell lines was detected by qRT-PCR and Western blot. The effect of GAPLINC on cell proliferation was investigated using CCK8-assay, colony formation assay, and xenograft model. The effects of GAPLINC on apoptosis and cell cycle were determined using flow cytometry. The mechanism of GAPLINC involved in NSCLC was explored using Western blot, luciferase reporter assay, and RNA fluorescence in situ hybridization. RESULTS: We found that GAPLINC expression was up-regulated in NSCLC tissues and cell lines. Overexpression of GAPLINC was associated with poor prognosis in patients with NSCLC. Silencing of GAPLINC significantly inhibited cell proliferation, promoted apoptosis, and induced cell cycle arrest in the G0/G1 phase. Results from xenograft transplantation showed that GAPLINC silencing inhibited the tumor growth in vivo. Interestingly, GAPLINC silencing decreased the expression of eukaryotic elongation factor-2 kinase (eEF2K) protein both in vivo and in vitro. Bioinformatic analysis and luciferase reporter confirmed that miR-661 targeted GAPLINC and eEF2K 3'-UTR and was negatively correlated with the expression of GAPLINC and eEF2K. CONCLUSION: Our findings indicate that GAPLINC promotes NSCLC tumorigenesis by regulating miR-661/eEF2K cascade and provide new insights for the pathogenesis underlying NSCLC and potential targets for therapeutic strategy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Quinase do Fator 2 de Elongação/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transdução de Sinais
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