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1.
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
2.
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
3.
Nanomedicine (Lond) ; 14(17): 2315-2338, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31432749

RESUMO

Aim: To investigate the role of EF2K in BRCA1-mutated breast cancer. Materials & methods: We developed silica coated cobalt-ferrite (CoFe) nanoparticles for in vivo delivery of small interfering RNAs (siRNAs) into BRCA1-mutated breast cancer. Results: Expression of EF2K is highly upregulated in the majority (78.5%) of BRCA1-mutated patients and significantly associated with poor patient survival and metastasis. Silencing of EF2K reduced cell proliferation, migration and invasion of the cancer cells. In vivo therapeutic targeting of EF2K by CoFe-siRNA-nanoparticles leads to sustained EF2K gene knockdown and suppressed tumor growth in orthotopic xenograft models of BRCA1-mutated breast cancer. Conclusion: EF2K is a potential novel molecular target in BRCA1-mutated tumors and CoFe-based siRNA nanotherapy may be used as a novel approach to target EF2K.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/terapia , Quinase do Fator 2 de Elongação/genética , RNA Interferente Pequeno/uso terapêutico , Terapêutica com RNAi/métodos , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Cobalto/química , Feminino , Compostos Férricos/química , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Camundongos Nus , Mutação , Nanomedicina/métodos , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem
4.
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
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.
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
7.
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
8.
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 , Proteínas dos Microfilamentos , Comportamento Social
9.
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
10.
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
11.
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
12.
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
13.
Lung Cancer ; 124: 31-39, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30268477

RESUMO

OBJECTIVES: Lung cancer is the leading cause of cancer related deaths in worldwide. Despite recent advances in treatment options, patient survival has not improved substantially due to lack of commonly expressed molecular targets and effective targeted therapeutics. Thus, better understanding of the biology of lung cancer and identification of novel therapeutic targets are urgently needed for development of highly effective molecularly targeted therapies. MATERIALS AND METHODS: Viability, proliferation and metastatic ability of lung cancer cells were evaluated using methylthiazoltetrazolium (MTT), colony formation and matrigel invasion assays, respectively. Western blotting, RT-PCR, and gene knockdown by siRNA transfections were carried out to investigate the effects of eEF-2K on lung cancer cells. Athymic Nu/Nu mice were treated with liposomal eEF-2KeEF-2K or control siRNA and tumor growth was evaluated in tumor xenograft models of lung cancer. RESULTS AND DISCUSSION: Here, we report that Eukaryotic Elongation Factor-2 kinase (eEF-2K), a member of an atypical alpha kinases family, is significantly upregulated in lung cancer cell lines and its expression is associated with shorter overall patient survival in lung cancer. Inhibition eEF-2K expression by siRNA or a chemical inhibitorsignificantly suppressed lung cancer cell proliferation, colony formation, survival, migration/invasion and tumorigenesis by inhibiting cyclin D1, Src and Mitogen-Activated Protein Kinases/Extracellular Signal-Regulated Kinase (MAPK/ERK) signaling. In vivo targeting of eEF-2K by systemically injected nanoliposomal eEF-2K siRNA resulted in a significant inhibition of lung cancer tumor xenografts in nude mice. Our results suggest, for the first time, that expression of eEF-2K is associated with poor patient prognosis and involved in regulation of critical pathways, including Src and MAPK/ERK and cyclin D1, promoting tumor growth and progression, and thus may be a novel potential therapeutic target in lung cancer.


Assuntos
Adenocarcinoma/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Neoplasias Pulmonares/metabolismo , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Animais , Movimento Celular , Proliferação de Células , Quinase do Fator 2 de Elongação/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Nus , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Breast Cancer Res Treat ; 171(3): 593-605, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29971628

RESUMO

BACKGROUND/PURPOSE: Triple-negative breast cancer (TNBC) is the most aggressive and chemoresistant subtype of breast cancer. Therefore, new molecular targets and treatments need to be developed to improve poor patient prognosis and survival. We have previously shown that eukaryotic elongation factor 2 kinase (eEF-2K) is highly expressed in TNBC cells, is associated with poor patient survival and prognosis, and promotes cell proliferation, migration, and invasion. In vivo targeting of eEF-2K significantly reduces the tumor growth of orthotopic TNBC xenograft mouse models, suggesting that eEF-2K may serve as a potential novel therapeutic target. METHODS/RESULTS: In the current study, we identified thymoquinone (TQ), an active ingredient of Nigella sativa, as a potential safe and effective eEF-2K inhibitor in TNBC. We demonstrated for the first time that TQ inhibits the protein and mRNA expression of eEF-2K, as well as the clinically relevant downstream targets, including Src/FAK and Akt, and induces the tumor suppressor miR-603, in response to NF-kB inhibition. This effect was associated with a significant decrease in the proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, systemic in vivo injection of TQ (20 and 100 mg/kg) significantly reduced the growth of MDA-MB-231 tumors and inhibited the eEF-2K expression in an orthotopic tumor model in mice. CONCLUSION: Our study provides first evidence that TQ treatment inhibits cell proliferation, migration/invasion, and tumor growth, in part through the inhibition of eEF-2K signaling in TNBC. Thus, our findings suggest that systemic TQ treatment may be used as a targeted therapeutic strategy for the inhibition of eEF-2K in TNBC tumor growth and progression.


Assuntos
Benzoquinonas/farmacologia , Proliferação de Células/efeitos dos fármacos , Quinase do Fator 2 de Elongação/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Benzoquinonas/efeitos adversos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Acta Neuropathol Commun ; 6(1): 54, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29961428

RESUMO

Parkinson disease (PD) is the second most common neurodegenerative disorder and the leading neurodegenerative cause of motor disability. Pathologic accumulation of aggregated alpha synuclein (AS) protein in brain, and imbalance in the nigrostriatal system due to the loss of dopaminergic neurons in the substantia nigra- pars compacta, are hallmark features in PD. AS aggregation and propagation are considered to trigger neurotoxic mechanisms in PD, including mitochondrial deficits and oxidative stress. The eukaryotic elongation factor-2 kinase (eEF2K) mediates critical regulation of dendritic mRNA translation and is a crucial molecule in diverse forms of synaptic plasticity. Here we show that eEF2K activity, assessed by immuonohistochemical detection of eEF2 phosphorylation on serine residue 56, is increased in postmortem PD midbrain and hippocampus. Induction of aggressive, AS-related motor phenotypes in a transgenic PD M83 mouse model also increased brain eEF2K expression and activity. In cultures of dopaminergic N2A cells, overexpression of wild-type human AS or the A53T mutant increased eEF2K activity. eEF2K inhibition prevented the cytotoxicity associated with AS overexpression in N2A cells by improving mitochondrial function and reduced oxidative stress. Furthermore, genetic deletion of the eEF2K ortholog efk-1 in C. elegans attenuated human A53T AS induced defects in behavioural assays reliant on dopaminergic neuron function. These data suggest a role for eEF2K activity in AS toxicity, and support eEF2K inhibition as a potential target in reducing AS-induced oxidative stress in PD.


Assuntos
Encéfalo/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Linhagem Celular Tumoral , Modelos Animais de Doenças , Quinase do Fator 2 de Elongação/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Neuroblastoma/patologia , Técnicas de Cultura de Órgãos , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Escleroproteínas/toxicidade , alfa-Sinucleína/genética
16.
Clin Cancer Res ; 24(17): 4225-4241, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29748184

RESUMO

Purpose: Recent studies indicated that dysregulation of noncoding RNAs (ncRNA) such as miRNAs is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC).Experimental Design: We performed in vitro functional assays on TNBC cell lines to investigate the role of miR-34a in FOXM1/eEF2K signaling axis. TNBC tumor xenograft models were used for in vivo therapeutic delivery of miR-34a.Results: In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival. We showed that miR-34a directly binds to the 3'-untranslated region of eEF2K and FOXM1 mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of eEF2K and FOXM1, the transcription factor for eEF2K and the direct target of p53, in TNBC cell lines, whereas overexpression of eEF2K and FOXM1 rescued the effects and signaling pathways mediated by miR-34a. Moreover, in vivo therapeutic delivery of miR-34a nanoparticles by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis.Conclusions: Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a-based gene therapy may be a potential therapeutic strategy in TNBC. Clin Cancer Res; 24(17); 4225-41. ©2018 AACR.


Assuntos
Quinase do Fator 2 de Elongação/genética , Proteína Forkhead Box M1/genética , MicroRNAs/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Apoptose/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Camundongos , Interferência de RNA , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/patologia
17.
J Mol Biol ; 430(17): 2802-2821, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29800565

RESUMO

Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM)-activated α-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca2+ ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry and small-angle X-ray scattering measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen/deuterium exchange mass spectrometry and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Quinase do Fator 2 de Elongação/química , Quinase do Fator 2 de Elongação/metabolismo , Calmodulina/química , Calmodulina/genética , Quinase do Fator 2 de Elongação/genética , Humanos , Cinética , Fosforilação , Conformação Proteica
18.
J Cell Sci ; 131(10)2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29700204

RESUMO

The rate at which ribosomes translate mRNAs regulates protein expression by controlling co-translational protein folding and mRNA stability. Many factors regulate translation elongation, including tRNA levels, codon usage and phosphorylation of eukaryotic elongation factor 2 (eEF2). Current methods to measure translation elongation lack single-cell resolution, require expression of multiple transgenes and have never been successfully applied ex vivo Here, we show, by using a combination of puromycilation detection and flow cytometry (a method we call 'SunRiSE'), that translation elongation can be measured accurately in primary cells in pure or heterogenous populations isolated from blood or tissues. This method allows for the simultaneous monitoring of multiple parameters, such as mTOR or S6K1/2 signaling activity, the cell cycle stage and phosphorylation of translation factors in single cells, without elaborated, costly and lengthy purification procedures. We took advantage of SunRiSE to demonstrate that, in mouse embryonic fibroblasts, eEF2 phosphorylation by eEF2 kinase (eEF2K) mostly affects translation engagement, but has a surprisingly small effect on elongation, except after proteotoxic stress induction.This article has an associated First Person interview with the first author of the paper.


Assuntos
Fibroblastos/citologia , Citometria de Fluxo/métodos , Elongação Traducional da Cadeia Peptídica , Análise de Célula Única/métodos , Animais , Quinase do Fator 2 de Elongação/genética , Quinase do Fator 2 de Elongação/metabolismo , Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biossíntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
19.
Cell Physiol Biochem ; 46(4): 1483-1492, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29689570

RESUMO

BACKGROUND/AIMS: Mitogen-activated protein kinases (MAPKs) are involved in the cellular response to hypoxia and their dysregulation may contribute to the progression and pathology of diverse human renal diseases. Recent studies suggest that the regulation of MAPK responses to hypoxic stress may be different in different cells, even within the same organ. However, it is unclear if MAPKs are differentially regulated in different renal cells in hypoxia. This work was carried out to clarify this fundamental issue. METHODS: We cultured normal rat kidney epithelial (NRK-52E) cells, human kidney epithelial (HK-2) cells and human renal cell adenocarcinoma (769-P) cells simultaneously under normoxia and hypoxia (1% O2) for 24-72 hours. The protein levels of P-ERK1/2, ERK1/2, P-p38, p38 and eEF2K were detected by western blotting. The morphology of all cells was examined using light microscopy. RESULTS: Under the same hypoxic condition, P-ERK1/2 was up-regulated in all renal cells. Meanwhile,P-p38 in NRK-52E cells was markedly increased after hypoxia for 24-72 hours, while it appeared to show no appreciable change in HK-2 and 769-P cells exposed to hypoxia for 24-48 hours and significantly decreased in these cells after 72 hours hypoxia. On the other hand, hypoxia markedly down-regulated the expression of eukaryotic elongation factor-2 kinase (eEF2K) in all three cells. Under microscopy, NRK-52E cells had no visible injury after 72 hours hypoxia, while HK-2 and 769-P cells were mostly damaged under the same condition. CONCLUSIONS: Our data suggest that in response to prolonged hypoxic stress, ERK1/2 and p38 are differentially regulated in three renal cells, while eEF2K is largely down-regulated in all of these cells.


Assuntos
Hipóxia Celular , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Quinase do Fator 2 de Elongação/genética , Quinase do Fator 2 de Elongação/metabolismo , Humanos , Rim/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ratos , Transdução de Sinais
20.
Biol Psychiatry ; 84(1): 65-75, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29395043

RESUMO

BACKGROUND: Ketamine is an N-methyl-D-aspartate receptor antagonist, which on administration produces fast-acting antidepressant responses in patients with major depressive disorder. Yet, the mechanism underlying the antidepressant action of ketamine remains unclear. METHODS: To unravel the mechanism of action of ketamine, we treated wild-type C57BL/6 mice with calcium/calmodulin-dependent protein kinase II (CaMKII) specific inhibitor tatCN21 peptide. We also used eukaryotic elongation factor 2 kinase (eEF2K) (also known as CaMKIII) knockout mice. We analyzed the effects biochemically and behaviorally, using the forced swim, tail suspension, and novelty suppressed feeding tests. RESULTS: Consistent with the literature, one of the major pathways mediating the antidepressant action of ketamine was reduction of phosphorylation of eEF2 via eEF2K. Specifically, knocking out eEF2K in mice eliminated phosphorylation of eEF2 at threonine at position 56, resulting in increased protein synthesis, and made mice resistant both biochemically and behaviorally to the antidepressant effects of ketamine. In addition, administration of ketamine led to differential regulation of CaMKII function, manifested as autoinhibition (pT305 phosphorylation) followed by autoactivation (pT286) of CaMKIIα in the hippocampus and cortex. The inhibition phase of CaMKII, which lasted 10 to 20 minutes after administration of ketamine, occurred concurrently with eEF2K-dependent increased protein synthesis. Moreover, ketamine administration-dependent delayed induction of GluA1 (24 hours) was regulated by the activation of CaMKII. Importantly, systemic administration of the CaMKII inhibitor tatCN21 increased global protein synthesis and induced behavioral resistance to ketamine. CONCLUSIONS: Our data suggest that drugs that selectively target CaMKs and regulate protein synthesis offer novel strategies for treatment of major depressive disorder.


Assuntos
Antidepressivos/uso terapêutico , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Depressão/tratamento farmacológico , Quinase do Fator 2 de Elongação/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ketamina/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quinase do Fator 2 de Elongação/genética , Inibidores Enzimáticos/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Elevação dos Membros Posteriores/psicologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Receptores de AMPA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Natação/psicologia
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