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1.
Nat Commun ; 11(1): 4676, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938922

RESUMO

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


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

RESUMO

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


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estresse do Retículo Endoplasmático/genética , Fatores de Iniciação em Eucariotos/genética , Biossíntese de Proteínas , Fatores de Transcrição/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Linhagem Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Mutação , Fases de Leitura Aberta , Interferência de RNA , Degeneração Retiniana/genética , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 11(1): 3603, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681107

RESUMO

Members of the PR/SET domain-containing (PRDM) family of zinc finger transcriptional regulators play diverse developmental roles. PRDM10 is a yet uncharacterized family member, and its function in vivo is unknown. Here, we report an essential requirement for PRDM10 in pre-implantation embryos and embryonic stem cells (mESCs), where loss of PRDM10 results in severe cell growth inhibition. Detailed genomic and biochemical analyses reveal that PRDM10 functions as a sequence-specific transcription factor. We identify Eif3b, which encodes a core component of the eukaryotic translation initiation factor 3 (eIF3) complex, as a key downstream target, and demonstrate that growth inhibition in PRDM10-deficient mESCs is in part mediated through EIF3B-dependent effects on global translation. Our work elucidates the molecular function of PRDM10 in maintaining global translation, establishes its essential role in early embryonic development and mESC homeostasis, and offers insights into the functional repertoire of PRDMs as well as the transcriptional mechanisms regulating translation.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Camundongos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Desenvolvimento Embrionário , Células-Tronco Embrionárias/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos/embriologia , Camundongos/genética , Biossíntese de Proteínas , Fatores de Transcrição/genética
4.
Anticancer Res ; 40(6): 3109-3118, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487605

RESUMO

BACKGROUND/AIM: Pancreatic cancer is one of the deadliest forms of cancer and ranks among the leading causes of cancer-related death worldwide. The most common histological type is ductal adenocarcinoma (PDAC), accounting for approximately 95% of cases. Deregulation of protein synthesis has been found to be closely related to cancer. The rate-limiting step of translation is initiation, which is regulated by a broad range of eukaryotic translation initiation factors (eIFs). PATIENTS AND METHODS: Human PDAC samples were biochemically analyzed for the expression of various eIF subunits on the protein level (immunohistochemistry, immunoblot analyses) in 174 cases of PDAC in comparison with non-neoplastic pancreatic tissue (n=10). RESULTS: Our investigation revealed a significant down-regulation of four specific eIF subunits, namely eIF1, eIF2D, eIF3C and eIF6. Concomitantly, the protein (immunoblot) levels of eIF1, eIF2D, eIF3C and eIF6 were reduced in PDAC samples as compared with non-neoplastic pancreatic tissue. CONCLUSION: Members of the eIF family are of relevance in pancreatic tumor biology and may play a major role in translational control in PDAC. Consequently, they might be useful as potential new biomarkers and therapeutic targets in PDAC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Fatores de Iniciação em Eucariotos/genética , Neoplasias Pancreáticas/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Regulação para Baixo , Fatores de Iniciação em Eucariotos/biossíntese , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Serina-Treonina Quinases TOR/metabolismo , Análise Serial de Tecidos
5.
Life Sci ; 255: 117845, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32470449

RESUMO

AIMS: Renal fibrosis is the typical manifestation of progressive kidney disease and causes a severe threat to human health. Surging evidence has illustrated that miRNA plays a core role in the genesis and development of kidney fibrosis. MiR-542-3p has been testified to function as a facilitator in hepatic stellate cell activation and fibrosis. The purpose of study is to investigate the potential of miR-542-3p in renal tubulointerstitial fibrosis. MATERIALS AND METHODS: In this study, to establish renal fibrosis model in vivo and in vitro, we first conducted unilateral ureteral obstruction (UUO) on rats and high glucose (HG) treatment on the HK-2 cells. Histological and western blot analyses were utilized for assessment of renal fibrosis model. Luciferase reporter assay was carried out to explore the regulatory mechanism underlying miR-542-3p in renal fibrosis. KEY FINDINGS: MiR-542-3p was found to be highly expressed in renal fibrosis. Functional experiments revealed that overexpression of miR-542-3p accelerated the deterioration of kidney fibrosis and inhibition of miR-542-3p led to the opposite result. Through the aid of bioinformatics tool, the speculated miR-542-3p binding sites were uncovered in the 3'UTR of argonaute RISC component 1 (AGO1). Mechanism study elucidated that AGO1 was a direct target of miR-542-3p. Lastly, our findings suggested that miR-542-3p played a promoting role in renal fibrosis via repression of AGO1. SIGNIFICANCE: We justified that miR-542-3p induced kidney fibrogenesis both in vivo and in vitro through targeting AGO1, unveiling that miR-542-3p might be a promising option for the treatment of patients with renal fibrosis.


Assuntos
Proteínas Argonauta/genética , Fatores de Iniciação em Eucariotos/genética , Nefropatias/patologia , Rim/patologia , MicroRNAs/genética , Animais , Sítios de Ligação , Linhagem Celular , Biologia Computacional , Modelos Animais de Doenças , Fibrose , Glucose/metabolismo , Humanos , Nefropatias/genética , Masculino , Ratos , Ratos Sprague-Dawley , Obstrução Ureteral/patologia
6.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32040547

RESUMO

Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extramitotic function of CDK1. Different pathways including eIF2α, 4EBP, and S6K1 signaling contribute to controlling global translation downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5'TOP mRNAs, which includes mRNAs encoding ribosomal proteins and several translation factors. This effect requires the 5'TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Thus, CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.


Assuntos
Proteína Quinase CDC2/metabolismo , Proliferação de Células , Neoplasias do Colo do Útero/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Feminino , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinética , Camundongos Endogâmicos C57BL , Fosforilação , Biossíntese de Proteínas , Sequência de Oligopirimidina na Região 5' Terminal do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
7.
Proc Natl Acad Sci U S A ; 117(3): 1429-1437, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31900355

RESUMO

Translation initiation controls protein synthesis by regulating the delivery of the first aminoacyl-tRNA to messenger RNAs (mRNAs). In eukaryotes, initiation is sophisticated, requiring dozens of protein factors and 2 GTP-regulated steps. The GTPase eIF5B gates progression to elongation during the second GTP-regulated step. Using electron cryomicroscopy (cryo-EM), we imaged an in vitro initiation reaction which is set up with purified yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog. A high-resolution reconstruction of a "dead-end" intermediate at 3.6 Šallowed us to visualize eIF5B in its ribosome-bound conformation. We identified a stretch of residues in eIF5B, located close to the γ-phosphate of GTP and centered around the universally conserved tyrosine 837 (Saccharomyces cerevisiae numbering), that contacts the catalytic histidine of eIF5B (H480). Site-directed mutagenesis confirmed the essential role that these residues play in regulating ribosome binding, GTP hydrolysis, and translation initiation both in vitro and in vivo. Our results illustrate how eIF5B transmits the presence of a properly delivered initiator aminoacyl-tRNA at the P site to the distant GTPase center through interdomain communications and underscore the importance of the multidomain architecture in translation factors to sense and communicate ribosomal states.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Guanosina Trifosfato/metabolismo , Iniciação Traducional da Cadeia Peptídica , Sítios de Ligação , Microscopia Crioeletrônica , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Hidrólise , Mutagênese Sítio-Dirigida , Ligação Proteica , Ribossomos/metabolismo , Saccharomyces cerevisiae
8.
Phys Chem Chem Phys ; 22(5): 2938-2948, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31951234

RESUMO

Eukaryotic translation initiation factor 4E binding protein 2 (4E-BP2) is an inhibitor of mRNA cap-dependent translations. Wild-type (WT) 4E-BP2 is intrinsically disordered under physiological conditions, while phosphorylation converts the disordered fragments 18-62 into a four-stranded ß-sheet structure. The regulation mechanism of phosphorylation on 4E-BP2 still remains ambiguous. In this study, replica-exchange molecular dynamics (REMD) simulations were utilized to sample the conformation spaces of WT, phosphorylated WT (pWT), and phosphorylated mutated (pMT) 4E-BP2. Starting from extended structures, the folded structures were only observed in pWT simulations. The folding pathway shows that the folded structures of pWT are formed in the order of ß1/ß4, ß3, and ß2. The formation of ß-turns on pWT, which are driven by hydrogen bonds between the phosphorylated residues and adjacent residues, are the rate-limiting steps in the folding process. The long-range electrostatic interactions contribute toward the stabilization of the folded structures. Moreover, the disruption of ß-turn structures induced by mutations would prevent the folding of pMT 4E-BP2. Our finding is helpful in understanding the regulation of the structural ensembles of intrinsically disordered proteins.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Sequência de Aminoácidos , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Humanos , Ligação de Hidrogênio , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Conformação Proteica em Folha beta , Dobramento de Proteína , Termodinâmica
9.
J Chem Theory Comput ; 16(1): 800-810, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31774674

RESUMO

Upon phosphorylation of specific sites, eukaryotic translation initiation factor 4E (eIF4E) binding protein 2 (4E-BP2) undergoes a fundamental structural transformation from a disordered state to a four-stranded ß-sheet, leading to decreased binding affinity for its partner. This change reflects the significant effects of phosphate groups on the underlying energy landscapes of proteins. In this study, we combine high-temperature molecular dynamics simulations and discrete path sampling to construct energy landscapes for a doubly phosphorylated 4E-BP218-62 and two mutants (a single site mutant D33K and a double mutant Y54A/L59A). The potential and free energy landscapes for these three systems are multifunneled with the folded state and several alternative states lying close in energy, suggesting perhaps a multifunneled and multifunctional protein. Hydrogen bonds between phosphate groups and other residues not only stabilize these low-lying conformations to different extents but also play an important role in interstate transitions. From the energy landscape perspective, our results explain some interesting experimental observations, including the low stability of doubly phosphorylated 4E-BP2 and its moderate binding to eIF4E and the inability of phosphorylated Y54A/L59A to fold.


Assuntos
Fatores de Iniciação em Eucariotos/química , Termodinâmica , Fatores de Iniciação em Eucariotos/genética , Temperatura Alta , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Fosforilação , Mutação Puntual , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica
10.
Int J Mol Sci ; 20(24)2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817792

RESUMO

Whilst the role of eukaryotic translation initiation factors (eIFs) has already been investigated in several human cancers, their role in endometrial cancer (EC) is relatively unknown. In the present retrospective study, 279 patients with EC (1180 samples) were included (mean age: 63.0 years, mean follow-up: 6.1 years). Samples were analysed for expression of 7 eIFs subunits (eIF2α, eIF3c, eIF3h, eIF4e, eIF4g, eIF5, eIF6) through immunohistochemistry and western blotting. Fifteen samples of healthy endometrium served as controls. Density and intensity were assessed and mean combined scores (CS) calculated for each patient. Upon immunohistochemistry, median eIF5 CS were significantly higher in EC as compared with non-neoplastic tissue (NNT, p < 0.001), whilst median eIF6 CS were significantly lower in EC (p < 0.001). Moreover, eIF5 (p = 0.002), eIF6 (p = 0.032) and eIF4g CS (p = 0.014) were significantly different when comparing NNT with EC grading types. Median eIF4g CS was higher in type II EC (p = 0.034). Upon western blot analysis, eIF4g (p < 0.001), peIF2α (p < 0.001) and eIF3h (p < 0.05) were significantly overexpressed in EC, while expression of eIF3c was significantly reduced in EC as compared with NNT (p < 0.001). The remaining eIFs were non-significant. Besides tumour stage (p < 0.001) and patient's age (p < 0.001), high eIF4g CS-levels were independently associated with poor prognosis (HR: 1.604, 95%CI: 1.037-2.483, p = 0.034). The other eIFs had no prognostic significance. Notably, the independent prognostic significance of eIF4g was lost when adding tumour type. Considering the difficulties in differentiating EC type I and II, eIF4g may serve as a novel prognostic marker indicating patient outcome.


Assuntos
Neoplasias do Endométrio/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Idoso , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4G em Eucariotos/genética , Fator de Iniciação 4G em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos
11.
Invest Ophthalmol Vis Sci ; 60(14): 4849-4857, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31747684

RESUMO

Purpose: We reported previously that retinas of mice with inherited retinal degeneration make less protein than retinas of normal mice. Despite recent studies suggesting that diminished protein synthesis rates may contribute to neurologic disorders, a direct link between protein synthesis rates and the progression of neurodegeneration has not been established. Moreover, it remains unclear whether reduced protein synthesis could be involved in retinal pathogenesis. Dysregulation of AKT/mTOR signaling has been reported in the retina during retinal degeneration, but to what extent this signaling contributes to translational attenuation in these mice remains uncertain. Methods: C57BL/6J and rd16 mice were subcutaneously injected with anisomycin to chronically inhibit protein synthesis rates. An AAV2 construct encoding constitutively active 4ebp1 was subretinally delivered in wildtype animals to lower protein synthesis rates. 4ebp1/2 were knocked out in rd16 mice. Results: Anisomycin treatment lowered retinal translation rates, accelerated retinal degeneration in rd16 mice, and initiated cell death in the retinas of C57BL/6J mice. AAV-mediated transfer of constitutively active 4ebp1-4A into the subretinal space of wildtype animals inhibited protein synthesis, and led to reduced electroretinography amplitudes and fewer ONL nuclei. Finally, we report that restoring protein synthesis rates by knocking out 4ebp1/2 was associated with an approximately 2-fold increase in rhodopsin levels and a delay in retinal degeneration in rd16 mice. Conclusions: Our study indicates that protein synthesis inhibition is likely not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but may be harmful to degenerating retinas, and that restoring protein synthesis may have therapeutic potential in delaying the progression of retinal degeneration.


Assuntos
Biossíntese de Proteínas/fisiologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Anisomicina/farmacologia , Proteínas de Ciclo Celular/genética , Morte Celular , Eletrorretinografia , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica/fisiologia , Marcação In Situ das Extremidades Cortadas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parvovirinae/genética , Inibidores da Síntese de Proteínas/farmacologia , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Transfecção
12.
BMC Plant Biol ; 19(1): 440, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640557

RESUMO

BACKGROUND: In plants, host factors encoded by susceptibility (S) genes are indispensable for viral infection. Resistance is achieved through the impairment or the absence of those susceptibility factors. Many S genes have been cloned from model and crop species and a majority of them are coding for members of the eukaryotic translation initiation complex, mainly eIF4E, eIF4G and their isoforms. The aim of this study was to investigate the role of those translation initiation factors in susceptibility of stone fruit species to sharka, a viral disease due to Plum pox virus (PPV). RESULTS: For this purpose, hairpin-inducing silencing constructs based on Prunus persica orthologs were used to generate Prunus salicina (Japanese plum) 4E and 4G silenced plants by Agrobacterium tumefaciens-mediated transformation and challenged with PPV. While down-regulated eIFiso4E transgenic Japanese plums were not regenerated in our conditions, eIFiso4G11-, but not the eIFiso4G10-, silenced plants displayed durable and stable resistance to PPV. We also investigated the alteration of the si- and mi-RNA profiles in transgenic and wild-type Japanese plums upon PPV infection and confirmed that the newly generated small interfering (si) RNAs, which are derived from the engineered inverted repeat construct, are the major contributor of resistance to sharka. CONCLUSIONS: Our results indicate that S gene function of the translation initiation complex isoform is conserved in Prunus species. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of the different isoforms of proteins involved in this complex to breed for resistance to sharka in fruit trees.


Assuntos
Resistência à Doença/genética , Fatores de Iniciação em Eucariotos/metabolismo , Doenças das Plantas/imunologia , Vírus Eruptivo da Ameixa/fisiologia , Prunus/genética , Fatores de Iniciação em Eucariotos/genética , Frutas/genética , Frutas/imunologia , Frutas/virologia , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Isoformas de Proteínas , Prunus/imunologia , Prunus/virologia , Interferência de RNA , RNA de Plantas/genética , RNA Interferente Pequeno/genética , Árvores
13.
J Cancer Res Clin Oncol ; 145(11): 2699-2711, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31586263

RESUMO

BACKGROUND: Gallbladder cancer (GBC) is a rare neoplasia of the biliary tract with high mortality rates and poor prognosis. Signs and symptoms of GBC are not specific and often arise at late stage of disease. For this reason, diagnosis is typically made when the cancer is already in advanced stages, and prognosis for survival is less than 5 years in 90% of cases. Biomarkers to monitor disease progression and novel therapeutic alternative targets for these tumors are strongly required. Commonly, dysregulated protein synthesis contributes to carcinogenesis and cancer progression. In this case, protein synthesis directs translation of specific mRNAs, and, in turn, promotes cell survival, invasion, angiogenesis, and metastasis of tumors. In eukaryotes, protein synthesis is regulated at its initiation, which is a rate-limiting step involving eukaryotic translation initiation factors (eIFs). We hypothesize that eIFs represent crossroads in the development of GBC, and might serve as potential biomarkers. The study focus was the role of eIF6 (an anti-association factor for the ribosomal subunits) in GBC. METHODS: In human GBC samples, the expression of eIF6 was analyzed biochemically at the protein (immunohistochemistry, immunoblot analyses) and mRNA levels (qRT-PCR). RESULTS: High levels of eIF6 correlated with shorter overall survival in biliary tract cancer (BTC) patients (n = 28). Immunohistochemical data from tissue microarrays (n = 114) demonstrated significantly higher expression levels of eIF6 in GBC compared to non-neoplastic tissue. Higher eIF6 expression on protein (immunoblot) and mRNA (qRT-PCR) level was confirmed by analyzing fresh frozen GBC patient samples (n = 14). Depletion of eIF6 (using specific siRNA-mediated knockdown) in Mz-ChA-2 and TFK-1 cell lines inhibited cell proliferation and induced apoptosis. CONCLUSION: Our data indicates that eIF6 overexpression plays a major role in the translational control of GBC, and indicates its potential as a new biomarker and therapeutic target in GBC.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Neoplasias da Vesícula Biliar/patologia , Regulação Neoplásica da Expressão Gênica , Idoso , Apoptose , Proliferação de Células , Fatores de Iniciação em Eucariotos/genética , Feminino , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/metabolismo , Humanos , Masculino , Prognóstico , Células Tumorais Cultivadas
14.
Nature ; 573(7775): 605-608, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31534220

RESUMO

Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors prepare ribosomes for polypeptide synthesis; however, the underlying dynamics of this process remain unclear1,2. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here we use in vitro single-molecule fluorescence microscopy approaches in a purified yeast Saccharomyces cerevisiae translation system to monitor directly, in real time, the pathways of late translation initiation and the transition to elongation. This transition was slower in our eukaryotic system than that reported for Escherichia coli3-5. The slow entry to elongation was defined by a long residence time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individual ribosomal subunits-a process that is catalysed by this universally conserved initiation factor. Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongation. Our findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition from initiation to elongation, and how its release may be governed by a change in the conformation of the ribosome complex that triggers GTP hydrolysis.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Elongação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ativação Enzimática , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Microscopia de Fluorescência , Ligação Proteica , Conformação Proteica , Ribossomos/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética
15.
EMBO J ; 38(16): e100727, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31330067

RESUMO

Translational readthrough generates proteins with extended C-termini, which often possess distinct properties. Here, we have used various reporter assays to demonstrate translational readthrough of AGO1 mRNA. Analysis of ribosome profiling data and mass spectrometry data provided additional evidence for translational readthrough of AGO1. The endogenous readthrough product, Ago1x, could be detected by a specific antibody both in vitro and in vivo. This readthrough process is directed by a cis sequence downstream of the canonical AGO1 stop codon, which is sufficient to drive readthrough even in a heterologous context. This cis sequence has a let-7a miRNA-binding site, and readthrough is promoted by let-7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post-transcriptional gene silencing, due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1.


Assuntos
Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , MicroRNAs/genética , Biossíntese de Proteínas , Proteínas Argonauta/química , Autoantígenos/metabolismo , Sítios de Ligação , Códon de Terminação , Fatores de Iniciação em Eucariotos/química , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais
16.
Dev Cell ; 50(2): 167-183.e8, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31336098

RESUMO

Genome integrity in primordial germ cells (PGCs) is a prerequisite for fertility and species maintenance. In C. elegans, PGCs require global-genome nucleotide excision repair (GG-NER) to remove UV-induced DNA lesions. Failure to remove the lesions leads to the activation of the C. elegans p53, CEP-1, resulting in mitotic arrest of the PGCs. We show that the eIF4E2 translation initiation factor IFE-4 in somatic gonad precursor (SGP) niche cells regulates the CEP-1/p53-mediated DNA damage response (DDR) in PGCs. We determine that the IFE-4 translation target EGL-15/FGFR regulates the non-cell-autonomous DDR that is mediated via FGF-like signaling. Using hair follicle stem cells as a paradigm, we demonstrate that the eIF4E2-mediated niche cell regulation of the p53 response in stem cells is highly conserved in mammals. We thus reveal that the somatic niche regulates the CEP-1/p53-mediated DNA damage checkpoint in PGCs. Our data suggest that the somatic niche impacts the stability of heritable genomes.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Dano ao DNA , Fatores de Iniciação em Eucariotos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Células Germinativas/patologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Reparo do DNA , Fatores de Iniciação em Eucariotos/genética , Feminino , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Proteína Supressora de Tumor p53/genética
17.
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
18.
Cells ; 8(7)2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31248014

RESUMO

After billions of years of evolution, mitochondrion retains its own genome, which gets expressed in mitochondrial matrix. Mitochondrial translation machinery rather differs from modern bacterial and eukaryotic cytosolic systems. Any disturbance in mitochondrial translation drastically impairs mitochondrial function. In budding yeast Saccharomyces cerevisiae, deletion of the gene coding for mitochondrial translation initiation factor 3 - AIM23, leads to an imbalance in mitochondrial protein synthesis and significantly delays growth after shifting from fermentable to non-fermentable carbon sources. Molecular mechanism underlying this adaptation to respiratory growth was unknown. Here, we demonstrate that slow adaptation from glycolysis to respiration in the absence of Aim23p is accompanied by a gradual increase of cytochrome c oxidase activity and by increased levels of Tma19p protein, which protects mitochondria from oxidative stress.


Assuntos
Adaptação Fisiológica , Fatores de Iniciação em Eucariotos/genética , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica/fisiologia , Genes Mitocondriais/genética , Glicólise/fisiologia , Mitocôndrias/genética , Estresse Oxidativo/fisiologia , Biossíntese de Proteínas/fisiologia
19.
J Immunol Res ; 2019: 9124326, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183394

RESUMO

Vaccination is the most effective tool against infectious diseases. Subunit vaccines are safer compared to live-attenuated vaccines but are less immunogenic and need to be delivered with an adjuvant. Adjuvants are essential for enhancing vaccine potency by improving humoral and cell-mediated immune responses. Only a limited number of adjuvants are licensed for human vaccines, and their mode of action is still not clear. Leishmania eukaryotic initiation factor (LeIF) has been described having a dual role, as a natural adjuvant and as an antigen that possesses advantageous immunomodulatory properties. In this study, we assessed the adjuvant properties of recombinant Leishmania infantum eukaryotic initiation factor (LieIF) through in vitro and in vivo assays. LieIF was intraperitoneally administered in combination with the protein antigen ovalbumin (OVA), and the widely used alum was used as a reference adjuvant. Our in vitro studies using J774A.1 macrophages showed that LieIF induced stimulatory effects as demonstrated by the enhanced surface expression of CD80 and CD86 co-stimulatory molecules and the induced production of the immune mediators NO and MIP-1α. Additionally, LieIF co-administration with OVA in an in vivo murine model induced a proinflammatory environment as demonstrated by the elevated expression of TNF-α, IL-1ß, and NF-κB2 genes in peritoneal exudate cells (PEC). Furthermore, PEC derived from OVA-LieIF-immunized mice exhibited elevated expression of CD80 molecule and production of NO and MIP-1α in culture supernatants. Moreover, LieIF administration in the peritoneum of mice resulted in the recruitment of neutrophils and monocytes at 24 h post-injection. Also, we showed that this immunopotentiating effect of LieIF did not depend on the induction of uric acid danger signal. These findings suggest the potential use of LieIF as adjuvant in new vaccine formulations against different infectious diseases.


Assuntos
Adjuvantes Imunológicos , Fatores de Iniciação em Eucariotos/imunologia , Inflamação/imunologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Proteínas de Protozoários/imunologia , Animais , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Fatores de Iniciação em Eucariotos/genética , Feminino , Humanos , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos , Ovalbumina/imunologia , Proteínas de Protozoários/genética , Fator de Necrose Tumoral alfa/metabolismo
20.
J Agric Food Chem ; 67(21): 6007-6018, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31060359

RESUMO

4EBP1 is a chief downstream factor of mTORC1, and PPARγ is a key lipogenesis-related transcription factor. mTORC1 and PPARγ are associated with lipid metabolism. However, it is unknown which effector protein connects mTORC1 and PPARγ. This study investigated the interaction between 4EBP1 with PPARγ as part of the underlying mechanism by which insulin-induced lipid synthesis and secretion are regulated by mTORC1 in primary bovine mammary epithelial cells (pBMECs). Rapamycin, a specific inhibitor of mTORC1, downregulated 4EBP1 phosphorylation and the expression of PPARγ and the following lipogenic genes: lipin 1, DGAT1, ACC, and FAS. Rapamycin also decreased the levels of intracellular triacylglycerol (TAG); 10 types of fatty acid; and the accumulation of TAG, palmitic acid (PA), and stearic acid (SA) in the cell culture medium. Inactivation of mTORC1 by shRaptor or shRheb attenuated the synthesis and secretion of TAG and PA. In contrast, activation of mTORC1 by Rheb overexpression promoted 4EBP1 phosphorylation and PPARγ expression and upregulated the mRNA and protein levels of lipin 1, DGAT1, ACC, and FAS, whereas the levels of intracellular and extracellular TAG, PA, and SA also rose. Further, 4EBP1 interacted directly with PPARγ. Inactivation of mTORC1 by shRaptor prevented the nuclear location of PPARγ. These results demonstrate that mTORC1 regulates lipid synthesis and secretion by inducing the expression of lipin 1, DGAT1, ACC, and FAS, which is likely mediated by the 4EBP1/PPARγ axis. This finding constitutes a novel mechanism by which lipid synthesis and secretion are regulated in pBMECs.


Assuntos
Células Epiteliais/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica , Insulina/metabolismo , Lipogênese , Glândulas Mamárias Animais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , PPAR gama/metabolismo , Animais , Carbono-Carbono Liases/genética , Carbono-Carbono Liases/metabolismo , Bovinos , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Fatores de Iniciação em Eucariotos/genética , Feminino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , PPAR gama/genética , Triglicerídeos/metabolismo
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