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1.
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
2.
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
3.
Gene ; 699: 54-61, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30858133

RESUMO

Epigenetic regulatory changes alter the gene regulation function of DNA repeat elements in cancer and consequently promote malignant phenotypes. Some short tandem repeat sequences, distributed throughout the human genome, can play a role as cis-regulatory elements of the genes. Distributions of tandem long (≥10) and short (<10) A-T repeats in the genome are different depending on gene functions. Long repeats are more commonly found in housekeeping genes and may regulate genes in harmonious fashion. Mononucleotide A-repeats around transcription start sites interact with Argonaute proteins (AGOs) to regulate gene expression. miRNA-bound AGO alterations in cancer have been reported; consequently, these changes would affect genes containing mononucleotide A- and T-repeats. Here, we showed an unprecedented hallmark of gene regulation in cancer. We evaluated the gene expression profiles reported in the Gene Expression Omnibus and found a high density of 13-27 A-T repeats in the up-regulated genes in malignancies derived from the bladder, cervix, head and neck, ovary, vulva, breast, colon, liver, lung, prostate, kidney, thyroid, adrenal gland, bone, blood cells, muscle and brain. Transfection of cell-penetrating protein tag AGO1 containing poly uracils (CPP-AGO1-polyUs) to the lung cancer cell lines altered gene regulation depending on the presence of long A-T repeats. CPP-AGO1-polyUs limited cell proliferation and the ability of a cancer cell to grow into a colony in lung cancer cell lines. In conclusion, long A-T repeats up-regulated many genes in cancer that can be targeted by AGO1 to change the expression of many genes and limited cancer growth.


Assuntos
Proteínas Argonauta/genética , Fatores de Iniciação em Eucariotos/genética , Repetições de Microssatélites/genética , Neoplasias/genética , Transcrição Genética/genética , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , MicroRNAs/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sítio de Iniciação de Transcrição/fisiologia , Transcriptoma/genética , Regulação para Cima/genética
4.
RNA ; 25(5): 620-629, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30770397

RESUMO

The small interfering RNAs (siRNA) or microRNAs (miRNA) incorporated into the RNA-induced silencing complex with the Argonaute (Ago) protein associates with target mRNAs through base-pairing, which leads to the cleavage or knockdown of the target mRNA. The seed region of the s(m)iRNA is crucial for target recognition. In this work, a molecular dynamic simulation was utilized to study the thermodynamics and kinetic properties of the third seed base binding to the target in the presence of the PIWI/MID domain of Ago. The results showed that in the presence of the PIWI/MID domain, the entropy and enthalpy changes for the association of the seed base with the target were smaller than those in the absence of protein. The binding affinity was increased due to the reduced entropy penalty, which resulted from the preorganization of the seed base into the A-helix form. In the presence of the protein, the association barrier resulting from the unfavorable entropy loss and the dissociation barrier coming from the destruction of hydrogen bonding and base-stacking interactions were lower than those in the absence of the protein. These results indicate that the seed region is crucial for fast recognition and association with the correct target.


Assuntos
Proteínas Argonauta/química , Fatores de Iniciação em Eucariotos/química , MicroRNAs/química , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Ligações de Hidrogênio , Cinética , MicroRNAs/genética , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Termodinâmica
5.
Int J Mol Sci ; 20(3)2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717141

RESUMO

Sea urchin early development is a powerful model to study translational regulation under physiological conditions. Fertilization triggers an activation of the translation machinery responsible for the increase of protein synthesis necessary for the completion of the first embryonic cell cycles. The cap-binding protein eIF4E, the helicase eIF4A and the large scaffolding protein eIF4G are assembled upon fertilization to form an initiation complex on mRNAs involved in cap-dependent translation initiation. The presence of these proteins in unfertilized and fertilized eggs has already been demonstrated, however data concerning the translational status of translation factors are still scarce. Using polysome fractionation, we analyzed the impact of fertilization on the recruitment of mRNAs encoding initiation factors. Strikingly, whereas the mRNAs coding eIF4E, eIF4A, and eIF4G were not recruited into polysomes at 1 h post-fertilization, mRNAs for eIF4B and for non-canonical initiation factors such as DAP5, eIF4E2, eIF4E3, or hnRNP Q, are recruited and are differentially sensitive to the activation state of the mechanistic target of rapamycin (mTOR) pathway. We discuss our results suggesting alternative translation initiation in the context of the early development of sea urchins.


Assuntos
Iniciação Traducional da Cadeia Peptídica , Polirribossomos/genética , RNA Mensageiro/genética , Ouriços-do-Mar/genética , Zigoto/metabolismo , Animais , Embrião não Mamífero , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A 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 , Fatores de Iniciação em Eucariotos/metabolismo , Feminino , Fertilização/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Masculino , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , Ouriços-do-Mar/crescimento & desenvolvimento , Ouriços-do-Mar/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Zigoto/citologia , Zigoto/crescimento & desenvolvimento
6.
Int J Mol Sci ; 20(3)2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30764584

RESUMO

The mTOR pathway is in the process of establishing itself as a key access-point of novel oncological drugs and targeted therapies. This is also reflected by the growing number of mTOR pathway genes included in commercially available next-generation sequencing (NGS) oncology panels. This review summarizes the portfolio of medium sized diagnostic, as well as research destined NGS panels and their coverage of the mTOR pathway, including 16 DNA-based panels and the current gene list of Foundation One as a major reference entity. In addition, we give an overview of interesting, mTOR-associated somatic mutations that are not yet incorporated. Especially eukaryotic translation initiation factors (eIFs), a group of mTOR downstream proteins, are on the rise as far as diagnostics and drug targeting in precision medicine are concerned. This review aims to raise awareness for the true coverage of NGS panels, which should be valuable in selecting the ideal platform for diagnostics and research.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Neoplasias/genética , Serina-Treonina Quinases TOR/genética
7.
Brain ; 142(1): 176-192, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30596903

RESUMO

MMP13 (matrix metallopeptidase 13) plays a key role in bone metabolism and cancer development, but has no known functions in Alzheimer's disease. In this study, we used high-throughput small molecule screening in SH-SY5Y cells that stably expressed a luciferase reporter gene driven by the BACE1 (ß-site amyloid precursor protein cleaving enzyme 1) promoter, which included a portion of the 5' untranslated region (5'UTR). We identified that CL82198, a selective inhibitor of MMP13, decreased BACE1 protein levels in cultured neuronal cells. This effect was dependent on PI3K (phosphatidylinositide 3-kinase) signalling, and was unrelated to BACE1 gene transcription and protein degradation. Further, we found that eukaryotic translation initiation factor 4B (eIF4B) played a key role, as the mutation of eIF4B at serine 422 (S422R) or deletion of the BACE1 5'UTR attenuated MMP13-mediated BACE1 regulation. In APPswe/PS1E9 mice, an animal model of Alzheimer's disease, hippocampal Mmp13 knockdown or intraperitoneal CL82198 administration reduced BACE1 protein levels and the related amyloid-ß precursor protein processing, amyloid-ß load and eIF4B phosphorylation, whereas spatial and associative learning and memory performances were improved. Collectively, MMP13 inhibition/CL82198 treatment exhibited therapeutic potential for Alzheimer's disease, via the translational regulation of BACE1.


Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Benzofuranos/uso terapêutico , Disfunção Cognitiva/tratamento farmacológico , Metaloproteinase 13 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Morfolinas/uso terapêutico , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Fatores de Iniciação em Eucariotos/genética , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Oligopeptídeos/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ratos
8.
Proc Natl Acad Sci U S A ; 116(2): 528-533, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30584092

RESUMO

The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR's MCT-1-binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.


Assuntos
Proteínas de Ciclo Celular/química , Fatores de Iniciação em Eucariotos/química , Proteínas Oncogênicas/química , Multimerização Proteica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Estrutura Quaternária de Proteína
9.
Med Sci Monit ; 24: 9151-9165, 2018 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-30556540

RESUMO

BACKGROUND Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA, which has also been identified in somatic tissues, and aberrant expression of piRNAs in tumor tissues may be implicated in carcinogenesis. piR-823 is increased in liver cirrhosis and hepatocellular carcinoma (HCC). However, there is no report on the function of piR-823 in hepatic stellate cells (HSCs) activation during hepatic fibrosis. The present study investigated the role of piR-823 in HSC activation. MATERIAL AND METHODS Liver fibrosis was induced in mice by carbon tetrachloride (CCL4) injection and bile duct ligation (BDL). The primary HSCs were isolated from mice and cultured. The expression of piR-823 was measured by real-time PCR. The effect of piR-823 on HSCs was evaluated by either sense sequence or antisense sequence of piR-823 carried by liposome. Proteins binding to piR-823 were assayed by RNA pull-down technique and liquid chromatography-mass spectrometry (LC-MS). RESULTS Our data for the first time show that piR-823 is significantly upregulated in activated HSCs. Overexpression of piR-823 promoted HSC proliferation, α-SMA and COL1a1 production, whereas inhibition of piR-823 suppressed the activity of HSCs. Interestingly, the combination of piR-823 and EIF3B promoted TGF-ß1 expression. CONCLUSIONS Our data illustrate a novel mechanism of piR-823 in HSC activities. The combination of piR-823 and EIF3B increased TGF-ß1 expression, which activates HSCs in liver fibrosis. piR-823 may be a new target in the treatment of liver fibrosis.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Células Estreladas do Fígado/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cirrose Hepática/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Tetracloreto de Carbono , Proliferação de Células/fisiologia , Fatores de Iniciação em Eucariotos/genética , Células Estreladas do Fígado/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Cultura Primária de Células , RNA Interferente Pequeno/genética , Transdução de Sinais , Ativação Transcricional , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima
10.
Cell Mol Life Sci ; 75(23): 4287-4300, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30019215

RESUMO

The initiator tRNA (Met-tRNA i Met ) at the P site of the small ribosomal subunit plays an important role in the recognition of an mRNA start codon. In bacteria, the initiator tRNA carrier, IF2, facilitates the positioning of Met-tRNA i Met on the small ribosomal subunit. Eukarya contain the Met-tRNA i Met carrier, eIF2 (unrelated to IF2), whose carrier activity is inhibited under stress conditions by the phosphorylation of its α-subunit by stress-activated eIF2α kinases. The stress-resistant initiator tRNA carrier, eIF2A, was recently uncovered and shown to load Met-tRNA i Met on the 40S ribosomal subunit associated with a stress-resistant mRNA under stress conditions. Here, we report that eIF2A interacts and functionally cooperates with eIF5B (a homolog of IF2), and we describe the functional domains of eIF2A that are required for its binding of Met-tRNA i Met , eIF5B, and a stress-resistant mRNA. The results indicate that the eukaryotic eIF5B-eIF2A complex functionally mimics the bacterial IF2 containing ribosome-, GTP-, and initiator tRNA-binding domains in a single polypeptide.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , RNA de Transferência de Metionina/metabolismo , eIF-2 Quinase/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Fator de Iniciação 2 em Eucariotos/genética , Fatores de Iniciação em Eucariotos/genética , Células HEK293 , Humanos , Mutação , Ligação Proteica , Interferência de RNA , RNA de Transferência de Metionina/genética , Homologia de Sequência de Aminoácidos , eIF-2 Quinase/genética
11.
Proc Natl Acad Sci U S A ; 115(26): E6075-E6084, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891689

RESUMO

Photosystem II (PSII), a multisubunit protein complex of the photosynthetic electron transport chain, functions as a water-plastoquinone oxidoreductase, which is vital to the initiation of photosynthesis and electron transport. Although the structure, composition, and function of PSII are well understood, the mechanism of PSII biogenesis remains largely elusive. Here, we identified a nuclear-encoded pentatricopeptide repeat (PPR) protein LOW PHOTOSYNTHETIC EFFICIENCY 1 (LPE1; encoded by At3g46610) in Arabidopsis, which plays a crucial role in PSII biogenesis. LPE1 is exclusively targeted to chloroplasts and directly binds to the 5' UTR of psbA mRNA which encodes the PSII reaction center protein D1. The loss of LPE1 results in less efficient loading of ribosome on the psbA mRNA and great synthesis defects in D1 protein. We further found that LPE1 interacts with a known regulator of psbA mRNA translation HIGH CHLOROPHYLL FLUORESCENCE 173 (HCF173) and facilitates the association of HCF173 with psbA mRNA. More interestingly, our results indicate that LPE1 associates with psbA mRNA in a light-dependent manner through a redox-based mechanism. This study enhances our understanding of the mechanism of light-regulated D1 synthesis, providing important insight into PSII biogenesis and the functional maintenance of efficient photosynthesis in higher plants.


Assuntos
Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Proteínas de Membrana Transportadoras/metabolismo , Complexo de Proteína do Fotossistema II/biossíntese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Iniciação em Eucariotos/genética , Proteínas de Membrana Transportadoras/genética , Complexo de Proteína do Fotossistema II/genética
12.
RNA ; 24(8): 1093-1105, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29844106

RESUMO

tRNA related RNA fragments (tRFs), also known as tRNA-derived RNAs (tdRNAs), are abundant small RNAs reported to be associated with Argonaute proteins, yet their function is unclear. We show that endogenous 18 nucleotide tRFs derived from the 3' ends of tRNAs (tRF-3) post-transcriptionally repress genes in HEK293T cells in culture. tRF-3 levels increase upon parental tRNA overexpression. This represses target genes with a sequence complementary to the tRF-3 in the 3' UTR. The tRF-3-mediated repression is Dicer-independent, Argonaute-dependent, and the targets are recognized by sequence complementarity. Furthermore, tRF-3:target mRNA pairs in the RNA induced silencing complex associate with GW182 proteins, known to repress translation and promote the degradation of target mRNAs. RNA-seq demonstrates that endogenous target genes are specifically decreased upon tRF-3 induction. Therefore, Dicer-independent tRF-3s, generated upon tRNA overexpression, repress genes post-transcriptionally through an Argonaute-GW182 containing RISC via sequence matches with target mRNAs.


Assuntos
Proteínas Argonauta/genética , Autoantígenos/metabolismo , RNA Helicases DEAD-box/genética , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica/genética , RNA de Transferência/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Linhagem Celular , Células HEK293 , Humanos , Processamento de Proteína Pós-Traducional/genética , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética
13.
Parasitol Res ; 117(4): 1291-1296, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29502294

RESUMO

Toxoplasma gondii deploys many effector proteins in order to hijack and manipulate host cell signaling pathways, allowing parasite colonization, subversion of immune responses, and disease progression. T. gondii effector protein 14-3-3 (Tg14-3-3) promotes parasite dissemination inside the body, by enhancing the migratory ability of infected microglia and dendritic cells. Understanding both the mechanism of action and the host targets of Tg14-3-3 effector is important because of their importance to the parasite's virulence. The aim of the present study was to explore the function of Tg14-3-3 by utilizing the yeast two-hybrid system (Y2HS) to identify novel Tg14-3-3 interactors/substrates in host cells. A human cDNA library was screened using Tg14-3-3 as the bait. Tg14-3-3 (RH strain, Type I) was cloned into the pGBKT7 vector and expressed in the Y2HGold yeast strain. The bait protein expression was validated by Western blotting analysis, auto-activation, and toxicity investigation compared with control (Y2HGold yeast strain transformed with empty pGBKT7 vector). Two positive Tg14-3-3 interactors identified by this screening, hCG1821272 and eIF5B (eukaryotic translation initiation factor 5B), were isolated and characterized. This approach made it possible to gain a better understanding of the function of Tg14-3-3 in regulating host proteins involved in key cellular processes, such as translational initiation and cell migration.


Assuntos
Proteínas 14-3-3/genética , Fatores de Iniciação em Eucariotos/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Western Blotting , Biblioteca Gênica , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Toxoplasma/patogenicidade , Toxoplasmose/parasitologia , Toxoplasmose/patologia , Técnicas do Sistema de Duplo-Híbrido
14.
Nat Commun ; 9(1): 829, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29483509

RESUMO

Altered lipid metabolism and aberrant protein translation are strongly associated with cancerous outgrowth; however, the inter-regulation of these key processes is still underexplored in diffuse large B-cell lymphoma (DLBCL). Although fatty acid synthase (FASN) activity is reported to positively correlate with PI3K-Akt-mTOR pathway that can modulate protein synthesis, the precise impact of FASN inhibition on this process is still unknown. Herein, we demonstrate that attenuating FASN expression or its activity significantly reduces eIF4B (eukaryotic initiation factor 4B) levels and consequently overall protein translation. Through biochemical studies, we identified eIF4B as a bonafide substrate of USP11, which stabilizes and enhances eIF4B activity. Employing both pharmacological and genetic approaches, we establish that FASN-induced PI3K-S6Kinase signaling phosphorylates USP11 enhancing its interaction with eIF4B and thereby promoting oncogenic translation.


Assuntos
Fatores de Iniciação em Eucariotos/genética , Ácido Graxo Sintase Tipo I/genética , Regulação Neoplásica da Expressão Gênica , Biossíntese de Proteínas , Proteínas Quinases S6 Ribossômicas/genética , Tioléster Hidrolases/genética , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Fatores de Iniciação em Eucariotos/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Feminino , Humanos , Metabolismo dos Lipídeos/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Tioléster Hidrolases/metabolismo
15.
Gene ; 651: 174-182, 2018 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-29414693

RESUMO

Eukaryotic cells evolved highly complex and accurate protein synthesis machinery that is finely tuned by various signaling pathways. Dysregulation of translation is a hallmark of many diseases, including cancer, and thus pharmacological approaches to modulate translation become very promising. While there has been much progress in our understanding of mammalian mRNA-specific translation control, surprisingly, relatively little is known about whether and how the protein components of the translation machinery shape translation of their own mRNAs. Here we analyze mammalian mRNAs encoding components of the translation initiation machinery for potential regulatory features such as 5'TOP motifs, TISU motifs, poor start codon nucleotide context and upstream open reading frames.


Assuntos
Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas , Animais , Humanos , Mamíferos , Biossíntese de Proteínas , Sequência de Oligopirimidina na Região 5' Terminal do RNA
16.
Cell Rep ; 21(6): 1507-1520, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29117557

RESUMO

Regular endurance training improves muscle oxidative capacity and reduces the risk of age-related disorders. Understanding the molecular networks underlying this phenomenon is crucial. Here, by exploiting the power of computational modeling, we show that endurance training induces profound changes in gene regulatory networks linking signaling and selective control of translation to energy metabolism and tissue remodeling. We discovered that knockdown of the mTOR-independent factor Eif6, which we predicted to be a key regulator of this process, affects mitochondrial respiration efficiency, ROS production, and exercise performance. Our work demonstrates the validity of a data-driven approach to understanding muscle homeostasis.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Exercício , Músculo Esquelético/metabolismo , Acetilação , Animais , Calorimetria , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Metabolismo Energético/fisiologia , Fatores de Iniciação em Eucariotos/deficiência , Fatores de Iniciação em Eucariotos/genética , Redes Reguladoras de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/metabolismo , Condicionamento Físico Animal , Proteoma/análise , Espécies Reativas de Oxigênio/metabolismo , Ribossomos/metabolismo , Espectrometria de Massas em Tandem , Transcrição Genética , Regulação para Cima
17.
PLoS Pathog ; 13(10): e1006694, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-29084265

RESUMO

Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5'UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5' end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity.


Assuntos
Evolução Molecular , Hepacivirus/metabolismo , Hepatite C/metabolismo , MicroRNAs/metabolismo , Tropismo Viral/fisiologia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Linhagem Celular Tumoral , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Hepacivirus/genética , Hepatite C/genética , Humanos , MicroRNAs/genética , Mutagênese
18.
Artigo em Inglês | MEDLINE | ID: mdl-28940716

RESUMO

Apoptotic hemocytes induced by Microplitis bicoloratus parasitism have been reported, and M. bicoloratus bracovirus (MbBV) is known to be the apoptosis inducer. However, the mechanism how MbBV regulates apoptosis remains unclear. eIF4A, one of translation initiation factors, was found from a Spodoptera litura transcriptome, the expression of which in the parasitized hemocytes of S. litura was inhibited in RT-qPCR analysis. The western blot also illustrated eIF4A at 6-day post-parasitization was inhibited in hemocytes. For testing interaction of MbBV-eIF4A-apoptosis, a cDNA clone encoding 1,266 bp of eIF4A was obtained from S. litura hemocytes and sequenced. Then, a 48 kDa V5-fusion protein of the eIF4A was detected by using the anti-V5 antibody at 72-h post-transfection in the High Five cells, which is located in the cell cytoplasm. In vitro, overexpression of eIF4A rescued the apoptotic High Five cells induced by MbBV. Conversely, in vivo, loss of eIF4A proteins by dsRNA feeding increased apoptosis of hemocytes. Furthermore, RNAi and parasitism significantly increased apoptosis of hemocytes in S. litura. These findings suggested that MbBV inhibited the expression of eIF4A, which was required for apoptosis mediated by MbBV. This study will contribute to biological pest control and enhance our understanding of molecular mechanisms underlying polydnavirus-parasitoid-host interaction.


Assuntos
Apoptose/fisiologia , Fatores de Iniciação em Eucariotos/metabolismo , Hemócitos/metabolismo , Vírus dos Insetos/fisiologia , Mariposas/virologia , Sequência de Aminoácidos , Animais , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Mariposas/metabolismo
19.
Mol Cell ; 67(6): 922-935.e5, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28918902

RESUMO

The mechanisms that link environmental and intracellular stimuli to mitochondrial functions, including fission/fusion, ATP production, metabolite biogenesis, and apoptosis, are not well understood. Here, we demonstrate that the nutrient-sensing mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates translation of mitochondrial fission process 1 (MTFP1) to control mitochondrial fission and apoptosis. Expression of MTFP1 is coupled to pro-fission phosphorylation and mitochondrial recruitment of the fission GTPase dynamin-related protein 1 (DRP1). Potent active-site mTOR inhibitors engender mitochondrial hyperfusion due to the diminished translation of MTFP1, which is mediated by translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Uncoupling MTFP1 levels from the mTORC1/4E-BP pathway upon mTOR inhibition blocks the hyperfusion response and leads to apoptosis by converting mTOR inhibitor action from cytostatic to cytotoxic. These data provide direct evidence for cell survival upon mTOR inhibition through mitochondrial hyperfusion employing MTFP1 as a critical effector of mTORC1 to govern cell fate decisions.


Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Dinâmica Mitocondrial , Serina-Treonina Quinases TOR/metabolismo , Apoptose , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Dinaminas/genética , Dinaminas/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/efeitos dos fármacos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Transfecção
20.
Fish Physiol Biochem ; 43(6): 1657-1675, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28913664

RESUMO

This study was conducted to evaluate the effect of enzyme-treated soy protein (ETSP) supplementation in the low-protein diet on growth performance, digestive and absorptive capacities, and related signaling molecules' gene expressions in juvenile Jian carp. The results showed that percent weight gain (PWG), specific growth rate (SGR), and feed intake (FI) were decreased by reducing dietary protein from 34 to 32% (P < 0.05). Supplying low-protein diet with optimal ETSP increased previously mentioned indices of juvenile Jian carp (P < 0.05), which also had no significant difference with the high-protein diet (34%CP) (P > 0.05). Compared with the low-protein diet, appropriate ETSP supplementation in the low-protein diet increased (P < 0.05) (1) the trypsin, lipase, and amylase activities in the hepatopancreas; (2) cholecystokinin concentration in the proximal intestine; (3) the γ-glutamyl transpeptidase (γ-GT), alkaline phosphatase (AKP), and Na+/K+-ATPase activities in all intestinal segments; and (4) the messenger RNA (mRNA) levels of trypsin, lipase, and amylase in hepatopancreas and γ-GT in the mid (MI) and distal (DI) intestine, alkaline phosphatase in MI, and Na+/K+-ATPase and target of rapamycin in all intestinal segments. At the same time, appropriate ETSP supplementation in the low-protein diet downregulated the mRNA levels of AKP in the DI and eIF4E-binding protein 2 in all intestinal segments (P < 0.05). In conclusion, adding 10 g ETSP/kg diet in the low-protein diet can restore the growth performance and digestive and absorptive abilities to the levels in group with 34% dietary protein. Supplementation of optimal ETSP in the low-protein diet enhanced the digestive and absorptive abilities and regulated the signaling molecules related to the TOR signaling pathway.


Assuntos
Carpas/fisiologia , Proteínas na Dieta/administração & dosagem , Digestão/efeitos dos fármacos , Proteínas de Soja/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Ração Animal , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta/veterinária , Suplementos Nutricionais , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética
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