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1.
Life Sci ; 241: 117187, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31863776

RESUMO

AIMS: Renal interstitial fibrosis (RIF) is marked by the epithelial-mesenchymal transition (EMT) and excessive extracellular matrix deposition. The long noncoding RNA myocardial infarction-associated transcript (MIAT) facilitates RIF; however, the molecular mechanism of MIAT in RIF remains unclear. Here, we explored the possible underlying mechanisms through which MIAT modulates RIF. MATERIALS AND METHODS: MIAT expression in human renal fibrotic tissues and unilateral ureteral obstruction (UUO) model mice was detected by qPCR. Transforming growth factor ß1 (TGF-ß1) was introduced to stimulate the EMT in human renal proximal tubular epithelial (HK-2) cells. CCK8, EdU, transwell and wound healing assays were employed to measure cell viability, proliferation, and migration respectively. RNA immunoprecipitation (RIP) assays and dual luciferase reporter assays were applied to determine the relationships among MIAT, miR-145, and EIF5A2. KEY FINDINGS: MIAT was upregulated in human renal fibrotic tissues and UUO model mice compared with normal tissue adjacent to renal tumors and sham operation mice, respectively. MIAT knockdown reduced cell viability, proliferation, migration, and the EMT in HK-2 cells. Additionally, MIAT served as an endogenous sponge for miR-145 in the TGF-ß1-induced-EMT in HK-2 cells, as demonstrated by dual luciferase reporter assays and RIP assays. EIF5A2 was confirmed as a target of miR-145, and MIAT knockdown suppressed EIF5A2 expression by sponging miR-145. Downregulation of EIF5A2 partly reversed induction of the EMT by miR-145 inhibitor transfection. SIGNIFICANCE: MIAT promoted cell viability, proliferation, migration, and the EMT via regulation of the miR-145/EIF5A2 axis. These data established a potential therapy for RIF.


Assuntos
Transição Epitelial-Mesenquimal , Fibrose/patologia , Nefropatias/patologia , MicroRNAs/genética , Fatores de Iniciação de Peptídeos/metabolismo , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Obstrução Ureteral/patologia , Animais , Estudos de Casos e Controles , Fibrose/genética , Fibrose/metabolismo , Regulação da Expressão Gênica , Humanos , Nefropatias/genética , Nefropatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo
2.
Mol Cell ; 76(1): 110-125.e9, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31474573

RESUMO

Failure to make adaptive immune responses is a hallmark of aging. Reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. Here we show that reduced autophagy is a central molecular mechanism underlying immune senescence. Autophagy levels are specifically reduced in mature lymphocytes, leading to compromised memory B cell responses in old individuals. Spermidine, an endogenous polyamine metabolite, induces autophagy in vivo and rejuvenates memory B cell responses. Mechanistically, spermidine post-translationally modifies the translation factor eIF5A, which is essential for the synthesis of the autophagy transcription factor TFEB. Spermidine is depleted in the elderly, leading to reduced TFEB expression and autophagy. Spermidine supplementation restored this pathway and improved the responses of old human B cells. Taken together, our results reveal an unexpected autophagy regulatory mechanism mediated by eIF5A at the translational level, which can be harnessed to reverse immune senescence in humans.


Assuntos
Autofagia/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Senescência Celular/efeitos dos fármacos , Imunossenescência/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Espermidina/farmacologia , Imunidade Adaptativa/efeitos dos fármacos , Fatores Etários , Envelhecimento , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/deficiência , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células HEK293 , Humanos , Memória Imunológica/efeitos dos fármacos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais
3.
J Appl Genet ; 60(3-4): 335-346, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31372832

RESUMO

MicroRNAs (miRNAs) are key regulators that play important biological roles in carcinogenesis and are promising biomarkers for cancer diagnosis and therapy. hsa-miR-375-3p (miR-375) has been suggested to serve as a tumor suppressor or oncogene in various tumor types; however, its specific expression and potential regulatory role in malignant breast cancer remain unclear. In this study, the results from noncoding RNA microarray analysis indicated that the miR-375 expression level is significantly decreased in malignant basal-like breast cancer compared with luminal-like breast cancer. A total of 1895 co-downregulated and 1645 co-upregulated genes were identified in miR-375 mimic-transfected basal-like breast cancer cell lines. Predicted miR-375 targets were obtained from the online databases TargetScan and DIANA-microT-CDS. Combined KEGG enrichment analysis for coregulated genes and predicted miR-375 targets provided information and revealed differences in potential dynamic signaling pathways regulated by miR-375 and also indicated specific regulatory pathways, such as RNA transport and processing, in basal-like breast cancer. Additionally, gene expression microarray analysis accompanied by UALCAN analysis was performed to screen upregulated genes in the basal-like subtype. Four potential key genes, including LDHB, CPNE8, QKI, and EIF5A2, were identified as candidate target genes of miR-375. Therefore, the present study demonstrated that miR-375 may be a potential key regulator and provide a promising direction for diagnostic and therapeutic developments for malignant breast cancer.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Redes Reguladoras de Genes/genética , MicroRNAs/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Biologia Computacional , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Isoenzimas/genética , L-Lactato Desidrogenase/genética , Células MCF-7 , Fatores de Iniciação de Peptídeos/genética , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética
4.
G3 (Bethesda) ; 9(9): 2951-2961, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31292157

RESUMO

Light is an important stimulus for fungi as it regulates many diverse and important biological processes. Metarhizium acridum is an entomopathogenic fungus currently used for the biological control of insect pests. The success of this approach is heavily dependent on tolerance to environmental stresses. It was previously reported that light exposure increases tolerance to ultraviolet radiation in M. acridum There is no information in the literature about how light globally influences gene expression in this fungus. We employed a combination of mRNA-Sequencing and high-throughput proteomics to study how light regulates gene expression both transcriptionally and post-transcriptionally. Mycelium was exposed to light for 5 min and changes at the mRNA and protein levels were followed in time-course experiments for two and four hours, respectively. After light exposure, changes in mRNA abundance were observed for as much as 1128 genes or 11.3% of the genome. However, only 57 proteins changed in abundance and at least 347 significant changes at the mRNA level were not translated to the protein level. We observed that light downregulated subunits of the eukaryotic translation initiation factor 3, the eIF5A-activating enzyme deoxyhypusine hydroxylase, and ribosomal proteins. We hypothesize that light is perceived as a stress by the cell that responds to it by reducing translational activity. Overall, our results indicate that light acts both as a signal and a stressor to M. acridum and highlight the importance of measuring protein levels in order to fully understand light responses in fungi.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Metarhizium/genética , Proteínas Fúngicas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Luz , Metarhizium/fisiologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Micélio/fisiologia , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteômica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico , Espectrometria de Massas em Tandem/métodos , Transcriptoma
5.
Mol Cell ; 74(5): 982-995.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31076285

RESUMO

PIWI-interacting RNAs (piRNAs) silence transposons in Drosophila ovaries, ensuring female fertility. Two coupled pathways generate germline piRNAs: the ping-pong cycle, in which the PIWI proteins Aubergine and Ago3 increase the abundance of pre-existing piRNAs, and the phased piRNA pathway, which generates strings of tail-to-head piRNAs, one after another. Proteins acting in the ping-pong cycle localize to nuage, whereas phased piRNA production requires Zucchini, an endonuclease on the mitochondrial surface. Here, we report that Armitage (Armi), an RNA-binding ATPase localized to both nuage and mitochondria, links the ping-pong cycle to the phased piRNA pathway. Mutations that block phased piRNA production deplete Armi from nuage. Armi ATPase mutants cannot support phased piRNA production and inappropriately bind mRNA instead of piRNA precursors. We propose that Armi shuttles between nuage and mitochondria, feeding precursor piRNAs generated by Ago3 cleavage into the Zucchini-dependent production of Aubergine- and Piwi-bound piRNAs on the mitochondrial surface.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Mitocôndrias/genética , Fatores de Iniciação de Peptídeos/genética , RNA Helicases/genética , RNA Interferente Pequeno/genética , Animais , Drosophila melanogaster/genética , Endorribonucleases/genética , Feminino , Fertilidade/genética , Células Germinativas/metabolismo , Mitocôndrias/metabolismo , Mutação , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Proteínas de Ligação a RNA/genética
6.
J Cell Biol ; 218(6): 1839-1852, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31023722

RESUMO

Importins ferry proteins into nuclei while exportins carry cargoes to the cytoplasm. In the accompanying paper in this issue (Vera Rodriguez et al. 2019. J. Cell Biol. https://doi.org/10.1083/jcb.201812091), we discovered that Pdr6 is a biportin that imports, e.g., the SUMO E2 ligase Ubc9 while depleting the translation factor eIF5A from the nuclear compartment. In this paper, we report the structures of key transport intermediates, namely, of the Ubc9•Pdr6 import complex, of the RanGTP•Pdr6 heterodimer, and of the trimeric RanGTP•Pdr6•eIF5A export complex. These revealed nonlinear transport signals, chaperone-like interactions, and how the RanGTPase system drives Pdr6 to transport Ubc9 and eIF5A in opposite directions. The structures also provide unexpected insights into the evolution of transport selectivity. Specifically, they show that recognition of Ubc9 by Pdr6 differs fundamentally from that of the human Ubc9-importer Importin 13. Likewise, Pdr6 recognizes eIF5A in a nonhomologous manner compared with the mammalian eIF5A-exporter Exportin 4. This suggests that the import of Ubc9 and active nuclear exclusion of eIF5A evolved in different eukaryotic lineages more than once and independently from each other.


Assuntos
Núcleo Celular/metabolismo , Carioferinas/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , beta Carioferinas/química , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Humanos , Carioferinas/química , Carioferinas/genética , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , beta Carioferinas/genética , Proteína ran de Ligação ao GTP/química , Proteína ran de Ligação ao GTP/genética
7.
J Cell Biol ; 218(6): 2006-2020, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31023724

RESUMO

Cleavage of affinity tags by specific proteases can be exploited for highly selective affinity chromatography. The SUMO/SENP1 system is the most efficient for such application but fails in eukaryotic expression because it cross-reacts with endogenous proteases. Using a novel selection system, we have evolved the SUMOEu/SENP1Eu pair to orthogonality with the yeast and animal enzymes. SUMOEu fusions therefore remain stable in eukaryotic cells. Likewise, overexpressing a SENP1Eu protease is nontoxic in yeast. We have used the SUMOEu system in an affinity-capture-proteolytic-release approach to identify interactors of the yeast importin Pdr6/Kap122. This revealed not only further nuclear import substrates such as Ubc9, but also Pil1, Lsp1, eIF5A, and eEF2 as RanGTP-dependent binders and thus as export cargoes. We confirmed that Pdr6 functions as an exportin in vivo and depletes eIF5A and eEF2 from cell nuclei. Thus, Pdr6 is a bidirectional nuclear transport receptor (i.e., a biportin) that shuttles distinct sets of cargoes in opposite directions.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Células HEK293 , Humanos , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , beta Carioferinas/genética
8.
J Biol Chem ; 294(18): 7528-7536, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30910813

RESUMO

The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in vitro analyses to characterize the interactions of one of these translational activators, the pentatricopeptide repeat protein Pet111p, with its presumed target, COX2 mRNA, which encodes subunit II of cytochrome c oxidase. Using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation analysis, we found that Pet111p binds directly and specifically to a 5'-end proximal region of the COX2 transcript. Further, we applied in vitro RNase footprinting and mapped two binding targets of the protein, of which one is located in the 5'-untranslated leader and the other is within the coding sequence. Combined with the available genetic data, these results suggest a plausible mechanism of translational activation, in which binding of Pet111p may prevent inhibitory secondary structures from forming in the translation initiation region, thus rendering the mRNA available for interaction with the ribosome.


Assuntos
Ciclo-Oxigenase 2/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Fatores de Iniciação de Peptídeos/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
9.
Int J Mol Sci ; 20(4)2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795538

RESUMO

Understanding molecular mechanisms of ribosomal translation sheds light on the emergence and evolution of protein synthesis in the three domains of life. Universally, ribosomal translation is described in three steps: initiation, elongation and termination. During initiation, a macromolecular complex assembled around the small ribosomal subunit selects the start codon on the mRNA and defines the open reading frame. In this review, we focus on the comparison of start codon selection mechanisms in eukaryotes and archaea. Eukaryotic translation initiation is a very complicated process, involving many initiation factors. The most widespread mechanism for the discovery of the start codon is the scanning of the mRNA by a pre-initiation complex until the first AUG codon in a correct context is found. In archaea, long-range scanning does not occur because of the presence of Shine-Dalgarno (SD) sequences or of short 5' untranslated regions. However, archaeal and eukaryotic translation initiations have three initiation factors in common: e/aIF1, e/aIF1A and e/aIF2 are directly involved in the selection of the start codon. Therefore, the idea that these archaeal and eukaryotic factors fulfill similar functions within a common structural ribosomal core complex has emerged. A divergence between eukaryotic and archaeal factors allowed for the adaptation to the long-range scanning process versus the SD mediated prepositioning of the ribosome.


Assuntos
Archaea/genética , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/química , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Eucariotos/genética , Evolução Molecular , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo
10.
RNA Biol ; 16(5): 675-685, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30777488

RESUMO

Translation factor a/eIF5A is highly conserved in Eukarya and Archaea. The eukaryal eIF5A protein is required for transit of ribosomes across consecutive proline codons, whereas the function of the archaeal orthologue remains unknown. Here, we provide a first hint for an involvement of Sulfolobus solfataricus (Sso) aIF5A in translation. CRISPR-mediated knock down of the aif5A gene resulted in strong growth retardation, underlining a pivotal function. Moreover, in vitro studies revealed that Sso aIF5A is endowed with endoribonucleolytic activity. Thus, aIF5A appears to be a moonlighting protein that might be involved in protein synthesis as well as in RNA metabolism.


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Sulfolobus solfataricus/crescimento & desenvolvimento , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sistemas CRISPR-Cas , Fatores de Iniciação de Peptídeos/genética , RNA Arqueal/metabolismo , Proteínas de Ligação a RNA/genética , Sulfolobus solfataricus/metabolismo
11.
Int J Biol Sci ; 15(3): 579-586, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30745844

RESUMO

Daunorubicin (Dnr) is at the forefront of acute myeloid leukemia (AML) therapy, but drug resistance poses a major threat to treatment success. MicroRNA (miR)-9 has been shown to have a pivotal role in AML development. However, little is known about the role of miR-9 in Dnr resistance in AML. We explored the potential role of miR-9 in Dnr resistance in AML cells and its mechanism of action. AML cell lines with high half-maximal inhibitory concentration to Dnr in vivo had significantly low miR-9 expression. miR-9 overexpresssion sensitized AML cells to Dnr, inhibited cell proliferation, and enhanced the ability of Dnr to induce apoptosis; miR-9 knockdown had the opposite effects. Mechanistic studies demonstrated that eukaryotic translation initiation factor 5A-2 (EIF5A2) was a putative target of miR-9, which was inversely correlated with the expression and role of miR-9 in AML cells. miR-9 improved the anti-tumor effects of Dnr by inhibiting myeloid cell leukemia-1 (MCL-1) expression, which was dependent on downregulation of EIF5A2 expression. These results suggest that miR-9 has an essential role in Dnr resistance in AML cells through inhibition of the EIF5A2/MCL-1 axis in AML cells. Our data highlight the potential application of miR-9 in chemotherapy for AML patients.


Assuntos
Leucemia Mieloide Aguda/metabolismo , MicroRNAs/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real
12.
Biosci Biotechnol Biochem ; 83(3): 400-408, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30551723

RESUMO

Recently, miR-221-3p expression has been reported to be down-regulated in medulloblastoma (MB), but its functional effects remains unclear. In this study, quantitative real-time PCR (qRT-PCR) revealed significantly decreased miR-221-3p in MB cell lines. Transfection of miR-221-3p mimics reduced, or inhibitor increased cell proliferation in MB cells using MTT assay. Flow cytometry analysis indicated miR-221-3p overexpression promoted, while knockdown alleviated G0/G1 arrest and apoptosis. Luciferase reporter assay confirmed miR-221-3p directly targets the EIF5A2 gene. Moreover, restoration of EIF5A2 in the miR-221-3p-overexpressing DAOY cells significantly alleviated the suppressive effects of miR-221-3p on cell proliferation, cell cycle and apoptosis. Furthermore, miR-221-3p overexpression decreased CDK4, Cyclin D1 and Bcl-2 and increased Bad expression, which was reversed by EIF5A2 overexpression. These results uncovered the tumor suppressive role of miR-221-3p in MB cell proliferation at least in part via targeting EIF5A2, suggesting that miR-221-3p might be a potential candidate target for diagnosis and therapeutics of MB.


Assuntos
Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Neoplasias Cerebelares/patologia , Regulação para Baixo/genética , Meduloblastoma/patologia , MicroRNAs/genética , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos
13.
Mol Biol Rep ; 46(1): 587-596, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30519811

RESUMO

The hormone insulin plays a central role in the metabolism of carbohydrates, lipids, and proteins. In relation to protein metabolism, insulin stimulates amino acid uptake and activates protein synthesis in responsive cells by modulation of signal transduction pathways, such as associated to Akt/PkB, mTOR, S6Ks, 4E-BP1, and several translation initiation/elongation factors. In this context, there is no information on direct cellular treatment with insulin and effects on eukaryotic translation initiation factor 5A (eIF5A) regulation. The eIF5A protein contains an exclusive amino acid residue denominated hypusine, which is essential for its activity and synthesized by posttranslational modification of a specific lysine residue using spermidine as substrate. The eIF5A protein is involved in cellular proliferation and differentiation processes, as observed for satellite cells derived from rat muscles, revealing that eIF5A has an important role in muscle regeneration. The aim of this study was to determine whether eIF5A expression and hypusination are influenced by direct treatment of insulin on L6 myoblast cells. We observed that insulin increased the content of eIF5A transcripts. This effect occurred in cells treated or depleted of fetal bovine serum, revealing a positive insulin effect independent of other serum components. In addition, it was observed that hypusination follows the maintenance of eIF5A protein content in the serum depleted cells and treated with insulin. These results demonstrate that eIF5A is modulated by insulin, contributing the protein synthesis machinery control, as observed by puromycin incorporation in nascent proteins.


Assuntos
Insulina/metabolismo , Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/efeitos dos fármacos , Proteínas de Ligação a RNA/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Insulina/farmacologia , Lisina/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/genética , Ratos , Transdução de Sinais/efeitos dos fármacos
14.
Biol Reprod ; 100(2): 561-572, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30295753

RESUMO

The expression of many genes during the postmeiotic stages of spermatogenesis is largely regulated by germ cell-specific RNA-binding proteins at the level of posttranscription. One of these RNA-binding proteins, YBX2, participates in mRNA storage and regulation of translation in haploid spermatids. How YBX2-stored mRNAs become translationally competent during spermiogenesis remains unknown. In the present study, we report for the first time that YBX2 interacts with PAIP1, a protein translation enhancer, in vitro and in vivo. In murine testes, PAIP1 is highly expressed and colocalizes with YBX2 in round spermatids. Using sequential RNA immunoprecipitation and sequence analysis, we identified a group of spermiogenic mRNAs indirectly bound by PAIP1 through YBX2. Translation of mRNAs bearing the YBX2 target sequence was significantly blocked by YBX2 protein, but was reinitiated when YBX2 was coexpressed with PAIP1 in vitro. Taken together, these results indicate that PAIP1 regulates the translation of YBX2-masked mRNAs during spermiogenesis, and we propose this YBX2-PAIP1 interaction plays an important role in male germ cell development.


Assuntos
Fator de Iniciação 4A em Eucariotos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermatogênese/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Fator de Iniciação 4A em Eucariotos/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Iniciação de Peptídeos/genética , Ligação Proteica , RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Testículo/metabolismo
15.
Int J Mol Sci ; 19(12)2018 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-30551605

RESUMO

A variety of cellular stresses lead to global translation attenuation due to phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2), which decreases the availability of the eIF2-GTP-Met-tRNAi ternary complex. However, a subset of mRNAs continues to be translated by non-canonical mechanisms under these conditions. In fact, although translation initiation of activating transcription factor 4 (ATF4) is normally repressed by an upstream open reading frame (uORF), a decreased availability of ternary complex leads to increased translation of the main ATF4-coding ORF. We show here that siRNA-mediated depletion of eIF5B-which can substitute for eIF2 in delivering Met-tRNAi-leads to increased levels of ATF4 protein in mammalian cells. This de-repression is not due to phosphorylation of eIF2α under conditions of eIF5B depletion. Although eIF5B depletion leads to a modest increase in the steady-state levels of ATF4 mRNA, we show by polysome profiling that the depletion of eIF5B enhances ATF4 expression primarily at the level of translation. Moreover, eIF5B silencing increases the expression of an ATF4-luciferase translational reporter by a mechanism requiring the repressive uORF2. Further experiments suggest that eIF5B cooperates with eIF1A and eIF5, but not eIF2A, to facilitate the uORF2-mediated repression of ATF4 translation.


Assuntos
Fator 4 Ativador da Transcrição/genética , Endonucleases/metabolismo , Fator de Iniciação 1 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Fatores de Iniciação de Peptídeos/genética , Fosforilação , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/genética
16.
Elife ; 72018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30475211

RESUMO

In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNAi in a 'PIN' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codonsin vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection.


Assuntos
Fator de Iniciação 1 em Eucariotos/genética , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação , Códon de Iniciação/genética , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/genética , Regulação Fúngica da Expressão Gênica , Subunidades Ribossômicas Menores de Eucariotos/genética , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura
17.
Physiol Rep ; 6(20): e13893, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30369085

RESUMO

The process of mitochondrial translation, in which mitochondrial (mt)DNA-encoded genes are translated into proteins, is crucial for mitochondrial function and biogenesis. In each phase, a series of mitochondrial translation factors is required for the synthesis of mtDNA-encoded mitochondrial proteins. Two mitochondrial initiation factors (mtIF2 and mtIF3), three mitochondrial elongation factors (mtEFTu, mtEFTs, and mtEFG1), one mitochondrial release factor (mtRF1L), and two mitochondrial recycling factors (mtRRF1 and mtRRF2) are mitochondrial translation factors that coordinate each translational phase. Exercise increases both nuclear DNA- and mtDNA-encoded mitochondrial proteins, resulting in mitochondrial biogenesis in skeletal muscles. Therefore, mitochondrial translation factors are likely regulated by exercise; however, it is unclear whether exercise affects mitochondrial translation factors in the skeletal muscles. We investigated whether exercise training comprehensively increases this series of mitochondrial translation factors, as well as mtDNA-encoded proteins, in the skeletal muscle. Mice were randomly assigned to either the sedentary or exercise group and housed in standard cages with or without a running wheel for 1 and 8 weeks. The expression levels of mitochondrial translation factors in the plantaris and soleus muscles were then measured. Exercise training concomitantly upregulated mitochondrial translation factors and mitochondrial proteins in the plantaris muscle. However, in the soleus muscle, these comprehensive upregulations were not detected. These results indicate that exercise-induced mitochondrial biogenesis coincides with the upregulation of mitochondrial translation factors.


Assuntos
Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Biogênese de Organelas , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Condicionamento Físico Animal , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/genética , Músculo Esquelético/fisiologia , Fatores de Alongamento de Peptídeos/genética , Fatores de Iniciação de Peptídeos/genética , Fatores de Terminação de Peptídeos/genética
18.
Cell Death Dis ; 9(9): 926, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206208

RESUMO

Trophoblast dysfunction is one mechanism implicated in the etiology of recurrent miscarriage (RM). Regulation of trophoblast function, however, is complex and the mechanisms contributing to dysregulation remain to be elucidated. Herein, we found EIF5A1 expression levels to be significantly decreased in cytotrophoblasts in RM villous tissues compared with healthy controls. Using the HTR-8/SVneo cell line as a model system, we found that overexpression of EIF5A1 promotes trophoblast proliferation, migration and invasion in vitro. Knockdown of EIF5A1 or inhibiting its hypusination with N1-guanyl-1,7-diaminoheptane (GC7) suppresses these activities. Similarly, mutating EIF5A1 to EIF5A1K50A to prevent hypusination abolishes its effects on proliferation, migration and invasion. Furthermore, upregulation of EIF5A1 increases the outgrowth of trophoblasts in a villous explant culture model, whereas knockdown has the opposite effect. Suppression of EIF5A1 hypusination also inhibits the outgrowth of trophoblasts in explants. Mechanistically, ARAF mediates the regulation of trophoblast migration and invasion by EIF5A1. Hypusinated EIF5A1 regulates the integrin/ERK signaling pathway via controlling the translation of ARAF. ARAF level is also downregulated in trophoblasts of RM villous tissues and expression of ARAF is positively correlated with EIF5A1. Together, our results suggest that EIF5A1 may be a regulator of trophoblast function at the maternal-fetal interface and low levels of EIF5A1 and ARAF may be associated with RM.


Assuntos
Movimento Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Integrinas/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas A-raf/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trofoblastos/metabolismo , Aborto Habitual/patologia , Linhagem Celular , Proliferação de Células , Feminino , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Fatores de Iniciação de Peptídeos/genética , Gravidez , Proteínas de Ligação a RNA/genética , Transdução de Sinais
19.
Biochem Biophys Res Commun ; 504(1): 6-12, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30170728

RESUMO

Human Papillomavirus (HPV) is considered as the major risk factor for the development and progression of cervical cancer. The high expression of HPV 16 E6 may be the causative factor for induction and maintenance of the transformed phenotype. These oncoproteins would interact with several intracellular proteins, such as eukaryotic translation initiation factor 5A-1 (eIF5A-1) that is essential for proliferation of eukaryotic cells. Our study explored the expression level of HPV 16 E6 and eIF5A-1 in human cervical squamous carcinoma samples and the adjacent non-cancerous cervix samples. Both C33a cells and SiHa cells transfected with a vector encoding HPV 16 E6 resulted in increase of eIF5A-1 expression level and enhancement of viability, migration and proliferation of these cells, furthermore, these effects in both C33a cells and SiHa cells could be abrogated by treatment with eIF5A-1 small-interfering RNA (siRNA) or the specific inhibitors ciclopirox (CPX) that was used to inhibit the function of eIF5A-1 via blocking the main enzymes deoxyhypusine hydroxylase (DOHH). Our results support that the silencing the eIF5A-1 gene or blocking the DOHH could induce the apoptosis of HPV 16 E6-infected cervical carcinoma cells. Thus might provide a new approach to preventing and treating cervical cancer.


Assuntos
Carcinoma de Células Escamosas/virologia , Proteínas Oncogênicas Virais/metabolismo , Fatores de Iniciação de Peptídeos/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/virologia , Apoptose , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Proteínas Oncogênicas Virais/genética , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
20.
J Genet ; 97(4): 953-964, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30262708

RESUMO

The eIF5 protein plays an important role in the fidelity of AUG start codon selection. However, the hyper GTPase eIF5G31R mutation in yeast causes preferential utilization of UUG as initiation codon and is termed as suppressor of initiation codon (Sui-) phenotype. The eIF5G31R mutant recognizes upUUG initiation codon from the 5' regulatory leader region of GCN4 transcript and dominantly represses GCN4 expression thereby conferring sensitivity to 3-amino-1,2,4-triazole (3AT)-induced starvation. The 3AT sensitivity was rescued by supplementing HIS4UUG allele. The eIF5G31R mutant has a better efficiency of UUG codon recognition from the HIS4UUG allele under starvation conditions. Moreover, the expression of HIS4UUG allele was significantly lower than the critical level causing additional derepression of GCN4 expression in eIF5G31R mutant to rescue its 3AT sensitivity. The overexpression of eIF1 improved expression of HIS4AUG allele and GCN4 transcript causing 3AT resistance, whereas overexpression of eIF1 resulted in diminished UUG codon recognition of HIS4UUG allele causing 3AT sensitivity, despite having higher GCN4 expression. This paper reports the critical role of HIS4 expression necessary in response to 3AT-induced starvation in the eIF5G31R mutant which is ostensibly not a direct target of 3AT inhibition.


Assuntos
Oxirredutases do Álcool/genética , Aminoidrolases/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Iniciação de Peptídeos/genética , Pirofosfatases/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Amitrol (Herbicida)/farmacologia , Códon/genética , Códon de Iniciação/genética , Proteínas Ativadoras de GTPase/genética , Regulação Fúngica da Expressão Gênica/genética , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos
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