RESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) ranks as the second leading cause of global cancer-related deaths and is characterized by a poor prognosis. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) have been proved to play important roles in various human cancers, whereas the deubiquitination of EEF1A1 was poorly understood. METHODS: The binding and regulatory relationship between Ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) and EEF1A1 was validated using clinical tissue samples, reverse transcription quantitative real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, co-immunoprecipitation, and immunofluorescence, as well as ubiquitin detection and cyclohexamide tracking experiments. Finally, the impact of the UCHL3/EEF1A1 axis on HCC malignant behavior was analyzed through functional experiments and nude mouse models. RESULTS: UCHL3 was found to have a high expression level in HCC tissues. Tissue samples from 60 HCC patients were used to evaluate the correlation between UCHL3 and EEF1A1. UCHL3 binds to EEF1A1 through the lysine site, which reduces the ubiquitination level of EEF1A1. Functional experiments and nude mouse models have demonstrated that the UCHL3/EEF1A1 axis promotes the migration, stemness, and drug resistance of HCC cells. Reducing the expression of EEF1A1 can reverse the effect of UCHL3 on the malignant behavior of HCC cells. CONCLUSION: Our findings revealed that UCHL3 binds and stabilizes EEF1A1 through deubiquitination. UCHL3 and EEF1A1 formed a functional axis in facilitating the malignant progression of HCC, proving new insights for the anti-tumor targeted therapy for HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Fator 1 de Elongação de Peptídeos , Ubiquitina Tiolesterase , Ubiquitinação , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Humanos , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Camundongos , Animais , Camundongos Nus , Progressão da Doença , Linhagem Celular Tumoral , Masculino , FemininoRESUMO
Cellular senescence is characterized by a decrease in protein synthesis, although the underlying processes are mostly unclear. Chemical modifications to transfer RNAs (tRNAs) frequently influence tRNA activity, which is crucial for translation. We describe how tRNA N7-methylguanosine (m7G46) methylation, catalyzed by METTL1-WDR4, regulates translation and influences senescence phenotypes. Mettl1/Wdr4 and m7G gradually diminish with senescence and aging. A decrease in METTL1 causes a reduction in tRNAs, especially those with the m7G modification, via the rapid tRNA degradation (RTD) pathway. The decreases cause ribosomes to stall at certain codons, impeding the translation of mRNA that is essential in pathways such as Wnt signaling and ribosome biogenesis. Furthermore, chronic ribosome stalling stimulates the ribotoxic and integrative stress responses, which induce senescence-associated secretory phenotype. Moreover, restoring eEF1A protein mitigates senescence phenotypes caused by METTL1 deficiency by reducing RTD. Our findings demonstrate that tRNA m7G modification is essential for preventing premature senescence and aging by enabling efficient mRNA translation.
Assuntos
Senescência Celular , Guanosina , Metiltransferases , Biossíntese de Proteínas , RNA de Transferência , Senescência Celular/genética , RNA de Transferência/metabolismo , RNA de Transferência/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Guanosina/análogos & derivados , Guanosina/metabolismo , Metilação , Humanos , Ribossomos/metabolismo , Envelhecimento/metabolismo , Envelhecimento/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Animais , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Estabilidade de RNARESUMO
Herein, we describe the total synthesis of the depsipeptide vioprolide B and of an analogue, in which the (E)-dehydrobutyrine amino acid was replaced by glycine. The compounds were studied in biological assays which revealed cytotoxicity solely for vioprolide B presumably by covalent binding to cysteine residues of elongation factor eEF1A1 and of chromatin assembly factor CHAF1A.
Assuntos
Depsipeptídeos , Glicina , Humanos , Depsipeptídeos/síntese química , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Glicina/análogos & derivados , Glicina/química , Glicina/síntese química , Glicina/farmacologia , Fator 1 de Elongação de Peptídeos/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Estrutura Molecular , AminobutiratosRESUMO
The pathological advancement of osteoporosis is caused by the uneven development of bone marrow-derived mesenchymal stem cells (BMSCs) in terms of osteogenesis and adipogenesis. While the role of EEF1B2 in intellectual disability and tumorigenesis is well established, its function in the bone-fat switch of BMSCs is still largely unexplored. During the process of osteogenic differentiation, we observed an increase in the expression of EEF1B2, while a decrease in its expression was noted during adipogenesis. Suppression of EEF1B2 hindered the process of osteogenic differentiation and mineralization while promoting adipogenic differentiation. On the contrary, overexpression of EEF1B2 enhanced osteogenesis and strongly inhibited adipogenesis. Furthermore, the excessive expression of EEF1B2 in the tibias has the potential to mitigate bone loss and decrease marrow adiposity in mice with osteoporosis. In terms of mechanism, the suppression of ß-catenin activity occurred when EEF1B2 function was suppressed during osteogenesis. Our collective findings indicate that EEF1B2 functions as a regulator, influencing the differentiation of BMSCs and maintaining a balance between bone and fat. Our finding highlights its potential as a therapeutic target for diseases related to bone metabolism.
Assuntos
Adipogenia , Diferenciação Celular , Células-Tronco Mesenquimais , Osteogênese , Osteoporose , Via de Sinalização Wnt , beta Catenina , Animais , Masculino , Camundongos , Adipogenia/genética , beta Catenina/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Células Cultivadas , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Osteogênese/genética , Osteoporose/metabolismo , Osteoporose/patologia , Fator 1 de Elongação de Peptídeos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismoRESUMO
Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.
Assuntos
Diferenciação Celular , Proliferação de Células , Desenvolvimento Muscular , Mioblastos , Fator 1 de Elongação de Peptídeos , Desenvolvimento Muscular/genética , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Animais , Camundongos , Proliferação de Células/genética , Diferenciação Celular/genética , Mioblastos/metabolismo , Mioblastos/citologia , Sistemas CRISPR-Cas , Linhagem Celular , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Given their critical role in plant reproduction and survival, seeds demand meticulous regulatory mechanisms to effectively store and mobilize reserves. Within seeds, the condition of storage reserves heavily depends on environmental stimuli and hormonal activation. Unlike non-protein reserves that commonly employ dedicated regulatory proteins for signaling, proteinaceous reserves may show a unique form of 'self-regulation', amplifying efficiency and precision in this process. Proteins rely on stability to carry out their functions. However, in specific physiological contexts, particularly in seed germination, protein instability becomes essential, fulfilling roles from signaling to regulation. In this study, the elongation factor 1-alpha has been identified as a main proteinaceous reserve in Nicotiana tabacum L. seeds and showed peculiar changes in stability based on tested chemical and physical conditions. A detailed biochemical analysis followed these steps to enhance our understanding of these protein attributes. The protein varied its behavior under different conditions of pH, temperature, and salt concentration, exhibiting shifts within physiological ranges. Notably, distinct solubility transitions were observed, with the elongation factor 1-alpha becoming insoluble upon reaching specific thresholds determined by the tested chemical and physical conditions. The findings are discussed within the context of seed signaling in response to environmental conditions during the key transitions of dormancy and germination.
Assuntos
Nicotiana , Sementes , Nicotiana/metabolismo , Nicotiana/fisiologia , Sementes/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Proteínas de Plantas/metabolismo , Germinação/fisiologia , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide, primarily due to its rapid progression. The current treatment options for PDAC are limited, and a better understanding of the underlying mechanisms responsible for PDAC progression is required to identify improved therapeutic strategies. In this study, we identified FBXO32 as an oncogenic driver in PDAC. FBXO32 was aberrantly upregulated in PDAC, and high FBXO32 expression was significantly associated with an unfavorable prognosis in patients with PDAC. FRG1 deficiency promoted FBXO32 upregulation in PDAC. FBXO32 promoted cell migration and invasion in vitro and tumor growth and metastasis in vivo. Mechanistically, FBXO32 directly interacted with eEF1A1 and promoted its polyubiquitination at the K273 site, leading to enhanced activity of eEF1A1 and increased protein synthesis in PDAC cells. Moreover, FBXO32-catalyzed eEF1A1 ubiquitination boosted the translation of ITGB5 mRNA and activated focal adhesion kinase (FAK) signaling, thereby facilitating focal adhesion assembly and driving PDAC progression. Importantly, interfering with the FBXO32-eEF1A1 axis or pharmaceutical inhibition of FAK by defactinib, an FDA-approved FAK inhibitor, substantially inhibited PDAC growth and metastasis driven by aberrantly activated FBXO32-eEF1A1 signaling. Overall, this study uncovers a mechanism by which PDAC cells rely on FBXO32-mediated eEF1A1 activation to drive progression and metastasis. FBXO32 may serve as a promising biomarker for selecting eligible patients with PDAC for treatment with defactinib. Significance: FBXO32 upregulation in pancreatic cancer induced by FRG1 deficiency increases eEF1A1 activity to promote ITGB5 translation and stimulate FAK signaling, driving cancer progression and sensitizing tumors to the FAK inhibitor defactinib.
Assuntos
Carcinoma Ductal Pancreático , Progressão da Doença , Proteínas F-Box , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Camundongos , Animais , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Linhagem Celular Tumoral , Camundongos Nus , Movimento Celular , Ubiquitinação , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica , Masculino , Feminino , Prognóstico , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Metástase Neoplásica , Proteínas Musculares , Proteínas Ligases SKP Culina F-BoxRESUMO
Colorectal cancer (CRC) is the third most common cancer and causes high mortality worldwide. Although CRC has been studied widely, the molecular mechanism is not completely known. Eukaryotic translation elongation factor 1 delta (EEF1D) participates in the progression of various tumors, however, the effect of EEF1D on CRC remains unclear. Here, we aimed to identify the potential mechanism of EEF1D in CRC. The expression levels of EEF1D were assessed in CRC samples. Functional analysis of EEF1D in CRC was detected in vitro and in vivo. The regulatory mechanism of EEF1D was identified with RNA immunoprecipitation, RNA pull-down assay, and proteomics analysis. Our findings confirmed that EEF1D was upregulated in human CRC tissues. Functionally, EEF1D overexpression accelerated cell proliferation and metastasis, whereas EEF1D knockdown inhibited cell proliferation and metastasis both in vitro and in vivo CRC models. Furthermore, we showed that EEF1D was upregulated by SRSF9 via binding to 3'UTR of EEF1D mRNA. EEF1D knockdown reversed the malignant phenotype induced by SRSF9 overexpression. These findings demonstrated that EEF1D promotes CRC progression, and EEF1D may be a molecular target against CRC.
Assuntos
Proliferação de Células , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Fator 1 de Elongação de Peptídeos , Fatores de Processamento de Serina-Arginina , Animais , Feminino , Humanos , Masculino , Camundongos , Regiões 3' não Traduzidas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Camundongos Nus , Metástase Neoplásica , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Regulação para CimaRESUMO
Rapid accumulation of boron (B) leads to toxicity in plant tissues, and the narrow gap between deficiency and toxicity makes it difficult to adjust essential B levels in soil for plant productivity. Therefore, understanding different aspects of B tolerance is necessary to provide new and valid solutions to B toxicity. Gypsophila perfoliata stands out as a remarkable example of a B-tolerant plant, with a natural propensity to thrive in environments such as B mines and soils enriched with high levels of B. In this study, a yeast functional screening experiment was conducted using cDNA libraries from G. perfoliata leaf and root cells for B tolerance. Ten colonies from the leaf library grew in 80 mm boric acid, while none emerged from the root library. Analysis of isolated cDNAs showed identical sequences and a unique motif related to B tolerance. The gene GpEF1A was identified in the tolerant yeast colonies, with predicted structural features suggesting its role, and RT-qPCR indicating increased expression under B stress. A regulatory role for EF1A lysine methylation was proposed in mammalian cells and fungi because of its dynamic and inducible nature under environmental constraints. This could also be relevant for plant cells, as the high similarity of the GpEF1A gene in some salt-tolerant plants might indicate the upregulation of EF1A as a conserved way to cope with abiotic stress conditions. This report represents the first instance of involvement of GpEF1A in B tolerance, and further detailed studies are necessary to understand other components of this tolerance mechanism.
Assuntos
Boro , Caryophyllaceae , Homeostase , Boro/metabolismo , Boro/toxicidade , Caryophyllaceae/genética , Caryophyllaceae/metabolismo , Caryophyllaceae/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Sequência de Aminoácidos , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Biblioteca GênicaRESUMO
Amino acids are essential for the activation of the mechanistic target of rapamycin (mTOR), but the corresponding molecular mechanism is not yet fully understood. We previously found that Met stimulated eukaryotic elongation factor α (eEF1Bα) nuclear localization in bovine mammary epithelial cells (MECs). Herein, we explored the role and molecular mechanism of eEF1Bα in methionine (Met)- and leucine (Leu)-stimulated mTOR gene transcription and milk synthesis in MECs. eEF1Bα knockdown decreased milk protein and fat synthesis, cell proliferation, and mTOR mRNA expression and phosphorylation, whereas eEF1Bα overexpression had the opposite effects. QE-MS analysis detected that eEF1Bα was phosphorylated at Ser106 in the nucleus and Met and Leu stimulated p-eEF1Bα nuclear localization. eEF1Bα knockdown abrogated the stimulation of Met and Leu by mTOR mRNA expression and phosphorylation, and this regulatory role was dependent on its phosphorylation. Akt knockdown blocked the stimulation of Met and Leu by eEF1Bα and p-eEF1Bα expression. ChIP-PCR detected that p-eEF1Bα bound only to the -548 to -793 nt site in the mTOR promoter, and ChIP-qPCR further detected that Met and Leu stimulated this binding. eEF1Bα mediated Met and Leu' stimulation on mTOR mRNA expression and phosphorylation through inducing AT-rich interaction domain 1A (ARID1A) ubiquitination degradation, and this process depended on eEF1Bα phosphorylation. p-eEF1Bα interacted with ARID1A and ubiquitin protein ligase E3 module N-recognition 5 (UBR5), and UBR5 knockdown rescued the decrease of the ARID1A protein level by eEF1Bα overexpression. Both eEF1Bα and p-eEF1Bα were highly expressed in mouse mammary gland tissues during the lactating period. In summary, we reveal that Met and Leu stimulate mTOR transcriptional activation and milk protein and fat synthesis in MECs through eEF1Bα-UBR5-ARID1A signaling.
Assuntos
Células Epiteliais , Leucina , Metionina , Leite , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Bovinos , Feminino , Camundongos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Leucina/farmacologia , Leucina/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/citologia , Metionina/metabolismo , Metionina/farmacologia , Leite/química , Leite/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoAssuntos
Oryza , Doenças das Plantas , Imunidade Vegetal , Proteínas de Plantas , Resistência à Doença/imunologia , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Oryza/imunologia , Oryza/genética , Oryza/microbiologia , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMO
Patients with triple-negative breast cancer (TNBC) have a poor prognosis due to the lack of effective molecular targets for therapeutic intervention. Here we found that the long noncoding RNA (lncRNA) MILIP supports TNBC cell survival, proliferation, and tumorigenicity by complexing with transfer RNAs (tRNA) to promote protein production, thus representing a potential therapeutic target in TNBC. MILIP was expressed at high levels in TNBC cells that commonly harbor loss-of-function mutations of the tumor suppressor p53, and MILIP silencing suppressed TNBC cell viability and xenograft growth, indicating that MILIP functions distinctively in TNBC beyond its established role in repressing p53 in other types of cancers. Mechanistic investigations revealed that MILIP interacted with eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) and formed an RNA-RNA duplex with the type II tRNAs tRNALeu and tRNASer through their variable loops, which facilitated the binding of eEF1α1 to these tRNAs. Disrupting the interaction between MILIP and eEF1α1 or tRNAs diminished protein synthesis and cell viability. Targeting MILIP inhibited TNBC growth and cooperated with the clinically available protein synthesis inhibitor omacetaxine mepesuccinate in vivo. Collectively, these results identify MILIP as an RNA translation elongation factor that promotes protein production in TNBC cells and reveal the therapeutic potential of targeting MILIP, alone and in combination with other types of protein synthesis inhibitors, for TNBC treatment. SIGNIFICANCE: LncRNA MILIP plays a key role in supporting protein production in TNBC by forming complexes with tRNAs and eEF1α1, which confers sensitivity to combined MILIP targeting and protein synthesis inhibitors.
Assuntos
Proliferação de Células , Fator 1 de Elongação de Peptídeos , Biossíntese de Proteínas , RNA Longo não Codificante , RNA de Transferência , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Humanos , Feminino , RNA de Transferência/genética , RNA de Transferência/metabolismo , Animais , Camundongos , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Regulação Neoplásica da Expressão GênicaRESUMO
Eukaryotic elongation factor 1A (eEF1A) is among the most abundant proteins in eukaryotic cells. Evolutionarily conserved across species, eEF1A is in charge of translation elongation for protein biosynthesis as well as a plethora of non-translational moonlighting functions for cellular homeostasis. In malignant cells, however, eEF1A becomes a pleiotropic driver of cancer progression via a broad diversity of pathways, which are not limited to hyperactive translational output. In the past decades, mounting studies have demonstrated the causal link between eEF1A and carcinogenesis, gaining deeper insights into its multifaceted mechanisms and corroborating its value as a prognostic marker in various cancers. On the other hand, an increasing number of natural and synthetic compounds were discovered as anticancer eEF1A-targeting inhibitors. Among them, plitidepsin was approved for the treatment of multiple myeloma whereas metarrestin was currently under clinical development. Despite significant achievements in these two interrelated fields, hitherto there lacks a systematic examination of the eEF1A protein in the context of cancer research. Therefore, the present work aims to delineate its clinical implications, molecular oncogenic mechanisms, and targeted therapeutic strategies as reflected in the ever expanding body of literature, so as to deepen mechanistic understanding of eEF1A-involved tumorigenesis and inspire the development of eEF1A-targeted chemotherapeutics and biologics.
Assuntos
Antineoplásicos , Neoplasias , Fator 1 de Elongação de Peptídeos , Humanos , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Relevância ClínicaRESUMO
The translation elongation factor 1α (EF1α) protein is a highly conserved G protein that is crucial for protein translation in all eukaryotic organisms. EF1α quickly became insoluble at temperatures 42 °C treatment for 2h in vitro, but generally remained soluble in vivo even after being exposed to temperatures as high as 45 °C for an extended period, which suggests that protective mechanisms exist for keeping EF1α soluble in plant cells under heat stress. EF1α had fast in vivo insolubilization when exposed to 45 °C, resulting in about 40% of the protein aggregating after 9 h. Given its established role in protein translation, heat-induced aggregation is most likely to impact the function of the elongation factor. Overexpression of constitutive mutants in both GTP-bound and GDP-bound forms of EF1α resulted in significantly decreased heat tolerance. These findings provide evidence to support the critical role of EF1α, a thermosensitive protein, in the heat tolerance of plants.
Assuntos
Fator 1 de Elongação de Peptídeos , Termotolerância , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Termotolerância/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Temperatura Alta , Agregados Proteicos , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Resposta ao Choque Térmico/fisiologiaRESUMO
BACKGROUND: Ischemic stroke (IS) is a serious cerebrovascular disease characterized by significantly elevated mortality and disability rates, and the treatments available for this disease are limited. Neuroinflammation and oxidative stress are deemed the major causes of cerebral ischemic injury. N-Cinnamoylpyrrole alkaloids form a small group of natural products from the genus Piper and have not been extensively analyzed pharmacologically. Thus, identifying the effect and mechanism of N-cinnamoylpyrrole-derived alkaloids on IS is worthwhile. PURPOSE: The present research aimed to explore the antineuroinflammatory and antioxidative stress effects of N-cinnamoylpyrrole-derived alkaloids isolated from the genus Piper and to explain the effects and mechanism on IS. METHODS: N-cinnamoylpyrrole-derived alkaloids were isolated from Piper boehmeriaefolium var. tonkinense and Piper sarmentosum and identified by various chromatographic methods. Lipopolysaccharide (LPS)-induced BV-2 microglia and a mouse model intracerebroventricularly injected with LPS were used to evaluate the antineuroinflammatory and antioxidative stress effects. Oxygenâglucose deprivation/reperfusion (OGD/R) and transient middle cerebral artery occlusion (tMCAO) models were used to evaluate the effect of PB-1 on IS. To elucidate the fundamental mechanism, the functional target of PB-1 was identified by affinity-based protein profiling (ABPP) strategy and verified by cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), and circular dichroism (CD) analyses. The effect of PB-1 on the NF-κB and NRF2 signaling pathways was subsequently evaluated via western blotting and immunofluorescence staining. RESULTS: The results showed that N-cinnamoylpyrrole-derived alkaloids significantly affected neuroinflammation and oxidative stress. The representative compound, PB-1 not only inhibited neuroinflammation and oxidative stress induced by LPS or OGD/R insult, but also alleviated cerebral ischemic injury induced by tMCAO. Further molecular mechanism research found that PB-1 promoted antineuroinflammatory and antioxidative stress activities via the NF-κB and NRF2 signaling pathways by targeting eEF1A1. CONCLUSION: Our research initially unveiled that the therapeutic impact of PB-1 on cerebral ischemic injury might rely on its ability to target eEF1A1, leading to antineuroinflammatory and antioxidative stress effects. The novel discovery highlights eEF1A1 as a potential target for IS treatment and shows that PB-1, as a lead compound that targets eEF1A1, may be a promising therapeutic agent for IS.
Assuntos
Alcaloides , AVC Isquêmico , Piper , Pirróis , Animais , Masculino , Camundongos , Alcaloides/farmacologia , Alcaloides/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Antioxidantes/farmacologia , Antioxidantes/química , Modelos Animais de Doenças , AVC Isquêmico/tratamento farmacológico , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Piper/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Pirróis/farmacologia , Pirróis/química , Cinamatos/química , Cinamatos/farmacologia , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Fator 1 de Elongação de Peptídeos/metabolismoRESUMO
The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3ß, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.
Assuntos
Aurora Quinase A , Neoplasias da Mama , Neoplasias Mamárias Animais , PTEN Fosfo-Hidrolase , Fator 1 de Elongação de Peptídeos , Animais , Feminino , Humanos , Camundongos , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína 7 com Repetições F-Box-WD/genética , Glicogênio Sintase Quinase 3 beta , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismoRESUMO
Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production in the absence of Chp1 triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult-to-fold protein that necessitates a biogenesis pathway starting with dedicated folding factor Chp1 at the ribosome to protect the eukaryotic cell from proteostasis collapse.
Assuntos
Proteínas de Ligação ao Cálcio , Chaperonas Moleculares , Fator 1 de Elongação de Peptídeos , Dobramento de Proteína , Ribossomos , Biossíntese de Proteínas , Proteostase , Ribossomos/genética , Ribossomos/metabolismo , Humanos , Proteínas de Ligação ao Cálcio/metabolismo , Chaperonas Moleculares/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismoRESUMO
Eukaryotic elongation factor 1A1 (EEF1A1) is canonically involved in protein synthesis but also has noncanonical functions in diverse cellular processes. Previously, we identified EEF1A1 as a mediator of lipotoxicity and demonstrated that chemical inhibition of EEF1A1 activity reduced mouse liver lipid accumulation. These findings suggested a link between EEF1A1 and metabolism. Therefore, we investigated its role in regulating metabolic substrate preference. EEF1A1-deficient Chinese hamster ovary (2E2) cells displayed reduced media lactate accumulation. These effects were also observed with EEF1A1 knockdown in human hepatocyte-like HepG2 cells and in WT Chinese hamster ovary and HepG2 cells treated with selective EEF1A inhibitors, didemnin B, or plitidepsin. Extracellular flux analyses revealed decreased glycolytic ATP production and increased mitochondrial-to-glycolytic ATP production ratio in 2E2 cells, suggesting a more oxidative metabolic phenotype. Correspondingly, fatty acid oxidation was increased in 2E2 cells. Both 2E2 cells and HepG2 cells treated with didemnin B exhibited increased neutral lipid content, which may be required to support elevated oxidative metabolism. RNA-seq revealed a >90-fold downregulation of a rate-limiting glycolytic enzyme, hexokinase 2, which we confirmed through immunoblotting and enzyme activity assays. Pathway enrichment analysis identified downregulations in TNFA signaling via NFKB and MYC targets. Correspondingly, nuclear abundances of RELB and MYC were reduced in 2E2 cells. Thus, EEF1A1 deficiency may perturb glycolysis by limiting NFKB- and MYC-mediated gene expression, leading to decreased hexokinase expression and activity. This is the first evidence of a role for a translation elongation factor, EEF1A1, in regulating metabolic substrate utilization in mammalian cells.
Assuntos
Hexoquinase , Fator 1 de Elongação de Peptídeos , Animais , Cricetinae , Humanos , Trifosfato de Adenosina , Linhagem Celular , Cricetulus , Hexoquinase/genética , Hexoquinase/metabolismo , Lipídeos , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/metabolismo , Glicólise , Oxirredução , Movimento Celular , Proliferação de Células , Metabolismo dos LipídeosRESUMO
Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. There is no specific treatment for FXS due to the lack of therapeutic targets. We report here that Elongation Factor 1α (EF1α) forms a complex with two other proteins: Tripartite motif-containing protein 3 (TRIM3) and Murine double minute (Mdm2). Both EF1α-Mdm2 and EF1α-TRIM3 protein complexes are increased in the brain of Fmr1 knockout mice as a result of FMRP deficiency, which releases the normal translational suppression of EF1α mRNA and increases EF1α protein levels. Increased EF1α-Mdm2 complex decreases PSD-95 ubiquitination (Ub-PSD-95) and Ub-PSD-95-C1q interaction. The elevated level of TRIM3-EF1α complex is associated with decreased TRIM3-Complement Component 3 (C3) complex that inhibits the activation of C3. Both protein complexes thereby contribute to a reduction in microglia-mediated phagocytosis and dendritic spine pruning. Finally, we created a peptide that disrupts both protein complexes and restores dendritic spine plasticity and behavioural deficits in Fmr1 knockout mice. The EF1α-Mdm2 and EF1α-TRIM3 complexes could thus be new therapeutic targets for FXS.
Assuntos
Espinhas Dendríticas , Proteína do X Frágil da Deficiência Intelectual , Camundongos Knockout , Microglia , Plasticidade Neuronal , Fator 1 de Elongação de Peptídeos , Fagocitose , Animais , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Microglia/metabolismo , Camundongos , Plasticidade Neuronal/fisiologia , Espinhas Dendríticas/metabolismo , Fagocitose/fisiologia , Fator 1 de Elongação de Peptídeos/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/genética , Camundongos Endogâmicos C57BL , Masculino , Encéfalo/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Ubiquitinação , Complemento C3/metabolismoRESUMO
The archaeal ancestor of eukaryotes apparently belonged to the phylum Asgardarchaeota, but the ecology and evolution of Asgard archaea are poorly understood. The optimal GDP-binding temperature of a translation elongation factor (EF-1A or EF-Tu) has been previously shown to correlate with the optimal growth temperature of diverse prokaryotes. Here, we reconstruct ancestral EF-1A sequences and experimentally measure the optimal GDP-binding temperature of EF-1A from ancient and extant Asgard archaea, to infer the evolution of optimal growth temperatures in Asgardarchaeota. Our results suggest that the Asgard ancestor of eukaryotes was a moderate thermophile, with an optimal growth temperature around 53 °C. The origin of eukaryotes appears to coincide with a transition from thermophilic to mesophilic lifestyle during the evolution of Asgard archaea.