RESUMEN
Diphthamide is a modified histidine residue unique for eukaryotic translation elongation factor 2 (eEF2), a key ribosomal protein. Loss of this evolutionarily conserved modification causes developmental defects through unknown mechanisms. In a patient with compound heterozygous mutations in Diphthamide Biosynthesis 1 (DPH1) and impaired eEF2 diphthamide modification, we observe multiple defects in neural crest (NC)-derived tissues. Knockin mice harboring the patient's mutations and Xenopus embryos with Dph1 depleted also display NC defects, which can be attributed to reduced proliferation in the neuroepithelium. DPH1 depletion facilitates dissociation of eEF2 from ribosomes and association with p53 to promote transcription of the cell cycle inhibitor p21, resulting in inhibited proliferation. Knockout of one p21 allele rescues the NC phenotypes in the knockin mice carrying the patient's mutations. These findings uncover an unexpected role for eEF2 as a transcriptional coactivator for p53 to induce p21 expression and NC defects, which is regulated by diphthamide modification.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Histidina , Histidina/análogos & derivados , Antígenos de Histocompatibilidad Menor , Cresta Neural , Factor 2 de Elongación Peptídica , Proteína p53 Supresora de Tumor , Proteínas Supresoras de Tumor , Animales , Cresta Neural/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Humanos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Ratones , Factor 2 de Elongación Peptídica/metabolismo , Factor 2 de Elongación Peptídica/genética , Histidina/metabolismo , Ribosomas/metabolismo , Mutación , Proliferación Celular , Xenopus laevis , Femenino , Técnicas de Sustitución del Gen , Xenopus , Masculino , Ratones NoqueadosRESUMEN
eEF2 post-translational modifications (PTMs) can profoundly affect mRNA translation dynamics. However, the physiologic function of eEF2K525 trimethylation (eEF2K525me3), a PTM catalyzed by the enzyme FAM86A, is unknown. Here, we find that FAM86A methylation of eEF2 regulates nascent elongation to promote protein synthesis and lung adenocarcinoma (LUAD) pathogenesis. The principal physiologic substrate of FAM86A is eEF2, with K525me3 modeled to facilitate productive eEF2-ribosome engagement during translocation. FAM86A depletion in LUAD cells causes 80S monosome accumulation and mRNA translation inhibition. FAM86A is overexpressed in LUAD and eEF2K525me3 levels increase through advancing LUAD disease stages. FAM86A knockdown attenuates LUAD cell proliferation and suppression of the FAM86A-eEF2K525me3 axis inhibits cancer cell and patient-derived LUAD xenograft growth in vivo. Finally, FAM86A ablation strongly attenuates tumor growth and extends survival in KRASG12C-driven LUAD mouse models. Thus, our work uncovers an eEF2 methylation-mediated mRNA translation elongation regulatory node and nominates FAM86A as an etiologic agent in LUAD.
Asunto(s)
Adenocarcinoma del Pulmón , Carcinogénesis , Neoplasias Pulmonares , Factor 2 de Elongación Peptídica , ARN Mensajero , Humanos , Animales , Metilación , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Factor 2 de Elongación Peptídica/genética , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proliferación Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Extensión de la Cadena Peptídica de Translación , Ratones Desnudos , Procesamiento Proteico-Postraduccional , FemeninoRESUMEN
One of the most critical steps of protein synthesis is coupled translocation of messenger RNA (mRNA) and transfer RNAs (tRNAs) required to advance the mRNA reading frame by one codon. In eukaryotes, translocation is accelerated and its fidelity is maintained by elongation factor 2 (eEF2)1,2. At present, only a few snapshots of eukaryotic ribosome translocation have been reported3-5. Here we report ten high-resolution cryogenic-electron microscopy (cryo-EM) structures of the elongating eukaryotic ribosome bound to the full translocation module consisting of mRNA, peptidyl-tRNA and deacylated tRNA, seven of which also contained ribosome-bound, naturally modified eEF2. This study recapitulates mRNA-tRNA2-growing peptide module progression through the ribosome, from the earliest states of eEF2 translocase accommodation until the very late stages of the process, and shows an intricate network of interactions preventing the slippage of the translational reading frame. We demonstrate how the accuracy of eukaryotic translocation relies on eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs. Our findings shed light on the mechanism of translation arrest by the anti-fungal eEF2-binding inhibitor, sordarin. We also propose that the sterically constrained environment imposed by diphthamide, a conserved eukaryotic posttranslational modification in eEF2, not only stabilizes correct Watson-Crick codon-anticodon interactions but may also uncover erroneous peptidyl-tRNA, and therefore contribute to higher accuracy of protein synthesis in eukaryotes.
Asunto(s)
Células Eucariotas , Biosíntesis de Proteínas , ARN Mensajero , Sistemas de Lectura , Ribosomas , Anticodón/genética , Anticodón/metabolismo , Codón/genética , Codón/metabolismo , Microscopía por Crioelectrón , Células Eucariotas/química , Células Eucariotas/metabolismo , Células Eucariotas/ultraestructura , Factor 2 de Elongación Peptídica/antagonistas & inhibidores , Factor 2 de Elongación Peptídica/metabolismo , Sistemas de Lectura/genética , Ribosomas/química , Ribosomas/metabolismo , Ribosomas/ultraestructura , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
Oxaliplatin is an important initial chemotherapy benefiting advanced-stage colorectal cancer patients. Frustratingly, acquired oxaliplatin resistance always occurs after sequential chemotherapy with diverse antineoplastic drugs. Therefore, an exploration of the mechanism of oxaliplatin resistance formation in-depth is urgently needed. We generated oxaliplatin-resistant colorectal cancer models by four representative compounds, and RNA-seq revealed that oxaliplatin resistance was mainly the result of cells' response to stimulus. Moreover, we proved persistent stimulus-induced endoplasmic reticulum stress (ERs) and associated cellular senescence were the core causes of oxaliplatin resistance. In addition, we screened diverse phytochemicals for ER inhibitors in silico, identifying inositol hexaphosphate (IP6), whose strong binding was confirmed by surface plasmon resonance. Finally, we confirmed the ability of IP6 to reverse colorectal cancer chemoresistance and investigated the mechanism of IP6 in the inhibition of diphthamide modification of eukaryotic elongation factor 2 (eEF2) and PERK activation. Our study demonstrated that oxaliplatin resistance contributed to cell senescence induced by persistently activated PERK and diphthamide modification of eEF2 levels, which were specifically reversed by combination therapy with IP6.
Asunto(s)
Neoplasias Colorrectales , Histidina/análogos & derivados , Ácido Fítico , Humanos , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Ácido Fítico/farmacología , Ácido Fítico/uso terapéutico , Factor 2 de Elongación Peptídica/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genéticaRESUMEN
Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the link to pathogenic bacteria. Diphthamide synthesis enzymes (DPH1 and DPH3) are associated with cancer, and DPH gene mutations can cause diphthamide deficiency syndrome (DDS). Finally, new analyses provide evidence that diphthamide may restrict propagation of viruses including SARS-CoV-2 and HIV-1, and that DPH enzymes are targeted by viruses for degradation to overcome this restriction. This review describes how diphthamide is synthesized and functions in translation, and covers its clinical relevance in human development, cancer, and infectious diseases.
Asunto(s)
Relevancia Clínica , Histidina/análogos & derivados , Neoplasias , Humanos , Factor 2 de Elongación Peptídica/metabolismo , Toxina Diftérica/metabolismoRESUMEN
In eukaryotes, the Dph1â¢Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis. Guided by evidence from archaeal orthologues, we searched for a putative SAM-binding pocket in Dph1â¢Dph2 from Saccharomyces cerevisiae. We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization through assay diagnostics for the loss of diphthamide reveal that the SAM pocket is essential for synthesis of the décor on EF2 in vivo. Further evidence from structural modeling suggests particularly critical residues close to the methionine moiety of SAM. Presumably, they facilitate a geometry specific for SAM cleavage and ACP radical formation that distinguishes Dph1â¢Dph2 from classical radical SAM enzymes, which generate canonical 5'-deoxyadenosyl (dAdo) radicals.
Asunto(s)
Histidina , Saccharomyces cerevisiae , Humanos , Histidina/química , Factor 2 de Elongación Peptídica/metabolismo , Saccharomyces cerevisiae/metabolismo , S-Adenosilmetionina/metabolismo , Mutación , Antígenos de Histocompatibilidad Menor , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The Intergenic Region Internal Ribosome Entry Sites (IGR IRESs) of Discistroviridae promote protein synthesis without initiation factors, with IRES translocation by elongation factor 2 (eEF2) being the first factor-catalysed reaction. Here, we developed a system that allows for the observation of intersubunit conformation of eukaryotic ribosomes at the single-molecule level by labeling rRNA. We used it to follow translation initiation and subsequent translocation of the cricket paralysis virus IRES (CrPV IRES). We observed that pre-translocation 80S-IRES ribosomes spontaneously exchanged between non-rotated and semi-rotated conformations, but predominantly occupied a semi-rotated conformation. In the presence of eEF2, ribosomes underwent forward and reverse translocation. Both reactions were eEF2 concentration dependent, indicating that eEF2 promoted both forward and reverse translocation. The antifungal, sordarin, stabilizes eEF2 on the ribosome after GTP hydrolysis in an extended conformation. 80S-CrPV IRES-eEF2-sordarin complexes underwent multiple rounds of forward and reverse translocations per eEF2 binding event. In the presence of sordarin, neither GTP hydrolysis nor a phosphate release were required for IRES translocation. Together, these results suggest that in the presence of sordarin, eEF2 promotes the mid and late stages of CrPV IRES translocation by unlocking ribosomal movements, with mid and late stages of translocation being thermally driven.
Asunto(s)
Sitios Internos de Entrada al Ribosoma , Biosíntesis de Proteínas , Sitios Internos de Entrada al Ribosoma/genética , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Guanosina Trifosfato/metabolismo , ARN Viral/metabolismoRESUMEN
INTRODUCTION AND OBJECTIVES: The development of hepatocellular carcinoma (HCC) is a multi-step process that accumulates genetic and epigenetic alterations, including changes in circular RNA (circRNA). This study aimed to understand the alterations in circRNA expression in HCC development and metastasis and to explore the biological functions of circRNA. MATERIALS AND METHODS: Ten pairs of adjacent chronic hepatitis tissues and HCC tissues from patients without venous metastases, and ten HCC tissues from patients with venous metastases were analyzed using human circRNA microarrays. Differentially expressed circRNAs were then validated by quantitative real-time PCR. In vitro and in vivo assays were performed to assess the roles of the circRNA in HCC progression. RNA pull-down assay, mass spectrometry analysis, and RNA-binding protein immunoprecipitation were conducted to explore the protein partners of the circRNA. RESULTS: CircRNA microarrays revealed that the expression patterns of circRNAs across the three groups were significantly different. Among these, hsa_circ_0098181 was validated to be lowly expressed and associated with poor prognosis in HCC patients. Ectopic expression of hsa_circ_0098181 delayed HCC metastasis in vitro and in vivo. Mechanistically, hsa_circ_0098181 sequestered eukaryotic translation elongation factor 2 (eEF2) and dissociated eEF2 from filamentous actin (F-actin) to prevent F-actin formation, which blocked activation of the Hippo signaling pathway. In addition, the RNA binding protein Quaking-5 bound directly to hsa_circ_0098181 and induced its biogenesis. CONCLUSIONS: Our study reveals changes in circRNA expression from chronic hepatitis, primary HCC, to metastatic HCC. Further, the QKI5-hsa_circ_0098181-eEF2-Hippo signaling pathway exerts a regulatory role in HCC.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , Humanos , Carcinoma Hepatocelular/patología , ARN Circular/genética , Neoplasias Hepáticas/patología , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Vía de Señalización Hippo , Actinas/metabolismo , Hepatitis Crónica , MicroARNs/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
The calmodulin-activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2K), serves as a master regulator of translational elongation by specifically phosphorylating and reducing the ribosome affinity of the guanosine triphosphatase, eukaryotic elongation factor 2 (eEF-2). Given its critical role in a fundamental cellular process, dysregulation of eEF-2K has been implicated in several human diseases, including those of the cardiovascular system, chronic neuropathies, and many cancers, making it a critical pharmacological target. In the absence of high-resolution structural information, high-throughput screening efforts have yielded small-molecule candidates that show promise as eEF-2K antagonists. Principal among these is the ATP-competitive pyrido-pyrimidinedione inhibitor, A-484954, which shows high specificity toward eEF-2K relative to a panel of "typical" protein kinases. A-484954 has been shown to have some degree of efficacy in animal models of several disease states. It has also been widely deployed as a reagent in eEF-2K-specific biochemical and cell-biological studies. However, given the absence of structural information, the precise mechanism of the A-484954-mediated inhibition of eEF-2K has remained obscure. Leveraging our identification of the calmodulin-activatable catalytic core of eEF-2K, and our recent determination of its long-elusive structure, here we present the structural basis for its specific inhibition by A-484954. This structure, which represents the first for an inhibitor-bound catalytic domain of a member of the α-kinase family, enables rationalization of the existing structure-activity relationship data for A-484954 variants and lays the groundwork for further optimization of this scaffold to attain enhanced specificity/potency against eEF-2K.
Asunto(s)
Adenosina Trifosfato , Calmodulina , Quinasa del Factor 2 de Elongación , Animales , Humanos , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Quinasa del Factor 2 de Elongación/química , Quinasa del Factor 2 de Elongación/genética , Quinasa del Factor 2 de Elongación/metabolismo , Factor 2 de Elongación Peptídica/química , Factor 2 de Elongación Peptídica/metabolismo , Fosforilación , Dominio Catalítico , Relación Estructura-Actividad , Extensión de la Cadena Peptídica de TranslaciónRESUMEN
FAM86A is a class I lysine methyltransferase (KMT) that generates trimethylation on the eukaryotic translation elongation factor 2 (EEF2) at Lys525. Publicly available data from The Cancer Dependency Map project indicate high dependence of hundreds of human cancer cell lines on FAM86A expression. This classifies FAM86A among numerous other KMTs as potential targets for future anticancer therapies. However, selective inhibition of KMTs by small molecules can be challenging due to high conservation within the S-adenosyl methionine (SAM) cofactor binding domain among KMT subfamilies. Therefore, understanding the unique interactions within each KMT-substrate pair can facilitate developing highly specific inhibitors. The FAM86A gene encodes an N-terminal FAM86 domain of unknown function in addition to its C-terminal methyltransferase domain. Here, we used a combination of X-ray crystallography, the AlphaFold algorithms, and experimental biochemistry to identify an essential role of the FAM86 domain in mediating EEF2 methylation by FAM86A. To facilitate our studies, we also generated a selective EEF2K525 methyl antibody. Overall, this is the first report of a biological function for the FAM86 structural domain in any species and an example of a noncatalytic domain participating in protein lysine methylation. The interaction between the FAM86 domain and EEF2 provides a new strategy for developing a specific FAM86A small molecule inhibitor, and our results provide an example in which modeling a protein-protein interaction with AlphaFold expedites experimental biology.
Asunto(s)
Lisina , Metiltransferasas , Modelos Moleculares , Dominios Proteicos , Humanos , Lisina/metabolismo , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , S-Adenosilmetionina/metabolismo , Especificidad por Sustrato , Estructura Terciaria de Proteína , Cristalografía por Rayos X , Mutación PuntualRESUMEN
Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α-kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway. Here, we provide structural, biochemical, and cell-biological evidence of a direct energy-sensing role of eEF-2K through its stimulation by ADP. A crystal structure of the nucleotide-bound complex between CaM and the functional core of eEF-2K phosphorylated at its primary stimulatory site (T348) reveals ADP bound at a unique pocket located on the face opposite that housing the kinase active site. Within this basic pocket (BP), created at the CaM/eEF-2K interface upon complex formation, ADP is stabilized through numerous interactions with both interacting partners. Biochemical analyses using wild-type eEF-2K and specific BP mutants indicate that ADP stabilizes CaM within the active complex, increasing the sensitivity of the kinase to CaM. Induction of energy stress through glycolysis inhibition results in significantly reduced enhancement of phosphorylated eEF-2 levels in cells expressing ADP-binding compromised BP mutants compared to cells expressing wild-type eEF-2K. These results suggest a direct energy-sensing role for eEF-2K through its cooperative interaction with CaM and ADP.
Asunto(s)
Calmodulina , Quinasa del Factor 2 de Elongación , Quinasa del Factor 2 de Elongación/metabolismo , Calmodulina/metabolismo , Regulación Alostérica , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Fosforilación , Eucariontes/metabolismo , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismoRESUMEN
BACKGROUND: Although molecular targets such as HER2, TP53 and PIK3CA have been widely studied in esophageal cancer, few of them were successfully applied for clinical treatment. Therefore, it is urgent to discover novel actionable targets and inhibitors. Eukaryotic translational elongation factor 2 (eEF2) is reported to be highly expressed in various cancers. However, its contribution to the maintenance and progression of cancer has not been fully clarified. METHODS: In the present study, we utilized tissue array to evaluate eEF2 protein expression and clinical significance in esophageal squamous cell carcinoma (ESCC). Next, we performed knockdown, overexpression, RNA-binding protein immunoprecipitation (RIP) sequence, and nascent protein synthesis assays to explore the molecular function of eEF2. Furthermore, we utilized compound screening, Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC) assay, cell proliferation and Patient derived xenograft (PDX) mouse model assays to discover an eEF2 inhibitor and assess its effects on ESCC growth. RESULTS: We found that eEF2 were highly expressed in ESCC and negatively associated with the prognosis of ESCC patients. Knocking down of eEF2 suppressed the cell proliferation and colony formation of ESCC. eEF2 bond with the mRNA of Topoisomerase II (TOP1) and Topoisomerase II (TOP2) and enhanced the protein biosynthesis of TOP1 and TOP2. We also identified Toosendanin was a novel inhibitor of eEF2 and Toosendanin inhibited the growth of ESCC in vitro and in vivo. CONCLUSIONS: Our findings show that Toosendanin treatment suppresses ESCC growth through targeting eEF2 and regulating downstream TOP1 and TOP2 biosynthesis. eEF2 could be supplied as a potential therapeutic target in the further clinical studies.
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Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , MicroARNs , Humanos , Animales , Ratones , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/patología , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas/patología , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs/genéticaRESUMEN
The principal mechanism underlying the reduced rate of protein synthesis in atrophied skeletal muscle is largely unknown. Eukaryotic elongation factor 2 kinase (eEF2k) impairs the ability of eukaryotic translation elongation factor 2 (eEF2) to bind to the ribosome via T56 phosphorylation. Perturbations in the eEF2k/eEF2 pathway during various stages of disuse muscle atrophy have been investigated utilizing a rat hind limb suspension (HS) model. Two distinct components of eEF2k/eEF2 pathway misregulation were demonstrated, observing a significant (P < 0.01) increase in eEF2k mRNA expression as early as 1-day HS and in eEF2k protein level after 3-day HS. We set out to determine whether eEF2k activation is a Ca2+-dependent process with involvement of Cav1.1. The ratio of T56-phosphorylated/total eEF2 was robustly elevated after 3-day HS, which was completely reversed by 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and decreased by 1.7-fold (P < 0.05) by nifedipine. Transfection of C2C12 with cytomegalovirus promoter (pCMV)-eEF2k and administration with small molecules were used to modulate eEF2k and eEF2 activity. More importantly, pharmacologic enhancement of eEF2 phosphorylation induced phosphorylated ribosomal protein S6 kinase (T389) up-regulation and restoration of global protein synthesis in the HS rats. Taken together, the eEF2k/eEF2 pathway was up-regulated during disuse muscle atrophy involving calcium-dependent activation of eEF2k partly via Cav1.1. The study provides evidence, in vitro and in vivo, of the eEF2k/eEF2 pathway impact on ribosomal protein S6 kinase activity as well as protein expression of key atrophy biomarkers, muscle atrophy F-box/atrogin-1 and muscle RING finger-1.
Asunto(s)
Quinasa del Factor 2 de Elongación , Músculo Esquelético , Ratas , Animales , Quinasa del Factor 2 de Elongación/genética , Quinasa del Factor 2 de Elongación/metabolismo , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Fosforilación , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismoRESUMEN
Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.
Asunto(s)
Alphavirus , Fiebre Chikungunya , Virus Chikungunya , Humanos , Alphavirus/metabolismo , Nucleósido-Trifosfatasa/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Eucariontes , Fosforilación , Virus Chikungunya/fisiología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Quinasa del Factor 2 de Elongación/metabolismoRESUMEN
An emerging body of research is revealing mutations in elongation factor eEF2 that are implicated in both inherited and de novo neurodevelopmental disorders. Previous structural analysis has revealed that most pathogenic amino acid substitutions map to the three main points of contact between eEF2 and critical large subunit rRNA elements of the ribosome, specifically to contacts with Helix 69, Helix 95, also known as the sarcin-ricin loop, and Helix 43 of the GTPase-associated center. In order to further investigate these eEF2-ribosome interactions, we identified a series of yeast eEF2 amino acid residues based on their proximity to these functionally important rRNA elements. Based on this analysis, we constructed mutant strains to sample the full range of amino acid sidechain biochemical properties, including acidic, basic, nonpolar, and deletion (alanine) residues. These were characterized with regard to their effects on cell growth, sensitivity to ribosome-targeting antibiotics, and translational fidelity. We also biophysically characterized one mutant from each of the three main points of contact with the ribosome using CD. Collectively, our findings from these studies identified functionally critical contacts between eEF2 and the ribosome. The library of eEF2 mutants generated in this study may serve as an important resource for biophysical studies of eEF2/ribosome interactions going forward.
Asunto(s)
Factor 2 de Elongación Peptídica , Ribosomas , Humanos , Aminoácidos/química , Aminoácidos/genética , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , MutaciónRESUMEN
Understanding the molecular signaling mechanisms underlying cognition and neuronal plasticity would provide insights into the pathogenesis of neuronal disorders characterized by cognitive syndromes such as Alzheimer disease (AD). Phosphorylation of the mRNA translational factor eukaryotic elongation factor 2 (eEF2) by its specific kinase eEF2K is critically involved in protein synthesis regulation. In this review, we discussed recent studies on the roles of eEF2K/eEF2 signaling in the context of regulation/dysregulation of cognitive function and synaptic plasticity. We specifically focus on the discussion of recent evidence indicating suppression of eEF2K signaling as a potential novel therapeutic avenue for AD and related dementias (ADRDs).
Asunto(s)
Enfermedad de Alzheimer , Quinasa del Factor 2 de Elongación , Humanos , Quinasa del Factor 2 de Elongación/genética , Quinasa del Factor 2 de Elongación/metabolismo , Enfermedad de Alzheimer/genética , Plasticidad Neuronal , Transducción de Señal/fisiología , Cognición , Fosforilación/fisiología , Factor 2 de Elongación Peptídica/metabolismoRESUMEN
Recent evidence links synaptic plasticity and mRNA translation, via the eukaryotic elongation factor 2 kinase (eEF2K) and its only known substrate, eEF2. However, the involvement of the eEF2 pathway in cocaine-induced neuroadaptations and cocaine-induced behaviours is not known. Knock-in (KI) mice and shRNA were used to globally and specifically reduce eEF2K expression. Cocaine psychomotor sensitization and conditioned place preference were used to evaluate behavioural outcome. Changes in eEF2 phosphorylation were determined by western blot analyses. No effect was observed on the AMPA/NMDA receptor current ratio in the ventral tegmental area, 24 h after cocaine injection in eEF2K-KI mice compared with WT. However, development and expression of cocaine psychomotor sensitization were decreased in KI mice. Phosphorylated eEF2 was decreased one day after psychomotor sensitization and returned to baseline at seven days in the nucleus accumbens (NAc) of WT mice, but not in eEF2K-KI mice. However, one day following cocaine challenge, phosphorylated eEF2 decreased in WT but not KI mice. Importantly, specific targeting of eEF2K expression by shRNA in the NAc decreased cocaine condition place preference. These results suggest that the eEF2 pathway play a role in cocaine-induced locomotor sensitization and conditioned place preference.
Asunto(s)
Cocaína , Quinasa del Factor 2 de Elongación , Animales , Ratones , Quinasa del Factor 2 de Elongación/genética , Quinasa del Factor 2 de Elongación/metabolismo , Cocaína/farmacología , ARN Interferente Pequeño/metabolismo , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Condicionamiento Clásico , Fosforilación , Núcleo Accumbens/metabolismoRESUMEN
Maintenance of energy level to drive movements and material exchange with the environment is a basic principle of life. AMP-activated protein kinase (AMPK) senses energy level and is a major regulator of cellular energy responses. The gamma subunit of AMPK senses elevated ratio of AMP to ATP and allosterically activates the alpha catalytic subunit to phosphorylate downstream effectors. Here, we report that knockout of AMPKγ, but not AMPKα, suppressed phosphorylation of eukaryotic translation elongation factor 2 (eEF2) induced by energy starvation. We identified PPP6C as an AMPKγ-regulated phosphatase of eEF2. AMP-bound AMPKγ sequesters PPP6C, thereby blocking dephosphorylation of eEF2 and thus inhibiting translation elongation to preserve energy and to promote cell survival. Further phosphoproteomic analysis identified additional targets of PPP6C regulated by energy stress in an AMPKγ-dependent manner. Thus, AMPKγ senses cellular energy availability to regulate not only AMPKα kinase, but also PPP6C phosphatase and possibly other effectors.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Biosíntesis de Proteínas , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Fosforilación , Factor 2 de Elongación Peptídica/metabolismoRESUMEN
Regulation of mRNA translation is essential for brain development and function. Translation elongation factor eEF2 acts as a molecular hub orchestrating various synaptic signals to protein synthesis control and participates in hippocampus-dependent cognitive functions. However, whether eEF2 regulates other behaviors in different brain regions has been unknown. Here, we construct a line of Eef2 heterozygous (HET) mice, which show a reduction in eEF2 and protein synthesis mainly in excitatory neurons of the prefrontal cortex. The mice also show lower spine density, reduced excitability, and AMPAR-mediated synaptic transmission in pyramidal neurons of the medial prefrontal cortex (mPFC). While HET mice exhibit normal learning and memory, they show defective social behavior and elevated anxiety. Knockdown of Eef2 in excitatory neurons of the mPFC specifically is sufficient to impair social novelty preference. Either chemogenetic activation of excitatory neurons in the mPFC or mPFC local infusion of the AMPAR potentiator PF-4778574 corrects the social novelty deficit of HET mice. Collectively, we identify a novel role for eEF2 in promoting prefrontal AMPAR-mediated synaptic transmission underlying social novelty behavior.
Asunto(s)
Factor 2 de Elongación Peptídica/metabolismo , Corteza Prefrontal , Transmisión Sináptica , Animales , Potenciales Postsinápticos Excitadores/fisiología , Ratones , Factores de Elongación de Péptidos/metabolismo , Corteza Prefrontal/fisiología , Conducta Social , Transmisión Sináptica/fisiologíaRESUMEN
The activation of memory T cells is a very rapid and concerted cellular response that requires coordination between cellular processes in different compartments and on different time scales. In this study, we use ribosome profiling and deep RNA sequencing to define the acute mRNA translation changes in CD8 memory T cells following initial activation events. We find that initial translation enables subsequent events of human and mouse T cell activation and expansion. Briefly, early events in the activation of Ag-experienced CD8 T cells are insensitive to transcriptional blockade with actinomycin D, and instead depend on the translation of pre-existing mRNAs and are blocked by cycloheximide. Ribosome profiling identifies â¼92 mRNAs that are recruited into ribosomes following CD8 T cell stimulation. These mRNAs typically have structured GC and pyrimidine-rich 5' untranslated regions and they encode key regulators of T cell activation and proliferation such as Notch1, Ifngr1, Il2rb, and serine metabolism enzymes Psat1 and Shmt2 (serine hydroxymethyltransferase 2), as well as translation factors eEF1a1 (eukaryotic elongation factor α1) and eEF2 (eukaryotic elongation factor 2). The increased production of receptors of IL-2 and IFN-γ precedes the activation of gene expression and augments cellular signals and T cell activation. Taken together, we identify an early RNA translation program that acts in a feed-forward manner to enable the rapid and dramatic process of CD8 memory T cell expansion and activation.