scholarly journals Repression of eEF2K transcription by NF-κB tunes translation elongation to inflammation and dsDNA-sensing

2019 ◽  
Vol 116 (45) ◽  
pp. 22583-22590 ◽  
Author(s):  
Christopher Bianco ◽  
Letitia Thompson ◽  
Ian Mohr
Keyword(s):  
Protein Synthesis ◽  
Transcriptional Repression ◽  
Elongation Factor ◽  
Mrna Translation ◽  
Bacterial Toxin ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Protein Levels ◽  
Cellular Translation ◽  
Infection And Inflammation

Gene expression is rapidly remodeled by infection and inflammation in part via transcription factor NF-κB activation and regulated protein synthesis. While protein synthesis is largely controlled by mRNA translation initiation, whether cellular translation elongation factors are responsive to inflammation and infection remains poorly understood. Here, we reveal a surprising mechanism whereby NF-κB restricts phosphorylation of the critical translation elongation factor eEF2, which catalyzes the protein synthesis translocation step. Upon exposure to NF-κB–activating stimuli, including TNFα, human cytomegalovirus infection, or double-stranded DNA, eEF2 phosphorylation on Thr56, which slows elongation to limit protein synthesis, and the overall abundance of eEF2 kinase (eEF2K) are reduced. Significantly, this reflected a p65 NF-κB subunit-dependent reduction in eEF2K pre-mRNA, indicating that NF-κB activation represses eEF2K transcription to decrease eEF2K protein levels. Finally, we demonstrate that reducing eEF2K abundance regulates protein synthesis in response to a bacterial toxin that inactivates eEF2. This establishes that NF-κB activation by diverse physiological effectors controls eEF2 activity via a transcriptional repression mechanism that reduces eEF2K polypeptide abundance to preclude eEF2 phosphorylation, thereby stimulating translation elongation and protein synthesis. Moreover, it illustrates how nuclear transcription regulation shapes translation elongation factor activity and exposes how eEF2 is integrated into innate immune response networks orchestrated by NF-κB.

scholarly journals Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Maxim V. Gerashchenko ◽  
Mikhail V. Nesterchuk ◽  
Elena M. Smekalova ◽  
Joao A. Paulo ◽  
Piotr S. Kowalski ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Mouse Liver ◽  
Negative Impact ◽  
Elongation Factor ◽  
Ribosome Profiling ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Elongation Factor 2

Abstract Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

scholarly journals A Chemical Genomic Screen in Saccharomyces cerevisiae Reveals a Role for Diphthamidation of Translation Elongation Factor 2 in Inhibition of Protein Synthesis by Sordarin

2008 ◽  
Vol 52 (5) ◽  
pp. 1623-1629 ◽  
Author(s):  
Javier Botet ◽  
María Rodríguez-Mateos ◽  
Juan P. G. Ballesta ◽  
José Luis Revuelta ◽  
Miguel Remacha
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Cellular Mechanisms ◽  
Translation Elongation ◽  
Chemical Genomic ◽  
Elongation Factor 2 ◽  
Fungal Organisms ◽  
Inhibitor Of Protein Synthesis

ABSTRACT Sordarin and its derivatives are antifungal compounds of potential clinical interest. Despite the highly conserved nature of the fungal and mammalian protein synthesis machineries, sordarin is a selective inhibitor of protein synthesis in fungal organisms. In cells sensitive to sordarin, its mode of action is through preventing the release of translation elongation factor 2 (eEF2) during the translocation step, thus blocking protein synthesis. To further investigate the cellular components required for the effects of sordarin in fungal cells, we have used the haploid deletion collection of Saccharomyces cerevisiae to systematically identify genes whose deletion confers sensitivity or resistance to the compound. Our results indicate that genes in a number of cellular pathways previously unknown to play a role in sordarin response are involved in its growth effects on fungal cells and reveal a specific requirement for the diphthamidation pathway of cells in causing eEF2 to be sensitive to the effects of sordarin on protein synthesis. Our results underscore the importance of the powerful genomic tools developed in yeast (Saccharomyces cerevisiae) to more comprehensively understanding the cellular mechanisms involved in the response to therapeutic agents.

scholarly journals In vivo characterization of the role of tissue‐specific translation elongation factor 1 A 2 in protein synthesis reveals insights into muscle atrophy

FEBS Journal ◽  
2013 ◽  
Vol 280 (24) ◽  
pp. 6528-6540 ◽  
Author(s):  
Jennifer Doig ◽  
Lowri A. Griffiths ◽  
David Peberdy ◽  
Permphan Dharmasaroja ◽  
Maria Vera ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Muscle Atrophy ◽  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
In Vivo Characterization ◽  
Elongation Factor 1

scholarly journals Overexpression of Translation Elongation Factor 1A Affects the Organization and Function of the Actin Cytoskeleton in Yeast

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1425-1436
Author(s):  
Raj Munshi ◽  
Kimberly A Kandl ◽  
Anne Carr-Schmid ◽  
Johanna L Whitacre ◽  
Alison E M Adams ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Elongation Factor ◽  
Nucleotide Exchange ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Function

Abstract The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1Bα, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

scholarly journals Eukaryotic Elongation Factor 3 Protects Saccharomyces cerevisiae Yeast from Oxidative Stress

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1432
Author(s):  
Karolina Gościńska ◽  
Somayeh Shahmoradi Ghahe ◽  
Sara Domogała ◽  
Ulrike Topf
Keyword(s):  
Oxidative Stress ◽  
Protein Synthesis ◽  
Elongation Factor ◽  
Cellular Protein ◽  
Stress Conditions ◽  
Translation Elongation ◽  
Protein Levels ◽  
Eukaryotic Elongation Factor ◽  
Elongation Factor 3 ◽  
Elongation Process

Translation is a core process of cellular protein homeostasis and, thus, needs to be tightly regulated. The production of newly synthesized proteins adapts to the current needs of the cell, including the response to conditions of oxidative stress. Overall protein synthesis decreases upon oxidative stress. However, the selective production of proteins is initiated to help neutralize stress conditions. In contrast to higher eukaryotes, fungi require three translation elongation factors, eEF1, eEF2, and eEF3, for protein synthesis. eEF1 and eEF2 are evolutionarily conserved, but they alone are insufficient for the translation elongation process. eEF3 is encoded by two paralogous genes, YEF3 and HEF3. However, only YEF3 is essential in yeast, whereas the function of HEF3 remains unknown. To elucidate the cellular function of Hef3p, we used cells that were depleted of HEF3 and treated with H2O2 and analyzed the growth of yeast, global protein production, and protein levels. We found that HEF3 is necessary to withstand oxidative stress conditions, suggesting that Hef3p is involved in the selective production of proteins that are necessary for defense against reactive oxygen species.

scholarly journals Translation Elongation Factor 4 (LepA) Contributes to Tetracycline Susceptibility by Stalling Elongating Ribosomes

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Bin Liu ◽  
Chunlai Chen
Keyword(s):  
Protein Synthesis ◽  
Ribosome Biogenesis ◽  
Molecular Mechanisms ◽  
Elongation Factor ◽  
Ribosome Profiling ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Content Type ◽  
Elongation Phase ◽  
Profiling Analysis

ABSTRACTEven though elongation factor 4 (EF4) is the third most conserved protein in bacteria, its physiological functions remain largely unknown and its proposed molecular mechanisms are conflicting among previous studies. In the present study, we show that the growth of anEscherichia colistrain is more susceptible to tetracycline than its EF4 knockout strain. Consistent with previous studies, our results suggested that EF4 affects ribosome biogenesis when tetracycline is present. Through ribosome profiling analysis, we discovered that EF4 causes 1-nucleotide shifting of ribosomal footprints on mRNA when cells have been exposed to tetracycline. In addition, when tetracycline is present, EF4 inhibits the elongation of protein synthesis, which leads to the accumulation of ribosomes in the early segment of mRNA. Altogether, when cells are exposed to tetracycline, EF4 alters both ribosome biogenesis and the elongation phase of protein synthesis.

scholarly journals Regulation of protein-synthesis elongation-factor-2 kinase by cAMP in adipocytes

1998 ◽  
Vol 336 (3) ◽  
pp. 525-529 ◽  
Author(s):  
Tricia A. DIGGLE ◽  
Nicholas T. REDPATH ◽  
Kate J. HEESOM ◽  
Richard M. DENTON
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Polypeptide Chain ◽  
Elongation Factor ◽  
Chain Elongation ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Elongation Factor 2 ◽  
Fold Activation

Treatment of primary rat epididymal adipocytes or 3T3-L1 adipocytes with various agents which increase cAMP led to the phosphorylation of eukaryotic translation elongation factor-2 (eEF-2). The increase in eEF-2 phosphorylation was a consequence of the activation of eEF-2 kinase (eEF-2K), which is a Ca2+/calmodulin-dependent kinase. eEF-2K was shown to be essentially inactive at less than 0.1 µM free Ca2+ when measured in cell-free extracts. Treatment of adipocytes with isoproterenol induced Ca2+-independent eEF-2K activity, and an 8–10-fold activation of eEF-2K was observed at Ca2+ concentrations of less than 0.1 µM. Increased cAMP in 3T3-L1 adipocytes led to the inhibition of total protein synthesis and decreased the rate of polypeptide-chain elongation. We also show that the phosphorylation of eEF-2 and the activity of eEF-2K are insulin-regulated in adipocytes. These results demonstrate a novel mechanism for the control of protein synthesis by hormones which act by increasing cytoplasmic cAMP.

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