A Complementary Mechanism of Bacterial mRNA Translation Inhibition by Tetracyclines

Tetracycline has positively impacted human health as well as the farming and animal industries. Its extensive usage and versatility led to the spread of resistance mechanisms followed by the development of new variants of the antibiotic. Tetracyclines inhibit bacterial growth by impeding the binding of elongator tRNAs to the ribosome. However, a small number of reports indicated that Tetracyclines could also inhibit translation initiation, yet the molecular mechanism remained unknown. Here, we use biochemical and computational methods to study how Oxytetracycline (Otc), Demeclocycline (Dem), and Tigecycline (Tig) affect the translation initiation phase of protein synthesis. Our results show that all three Tetracyclines induce Initiation Factor IF3 to adopt a compact conformation on the 30S ribosomal subunit, similar to that induced by Initiation Factor IF1. This compaction was faster for Tig than Dem or Otc. Furthermore, all three tested tetracyclines affected IF1-bound 30S complexes. The dissociation rate constant of IF1 in early 30S complexes was 14-fold slower for Tig than Dem or Otc. Late 30S initiation complexes (30S pre-IC or IC) exhibited greater IF1 stabilization by Tig than for Dem and Otc. Tig and Otc delayed 50S joining to 30S initiation complexes (30S ICs). Remarkably, the presence of Tig considerably slowed the progression to translation elongation and retained IF1 in the resulting 70S initiation complex (70S IC). Molecular modeling of Tetracyclines bound to the 30S pre-IC and 30S IC indicated that the antibiotics binding site topography fluctuates along the initiation pathway. Mainly, 30S complexes show potential contacts between Dem or Tig with IF1, providing a structural rationale for the enhanced affinity of the antibiotics in the presence of the factor. Altogether, our data indicate that Tetracyclines inhibit translation initiation by allosterically perturbing the IF3 layout on the 30S, retaining IF1 during 70S IC formation, and slowing the transition toward translation elongation. Thus, this study describes a new complementary mechanism by which Tetracyclines may inhibit bacterial protein synthesis.

Download Full-text

Correction of eIF2-dependent defects in brain protein synthesis, synaptic plasticity, and memory in mouse models of Alzheimer’s disease

Science Signaling ◽

10.1126/scisignal.abc5429 ◽

2021 ◽

Vol 14 (668) ◽

pp. eabc5429

Author(s):

Mauricio M. Oliveira ◽

Mychael V. Lourenco ◽

Francesco Longo ◽

Nicole P. Kasica ◽

Wenzhong Yang ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Protein Synthesis ◽

Synaptic Plasticity ◽

Translation Initiation ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Postmortem Brain Tissue ◽

Phosphorylated Eif2α

Neuronal protein synthesis is essential for long-term memory consolidation, and its dysregulation is implicated in various neurodegenerative disorders, including Alzheimer’s disease (AD). Cellular stress triggers the activation of protein kinases that converge on the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which attenuates mRNA translation. This translational inhibition is one aspect of the integrated stress response (ISR). We found that postmortem brain tissue from AD patients showed increased phosphorylation of eIF2α and reduced abundance of eIF2B, another key component of the translation initiation complex. Systemic administration of the small-molecule compound ISRIB (which blocks the ISR downstream of phosphorylated eIF2α) rescued protein synthesis in the hippocampus, measures of synaptic plasticity, and performance on memory-associated behavior tests in wild-type mice cotreated with salubrinal (which inhibits translation by inducing eIF2α phosphorylation) and in both β-amyloid-treated and transgenic AD model mice. Thus, attenuating the ISR downstream of phosphorylated eIF2α may restore hippocampal protein synthesis and delay cognitive decline in AD patients.

Download Full-text

Inferring efficiency of translation initiation and elongation from ribosome profiling

Nucleic Acids Research ◽

10.1093/nar/gkaa678 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9478-9490

Author(s):

Juraj Szavits-Nossan ◽

Luca Ciandrini

Keyword(s):

Protein Synthesis ◽

Mathematical Modelling ◽

Translation Initiation ◽

Mrna Translation ◽

Elongation Rate ◽

Ribosome Profiling ◽

Translation Elongation ◽

Recent Advances ◽

Two Stages

Abstract One of the main goals of ribosome profiling is to quantify the rate of protein synthesis at the level of translation. Here, we develop a method for inferring translation elongation kinetics from ribosome profiling data using recent advances in mathematical modelling of mRNA translation. Our method distinguishes between the elongation rate intrinsic to the ribosome’s stepping cycle and the actual elongation rate that takes into account ribosome interference. This distinction allows us to quantify the extent of ribosomal collisions along the transcript and identify individual codons where ribosomal collisions are likely. When examining ribosome profiling in yeast, we observe that translation initiation and elongation are close to their optima and traffic is minimized at the beginning of the transcript to favour ribosome recruitment. However, we find many individual sites of congestion along the mRNAs where the probability of ribosome interference can reach $50\%$. Our work provides new measures of translation initiation and elongation efficiencies, emphasizing the importance of rating these two stages of translation separately.

Download Full-text

Expression of Ribosomal Protein S6 (R6SP) and Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1) Is Correlated in Acute Myelogenous Leukemia (AML) and Is Highly Prognostic, Especially in NPM1 and FLT3 Wildtype Patients

Blood ◽

10.1182/blood.v124.21.2369.2369 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2369-2369

Author(s):

Steven M. Kornblau ◽

Chenyue W Hu ◽

Yihua Qiu ◽

Suk Young Yoo ◽

Rebecca A Murray ◽

...

Keyword(s):

Translation Initiation ◽

Ribosomal Subunit ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Npm1 Mutation ◽

Eukaryotic Translation ◽

Ribosomal Protein S6 ◽

40S Ribosomal Subunit ◽

Eukaryotic Translation Initiation

Abstract Background. Conceptually mRNA processing and ribosomal regulation should interact as both affect mRNA translation and protein production. We studied protein expression and functional relationships between proteins in AML using a custom made reverse phase protein array (RPPA), probed with 231 strictly validated antibodies. We found a relationship between expression of Ribosomal Protein S6 (HUGO name R6SP, a.k.a. S6RP) and Eukaryotic Translation Initiation Factor 4EBinding Protein 1, (HUGO name EIF4EBP1). R6SP, a 40S ribosomal subunit component, activated by phosphorylation, regulates cell growth via selective mRNA translation. EIF4EBP1 interacts with eIF4E to recruit the 40S ribosomal subunit, thereby affecting ribosomal assembly. When phosphorylated, in response to cellular signaling, it releases eIF4E allowing transcription. Methods. Our RPPA has protein from leukemia enriched cells from 511 newly diagnosed AML patients and was probed with 231 strictly validated antibodies, including antibodies against total RPS6 and forms phosphorylated on S235-236 and S240-244, and against total EIF4EBP1 and forms phosphorylated on T37 & 46, T70 and S65. Expression was compared to normal bone marrow derived CD34+ cells. Interaction networks with the other 224 proteins were generated from the RPPA data using glasso and supplemented by the literature of known interactions. Results. A heatmap of expression of the 3 R6SP and 4 PA2 forms was generated and hierarchical k-and means clustering performed (Fig A). Using the Prototype Clustering method an optimal division into four clusters (Fig B) was determined. This includes an All-Off state (18%), a state characterized by weak activation of RPS6 alone (RP-Only, 36%) activation of only EIF4EBP1 (EIF4EBP1-Only, 26%) and a group where both were on simultaneously (Both-On). The RPS6 interactome (Fig B) showed the expected positive correlation with mTOR, and P70 (Hugo RPS6KB1) and a previously unknown, but very strong, negative correlation with transcription factor ZNF296. The EIF4EBP1 interactome showed the expected strong positive correlation with many signal transduction pathways (MAP2K1, MAPK14) and proliferation related proteins (pRB, EIF2AK, EIF2S1, FOXO3) and negative correlation with several transcription factors (GATA3, SPI1, CREB). Cluster membership was unassociated with most clinical features including cytogenetics, FLT3 , RAS and NPM1 mutation, excluding gender (more F in All-Off, more M in Both-On, p=0.01). EIF4EBP1 and Both-On had higher WBC (p=0.0001) and % marrow (p=0.0001) and blood blasts (0.0007) and lower platelet counts (p=0.025). Response rates did not differ, although fewer All-Off were primary refractory. Relapse was more common in EIF4EBP1-Only and Both-On clusters. Overall survival (OS) and remission duration (RemDur) (Fig C) of the EIF4EBP1-Only and Both-On clusters was inferior to that of the All-Off and RP-Only clusters (OS median 41 & 45 vs. 52 &63,p=0.06, RemDur 39 & 27 weeks vs. 63 & 53, p=0.008) but this was restricted to Intermediate cytogenetics cases (Fig C IntCyto OS 49 & 55 weeks vs. 107& 79 p=0.01, RemDur 37 & 35 weeks vs. 89 & 53 , p = 0.005) that were FLT3 mutation ((Fig C FLT3-WT OS p=0.006, RemDur p0.007) and NPM1 mutation negative (Fig C NPM1-WT, OS p=0.006, RemDur p=0.001). Conclusions. Activation of EIF4EBP1, with or without RPS6 activation is prognostically adverse in AML, particularly in intermediate cytogenetic cases with wildtype FLT3 and NPM1. This is associated with increased proliferation. Therapy directed against EIF4EBP1 activity, e.g. that block it's phosphorylation, may have utility in the ~46% of cases of AML that demonstrate high levels of EIF4EBP1 phosphorylation, especially in FLT3/NPM1 wildtype cases. Many agents that inhibit signal transduction pathways are in clinical development, analyzing them for the ability to inhibition the activation of EIF4EBP1 might identify clinically useful molecules. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates

10.1101/465914 ◽

2018 ◽

Author(s):

Andrea Riba ◽

Noemi Di Nanni ◽

Nitish Mittal ◽

Erik Arhné ◽

Alexander Schmidt ◽

...

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Amino Acid Composition ◽

Acid Composition ◽

Translation Initiation ◽

Mrna Translation ◽

Elongation Rate ◽

Translation Elongation ◽

Lower Protein ◽

Protein Output

AbstractAlthough protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a thousand ORFs in exponentially-growing wildtype yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and tRNA adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output than other transcripts with equally high ribosome densities.Significance StatementAlthough sequencing of ribosome footprints has uncovered new aspects of mRNA translation, the determinants of ribosome flux remain incompletely understood. Combining ribosome footprint data with measurements of protein synthesis rates, we here inferred translation initiation and elongation rates for over a thousand ORFs in yeast strains with varying translation capacity. We found that the translation elongation rate varies up to ~20-fold among yeast transcripts, and is significantly correlated with the rate of translation initiation. Furthermore, the amino acid composition of synthesized proteins impacts the rate of translation elongation to the same extent as measures of codon and tRNA adaptation. Transcripts encoding ribosomal proteins are translated especially slow, having markedly lower protein output than other transcripts with equally high ribosome densities.

Download Full-text

The 39-kilodalton subunit of eukaryotic translation initiation factor 3 is essential for the complex's integrity and for cell viability in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.17.1.145 ◽

1997 ◽

Vol 17 (1) ◽

pp. 145-153 ◽

Cited By ~ 33

Author(s):

T Naranda ◽

M Kainuma ◽

S E MacMillan ◽

J W Hershey

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Synthesis ◽

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Initiation Phase ◽

Eukaryotic Translation Initiation ◽

Translation Initiation Factor 3

Eukaryotic translation initiation factor 3 (eIF3) in the yeast Saccharomyces cerevisiae comprises about eight polypeptides and plays a central role in the binding of methionyl-tRNAi and mRNA to the 40S ribosomal subunit. The fourth largest subunit, eIF3-p39, was gel purified, and a 12-amino-acid tryptic peptide was sequenced, enabling the cloning of the TIF34 gene. TIF34 encodes a 38,753-Da protein that corresponds to eIF3-p39 in size and antigenicity. Disruption of TIF34 is lethal, and depletion of eIF3-p39 by glucose repression of TIF34 expressed from a GAL promoter results in cessation of cell growth. As eIF3-p39 levels fall, polysomes become smaller, indicating a role for eIF3-p39 in the initiation phase of protein synthesis. Unexpectedly, depletion results in degradation of all of the subunit proteins of eIF3 at a rate much faster than the normal turnover rates of these proteins. eIF3-p39 has 46% sequence identity with the p36 subunit of human eIF3. Both proteins are members of the WD-repeat family of proteins, possessing five to seven repeat elements. Taken together, the results indicate that eIF3-p39 plays an important, although not necessarily direct, role in the initiation phase of protein synthesis and suggest that it may be required for the assembly and maintenance of the eIF3 complex in eukaryotic cells.

Download Full-text

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells

Biochemical Journal ◽

10.1042/bcj20160845 ◽

2016 ◽

Vol 473 (24) ◽

pp. 4651-4664 ◽

Cited By ~ 22

Author(s):

Lyne Jossé ◽

Jianling Xie ◽

Christopher G. Proud ◽

C. Mark Smales

Keyword(s):

Protein Synthesis ◽

Cell Growth ◽

Recombinant Protein ◽

Cell Lines ◽

Translation Initiation ◽

Mammalian Cells ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Cho Cell

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.

Download Full-text

Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1521156113 ◽

2016 ◽

Vol 113 (16) ◽

pp. E2286-E2295 ◽

Cited By ~ 14

Author(s):

Attilio Fabbretti ◽

Andreas Schedlbauer ◽

Letizia Brandi ◽

Tatsuya Kaminishi ◽

Anna Maria Giuliodori ◽

...

Keyword(s):

Protein Synthesis ◽

Translation Initiation ◽

Dynamic Equilibrium ◽

Ribosomal Subunit ◽

Structural Level ◽

Initiation Complex ◽

Stem Loop ◽

Initiation Factors ◽

Initiation Phase

In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon–anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.

Download Full-text

Effect of dietary protein on translation initiation in rat skeletal muscle and liver

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1998.275.5.e814 ◽

1998 ◽

Vol 275 (5) ◽

pp. E814-E820 ◽

Cited By ~ 57

Author(s):

Fumiaki Yoshizawa ◽

Scot R. Kimball ◽

Thomas C. Vary ◽

Leonard S. Jefferson

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Translation Initiation ◽

Dietary Protein ◽

Plasma Insulin ◽

Mrna Translation ◽

Initiation Factor ◽

Translational Repressor ◽

Isocaloric Diets ◽

Induced Changes

The effect of dietary protein on the initiation of mRNA translation was examined in rats starved for 18 h and then fed isocaloric diets containing either 20% protein (20P) or no added protein (0P). Feeding the 20P diet, but not the 0P diet, stimulated protein synthesis in skeletal muscle and liver by 38 and 41%, respectively. The stimulation was associated with reduced binding of eukaryotic initiation factor (eIF) 4E to the translational repressor 4E-BP1, increased formation of the active eIF4E-eIF4G complex, and increased phosphorylation of 4E-BP1. In contrast, feeding a 0P diet had no effect on any of these parameters. Feeding a 20P diet resulted in partial dephosphorylation of eIF4E in both tissues. In liver, refeeding a 0P diet also resulted in partial eIF4E dephosphorylation, suggesting that the phosphorylation state of eIF4E is not important in the stimulation of protein synthesis under these conditions. Finally, plasma insulin concentrations were the same in rats fed either diet (14.8 ± 4.9 vs. 15.5 ± 4.5 μU/ml for 20P and 0P groups, respectively), suggesting that feeding-induced changes in plasma insulin are not sufficient to stimulate protein synthesis. Instead, a combination of dietary protein and insulin may be required to stimulate translation initiation.

Download Full-text

Migration of Small Ribosomal Subunits on the 5′ Untranslated Regions of Capped Messenger RNA

International Journal of Molecular Sciences ◽

10.3390/ijms20184464 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4464 ◽

Cited By ~ 3

Author(s):

Nikolay E. Shirokikh ◽

Yulia S. Dutikova ◽

Maria A. Staroverova ◽

Ross D. Hannan ◽

Thomas Preiss

Keyword(s):

Translation Initiation ◽

Ribosomal Subunit ◽

Mrna Translation ◽

Primer Extension ◽

Initiation Factor ◽

Start Codon ◽

Untranslated Regions ◽

Specific Rate ◽

Messenger Ribonucleic Acid ◽

Free Translation

Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential ‘scanning’ of the 5′ untranslated regions (UTRs). Scanning through the 5′UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5′UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured β-globin sequences in the 5′UTR but not that of an mRNA with a poly(A) sequence as the 5′UTR. By contrast, the nonhydrolysable ATP analogue β,γ-imidoadenosine 5′-triphosphate (AMP-PNP) inhibited translation irrespective of the 5′UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA (‘toeprinting’), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This ‘−8 nt toeprint’ was seen with mRNA 5′UTRs of different length, sequence and structure potential. Importantly, the ‘−8 nt toeprint’ was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5′UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5′UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes.

Download Full-text

Insulin fails to stimulate muscle protein synthesis in sepsis despite unimpaired signaling to 4E-BP1 and S6K1

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2001.281.5.e1045 ◽

2001 ◽

Vol 281 (5) ◽

pp. E1045-E1053 ◽

Cited By ~ 44

Author(s):

Thomas C. Vary ◽

Leonard S. Jefferson ◽

Scot R. Kimball

Keyword(s):

Protein Synthesis ◽

Translation Initiation ◽

Insulin Signaling ◽

Insulin Action ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Mrna Translation ◽

Initiation Factor ◽

S6 Kinase ◽

Eif4f Complex

Induction of sepsis in rats causes an inhibition of protein synthesis in skeletal muscle that is resistant to the stimulatory actions of insulin. To gain a better understanding of the underlying reason for this lack of response, the present study was undertaken to investigate sepsis-induced alterations in insulin signaling to regulatory components of mRNA translation. Experiments were performed in perfused hindlimb preparations from rats 5 days after induction of a septic abscess. Sepsis resulted in a 50% reduction in protein synthesis in the gastrocnemius. Protein synthesis in muscles from septic rats, but not controls, was unresponsive to stimulation by insulin. The insulin-induced hyperphosphorylation response of the translation repressor protein 4E-binding protein 1 (4E-BP1) and of the 70-kDa S6 kinase (S6K1) (1), two targets of insulin action on mRNA translation, was unimpaired in gastrocnemius of septic rats. Hyperphosphorylation of 4E-BP1 in response to insulin resulted in its dissociation from the inactive eukaryotic initiation factor (eIF)4E · 4E-BP1 complex in both control and septic rats. However, assembly of the active eIF4F complex as assessed by the association of eIF4E with eIF4G did not follow the pattern predicted by the increased availability of eIF4E resulting from changes in the phosphorylation of 4E-BP1. Indeed, sepsis caused a dramatic reduction in the amount of eIF4G associated with eIF4E in the presence or absence of insulin. Thus the inability of insulin to stimulate protein synthesis during sepsis may be related to a defect in signaling to a step in translation initiation involved in assembly of an active eIF4F complex.

Download Full-text