Initiation Of Protein Synthesis Research Articles

Translocation and protein complex co-localization of mTOR is associated with postprandial myofibrillar protein synthesis at rest and after endurance exercise.

Translocation and colocalization of mechanistic target of rapamycin complex 1 (mTORC1) with regulatory proteins represents a critical step in translation initiation of protein synthesis in vitro. However, mechanistic insight into the control of postprandial skeletal muscle protein synthesis rates at rest and after an acute bout of endurance exercise in humans is lacking. In crossover trials, eight endurance‐trained men received primed‐continuous infusions of L‐[ring‐2H5]phenylalanine and consumed a mixed‐macronutrient meal (18 g protein, 60 g carbohydrates, 17 g fat) at rest (REST) and after 60 min of treadmill running at 70% VO2peak (EX). Skeletal muscle biopsies were collected to measure changes in phosphorylation and colocalization in the mTORC1‐pathway, in addition to rates of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis. MyoPS increased (P < 0.05) above fasted in REST (~2.1‐fold) and EX (~twofold) during the 300 min postprandial period, with no corresponding changes in MitoPS (P > 0.05). TSC2/Rheb colocalization decreased below fasted at 60 and 300 min after feeding in REST and EX (P < 0.01). mTOR colocalization with Rheb increased above fasted at 60 and 300 min after feeding in REST and EX (P < 0.01), which was consistent with an increased phosphorylation 4E‐BP1Thr37/46 and rpS6ser240/244 at 60 min. Our data suggest that MyoPS, but not MitoPS, is primarily nutrient responsive in trained young men at rest and after endurance exercise. The postprandial increase in MyoPS is associated with an increase in mTOR/Rheb colocalization and a reciprocal decrease in TSC2/Rheb colocalization and thus likely represent important regulatory events for in vivo skeletal muscle myofibrillar mRNA translation in humans.

Rocaglates as dual-targeting agents for experimental cerebral malaria

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.

Eukaryotic initiation factor 4A2 (EIF4A2) expression in colorectal cancer and prediction of prognosis.

664 Background: Eukaryotic initiation factor 4A2(EIF4A2) is a member of the eukaryotic initiation factor 4A family and it is one of the protein-synthesis initiation factors, which are involved in the binding of messenger RNA to the ribosome. The prognostic roles of EIF4A2 in breast cancer, melanoma and lung cancer have been studied. However, the clinical significance and biologic role of EIF4A2 in colorectal cancer remain unknown. Methods: We used quantitative real-time polymerase chain reaction to compare expression of eIF4A2 mRNA between 72 paired colorectal cancer tissues and non-tumor colorectal tissues. We used immunohistochemistry to study the expression of EIF4A2 in 310 colorectal cancer patients under surgical resection, 247 of them received curative surgery. We also analyzed correlation between EIF4A2 expression and clinicopathological characteristics. The biological function of EIF4A2 on colorectal cancer cells were determined in vitro by knock-downing EIF4A2 in HCT116 and DLD1 cell lines. We performed MTT tests, colony formation assays, transwell and wound healing assays. Results: EIF4A2 mRNA was significantly higher in colorectal cancer tissues compared to the paired non-tumor colorectal tissues(P = 0.0034). Over-expression of EIF4A2 was prognostic of shorter overall survival(P = 0.002) and shorter disease-free survival(P = 0.042). Over-expression of EIF4A2 was significantly correlated with more TNM stage IV(P = 0.012). Multivariate analysis suggested EIF4A2 as an independent predictor of overall survival (hazard ratio 2.041[95% CI 1.385 to 3.008], P &lt; 0.001) and disease-free survival (hazard ratio 1.537[95% CI 1.003 to 2.355], P = 0.049). Functionally, knockdown of EIF4A2 in HCT116 and DLD1 cell lines exerted tumor-suppressive effects by significantly inhibiting cell proliferation, colony formation,migration and invasion(P &lt; 0.05). Conclusions: EIF4A2 is highly expressed in colorectal cancer and predicts poor prognosis. EIF4A2 could be served as a potential prognostic marker for colorectal cancer patients.

Plant growth and fertility requires functional interactions between specific PABP and eIF4G gene family members.

The initiation of protein synthesis requires the involvement of the eukaryotic translation initiation factor (eIF) 4G to promote assembly of the factors needed to recruit a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, those in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization. Species of the Brassicaceae and the Cleomaceae also express a divergent eIFiso4G isoform, referred to as eIFiso4G2, not found elsewhere in the plant kingdom. Despite their divergence, eIF4G and eIFiso4G interact with eIF4A, eIF4B, and eIF4E isoforms needed for binding an mRNA. eIF4G and eIFiso4G also interact with the poly(A)-binding protein (PABP) which promotes ribosome recruitment to an mRNA. Increasing the complexity of such an interaction, however, Arabidopsis also expresses three PABP isoforms (PAB2, PAB4, and PAB8) in vegetative and reproductive tissues. In this study, the functional interactions among the eIF4G and the widely-expressed PABP isoforms were examined. Loss of PAB2 or PAB8 in combination with loss of eIF4G or eIFiso4G had little to no effect on growth or fertility whereas pab2 pab8 eif4g or pab2 pab8 eifiso4g1/2 mutants exhibited smaller stature and reduced fertility. Although the pab4 eifiso4g1 mutant grows normally and is fertile, pab4 eif4g or pab4 eifiso4g2 mutants could not be isolated. Even pab4/PAB4 eif4g/eIF4G heterozygous plants exhibited growth defects and low fertility. Mutant co-inheritance analysis in reciprocal crosses with wild-type plants revealed that most ovaries and pollen from pab4/PAB4 eif4g/eIF4G plants were PAB4 eif4g. Similarly, co-inheritance studies with pab4/PAB4 eifiso4g2/eIFiso4G2 plants suggested most ovaries were PAB4 eifiso4g2. These results suggest that a functional interaction between PAB4 and eIF4G and between PAB4 and eIFiso4G2 is required for growth and normal fertility.

Differential effects of SUMO1 and SUMO3 on PKR activation and stability

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a serine/threonine kinase that exerts its own phosphorylation and the phosphorylation of the α subunit of the protein synthesis initiation factor eIF-2α. PKR was identified as a target of SUMOylation and the triple PKR-SUMO deficient mutant on Lysine residues K60-K150-K440 has reduced PKR activity. We report that SUMO1 and SUMO3 expression exert differential effects on PKR localization, activation and stability. SUMO1 or SUMO3 did not alter the repartition of PKR in the cytoplasm and the nucleus. However, in SUMO3-expressing cells PKR was found more concentrated around the perinuclear membrane and was recruited from small speckles to nuclear dots. Interestingly, SUMO1 expression alone resulted in PKR and eIF-2α activation, whereas SUMO3 reduced PKR and eIF-2α activation upon viral infection or dsRNA transfection. In addition, encephalomyocarditis virus (EMCV) enhanced PKR conjugation to SUMO1 and SUMO3 but only SUMO3 expression promoted caspase-dependent EMCV-induced PKR degradation. Furthermore, the higher EMCV-induced PKR activation by SUMO1 was correlated with an inhibition of EMCV. Importantly SUMO1, by inducing PKR activation in the absence of viral infection, and SUMO3, by counteracting both PKR activation and stability upon viral infection, shed a new light on the differential effects of SUMO-modified PKR.

Insights into Structural and Mechanistic Features of Viral IRES Elements.

Internal ribosome entry site (IRES) elements are cis-acting RNA regions that promote internal initiation of protein synthesis using cap-independent mechanisms. However, distinct types of IRES elements present in the genome of various RNA viruses perform the same function despite lacking conservation of sequence and secondary RNA structure. Likewise, IRES elements differ in host factor requirement to recruit the ribosomal subunits. In spite of this diversity, evolutionarily conserved motifs in each family of RNA viruses preserve sequences impacting on RNA structure and RNA–protein interactions important for IRES activity. Indeed, IRES elements adopting remarkable different structural organizations contain RNA structural motifs that play an essential role in recruiting ribosomes, initiation factors and/or RNA-binding proteins using different mechanisms. Therefore, given that a universal IRES motif remains elusive, it is critical to understand how diverse structural motifs deliver functions relevant for IRES activity. This will be useful for understanding the molecular mechanisms beyond cap-independent translation, as well as the evolutionary history of these regulatory elements. Moreover, it could improve the accuracy to predict IRES-like motifs hidden in genome sequences. This review summarizes recent advances on the diversity and biological relevance of RNA structural motifs for viral IRES elements.

Up-regulation Of EIF3e Is Associated with The Progression of Esophageal Squamous Cell Carcinoma and Poor Prognosis in Patients.

Introduction: Esophageal cancer is one of the most common malignant tumors in the world. Eukaryotic translation initiation factors 3e (eIF3e) makes a notable difference in the initiation of protein synthesis and tumor progression. However, the role of eIF3e in ESCC has not been revealed yet. This study aims to investigate the bio-functional and prognostic role of eIF3e in human ESCC tissues and cells.Methods: Immunohistochemical staining and Western blot were performed to detect the eIF3e expression in ESCC patients' tissues. The Kaplan-Meier product limit method and Cox regression were conducted to analyze the association between eIF3e expression, together with other related clinical/pathological features, and patients' prognosis. In the analysis of bio-functional role of eIF3e, CCK-8 and Transwell assay were performed to compare the proliferative and migrative ability after knockdown of eIF3e.Results: Up-regulation of eIF3e were demonstrated in ESCC tissues compared with the corresponding para-cancerous tissues. Overexpression of eIF3e was associated with deep tumor depth, lymph nodes metastasis, and advanced TNM stage. Importantly, the patients with high eIF3e expression suffered shorter overall and disease-free survival. Lymph node metastasis and histological grade served as independent prognostic predictors in patients' prognosis. Knockdown of eIF3e could inhibit cell proliferation and migration, in vitro.Conclusions: The eIF3e expression, or combined with other members of eIF3 complex, might predict poor prognosis of ESCC and serve as a potential breakthrough in the multimodality therapy of ESCC.

Trypanosoma brucei EIF4E2 cap-binding protein binds a homolog of the histone-mRNA stem-loop-binding protein.

Trypanosomatids are parasitic protozoans characterized by several unique structural and metabolic processes that include exquisite mechanisms associated with gene expression and regulation. During the initiation of protein synthesis, for instance, mRNA selection for translation seems to be mediated by different eIF4F-like complexes, which may play a significant role in parasite adaptation to different hosts. In eukaryotes, the heterotrimeric eIF4F complex (formed by eIF4E, eIF4G, and eIF4A) mediates mRNA recognition and ribosome binding and participates in various translation regulatory events. Six eIF4Es and five eIF4Gs have been described in trypanosomatids with several of these forming different eIF4F-like complexes. This has raised questions about their role in differential mRNA translation. Here we have studied further TbEIF4E2, the least known eIF4E homologue from Trypanosoma brucei, and found that it is not associated with an eIF4G homolog. It is, however, associated with mature mRNAs and binds to a histone mRNA stem-loop-binding protein (SLBP), one of two Trypanosoma SLBP homologs (TbSLBP1 and TbSLBP2). TbSLBP1 is more similar to the mammalian counterpart while TbSLBP2 is exclusive to trypanosomatids and related organisms. TbSLBP2 binds to TbEIF4E2 through a conserved central region missing in other SLBP homologs. Both SLBPs, as well as TbEIF4E2, were found to localize to the cytoplasm. TbEIF4E2 and TbSLBP2 are differentially expressed during cell culture, being more abundant in early-log phase, with TbSLBP2 also showing cell-cycle dependent expression. The new data reinforce unique aspects of the trypanosomatid eIF4Es, with the TbEIF4E2-TbSLBP complex possibly having a role in differential selection of mRNAs containing stem-loop structures.

Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus internal ribosome entry sites.

Viral internal ribosomes entry site (IRES) elements coordinate the recruitment of the host translation machinery to direct the initiation of viral protein synthesis. Within hepatitis C virus (HCV)-like IRES elements, the sub-domain IIId(1) is crucial for recruiting the 40S ribosomal subunit. However, some HCV-like IRES elements possess an additional sub-domain, termed IIId2, whose function remains unclear. Herein, we show that IIId2 sub-domains from divergent viruses have different functions. The IIId2 sub-domain present in Seneca valley virus (SVV), a picornavirus, is dispensable for IRES activity, while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting the assembly of 80S ribosomes. Consequently, this negatively affects the replication of CSFV and BDV. Finally, we show that the SVV IIId2 sub-domain is required for efficient viral RNA synthesis and growth of SVV, but not for IRES function. This study sheds light on the molecular evolution of viruses by clearly demonstrating that conserved RNA structures, within distantly related RNA viruses, have acquired different roles in the virus life cycles.

Heterologous Gene Expression of N-Terminally Truncated Variants of LipPks1 Suggests a Functionally Critical Structural Motif in the N-terminus of Modular Polyketide Synthase.

Streptomyces genomes have a high G + C content and typically use an ATG or GTG codon to initiate protein synthesis. Although gene-finding tools perform well in low GC genomes, it is known that the accuracy in predicting a translational start site (TSS) is much less for high GC genomes. LipPks1 is a Streptomyces-derived, well-characterized modular polyketide synthase (PKS). Using this enzyme as a model, we experimentally investigated the effects of alternative TSSs using a heterologous host, Streptomyces venezuelae. One of the TSSs employed boosted the protein level by 59-fold and the product yield by 23-fold compared to the originally annotated start codon. Interestingly, a structural model of the PKS indicated the presence of a structural motif in the N-terminus, which may explain the observed different protein levels together with a proline and arginine-rich sequence that may inhibit translational initiation. This structure was also found in six other modular PKSs that utilize noncarboxylated starter substrates, which may guide the selection of optimal TSSs in conjunction with start-codon prediction software.

Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment.

The actual strategy to improve current therapies in advanced prostate cancer involves targeting genes activated by androgen withdrawal, either to delay or prevent the emergence of the castration-refractory phenotype. However, these genes are often implicated in several physiological processes, and long-term inhibition of survival proteins might be accompanied with cytotoxic effects. To avoid this problem, an alternative therapeutic strategy relies on the identification and use of compounds that disrupt specific protein-protein interactions involved in androgen withdrawal. Specifically, the interaction of the chaperone protein Hsp27 with the initiation factor eIF4E leads to the protection of protein synthesis initiation process and enhances cell survival during cell stress induced by castration or chemotherapy. Thus, in this work we aimed at i) identifying the interaction site of the Hsp27/eIF4E complex and ii) interfere with the relevant protein/protein association mechanism involved in castration-resistant progression of prostate cancer. By a combination of experimental and modeling techniques, we proved that eIF4E interacts with the C-terminal part of Hsp27, preferentially when Hsp27 is phosphorylated. We also observed that the loss of this interaction increased cell chemo-and hormone-sensitivity. In order to find a potential inhibitor of Hsp27/eIF4E interaction, BRET assays in combination with molecular simulations identified the phenazine derivative 14 as the compound able to efficiently interfere with this protein/protein interaction, thereby inhibiting cell viability and increasing cell death in chemo- and castration-resistant prostate cancer models in vitro and in vivo.

Kinetic analyses of phosphorylated and non-phosphorylated eIFiso4E binding to mRNA cap analogues

Phosphorylation of eukaryotic initiation factors was previously shown to interact with m7G cap and play an important role in the regulation of translation initiation of protein synthesis. To gain further insight into the phosphorylation process of plant protein synthesis, the kinetics of phosphorylated wheat eIFiso4E binding to m7G cap analogues were examined. Phosphorylation of wheat eIFiso4E showed similar kinetic effects to human eIF4E binding to m7-G cap. Phosphorylation of eIFiso4E decreased the kinetic rate (2-fold) and increased the dissociation rate (2-fold) as compared to non-phosphorylated eIFiso4E binding to both mono- and di-nucleotide analogues at 22°C. Phosphorylated and non-phosphorylated eIFiso4E-m7G cap binding rates were found to be independent of concentration, suggesting conformational changes were rate limiting. Rate constant for phosphorylated and non-phosphorylated eIFiso4E binding to m7-G cap increased with temperature. Phosphorylation of eIFiso4E decreased (2-fold) the activation energy for both m7-G cap analogues binding as compared to non-phosphorylated eIFiso4E. The reduced energy barrier for the formation of eIFiso4E-m7-G cap complex suggests a more stable platform for further initiation complex formation and possible means of adapting variety of environmental conditions. Furthermore, the formation of phosphorylated eIFiso4E-cap complex may contribute to modulation of the initiation of protein synthesis in plants.

Abstract 1335: Deubiquitinase OTUD6B isoforms are important regulators of growth and proliferation

Abstract Deubiquitinases (DUBs) are increasingly linked to the regulation of fundamental processes in normal and cancer cells, including DNA replication and repair, programmed cell death, and oncogenes and tumor suppressors signaling. Here evidence is presented that the deubiquitinase OTUD6B regulates protein synthesis in non-small cell lung cancer (NSCLC) cells, operating downstream from mTORC1. OTUD6B associates with the protein synthesis initiation complex and modifies components of the 48S preinitiation complex. The two main OTUD6B splicing isoforms seem to regulate protein synthesis in opposing fashions: the long OTUD6B-1 isoform is inhibitory, while the short OTUD6B-2 isoform stimulates protein synthesis. These properties affect NSCLC cell proliferation, since OTUD6B-1 represses DNA synthesis while OTUD6B-2 promotes it. Mutational analysis and downstream mediators suggest that the two OTUD6B isoforms modify different cellular targets. OTUD6B-2 influences the expression of cyclin D1 by promoting its translation while regulating (directly or indirectly) c-Myc protein stability. This phenomenon appears to have clinical relevance as NSCLC cells and human tumor specimens have a reduced OTUD6B-1/OTUD6B-2 mRNA ratio compared to normal samples. The global OTUD6B expression level does not change significantly between non-neoplastic and malignant tissues, suggesting that modifications of splicing factors during the process of transformation are responsible for this isoform switch. Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Maurizio Bocchetta, Clodia Osipo, Anna Sobol. Deubiquitinase OTUD6B isoforms are important regulators of growth and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1335. doi:10.1158/1538-7445.AM2017-1335

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits.

Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N- and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, the in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli and by complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3.IMPORTANCE Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.

Cut in translation: ribosome-dependent mRNA decay.

Transcription and translation are two complex mechanisms that are tightly coupled in prokaryotic cells. Even before the completion of transcription, ribosomes attach to the nascent mRNA and initiate protein synthesis. Remarkably, recent publications have indicated an association between translation and decay of certain mRNAs. In this issue of The EMBO Journal, Leroy etal (2017) depicts a fascinating mechanism of mRNA degradation, which involves the ribosome-associated ribonuclease Rae1 in Bacillus subtilis In a translation-dependent manner, Rae1 binds the ribosomal aminoacylation (A)-site and cleaves between specific codons of the targeted mRNA.

Prematurity Blunts Feeding‐Induced Stimulation of Translation Initiation Signaling and Protein Synthesis in Muscle of Neonatal Piglets

BackgroundPreterm infants commonly experience extrauterine growth faltering, with many weighing less than the 10th percentile for age at hospital discharge. Growth restriction during the neonatal period may contribute to both short‐ and long‐term morbidities.ObjectiveTo determine how preterm birth alters the protein anabolic response of muscle to feeding in a neonatal piglet model.MethodsPiglets were delivered at term (112 d) or preterm (103 d) gestation by Cesarean section and immediately fitted with a jugular catheter for delivery of total parenteral nutrition (240 mL/kg/d). Following a 4 hr fast at 3 d of age, pigs were fasted an additional hour or fed an enteral elemental meal (4 groups: PF, preterm fasted; PP, preterm postprandial; TF, term fasted; TP, term postprandial). Flooding dose of L[4‐3H]phenylalanine was injected at 30 min relative to the meal for measurement of protein synthesis, and tissues collected at 60 min.ResultsBirth weight of preterm (n = 48, 4 litters) pigs was lower (P &lt; 0.01) than that of term (n = 42, 4 litters) pigs. Body weight gain was also lower (g/kg/d; P = 0.01) in preterm than term pigs despite equivalent nutrient delivery. Preterm pigs had greater kidney (g/kg; P = 0.03) and liver (P &lt; 0.01) masses, but lower (P = 0.04) longissimus dorsi (LD) mass than term pigs. Gestational age at birth (GAB) did not affect gastrocnemius, soleus, or heart masses. GAB did not affect plasma glucose or insulin of fasted pigs, but among fed pigs both plasma insulin and glucose rose more rapidly, and reached a higher peak, in term than preterm pigs. Plasma insulin‐like growth factor‐1 (IGF‐1) was lower (P = 0.045) in preterm than term pigs at 60 min. Feeding increased LD fractional protein synthesis rates (Ks) in both GAB groups, but the response was lower in preterm than term pigs (PF = TF &lt; PP &lt; TP; P &lt; 0.01). LD protein kinase B phosphorylation followed a similar pattern (PF = TF &lt; PP &lt; TP; P &lt; 0.01), indicating GAB‐dependent insulin signaling pathway activation. Formation of active eukaryotic initiation factor (eIF) eIF4E·eIF4G complex, and phosphorylation of S6 kinase (S6K1) and eIF4E‐binding protein 1 (4EBP1) in the LD also reflected this trend (all proteins PF = TF &lt; PP &lt; TP; P &lt; 0.01), indicating enhanced translation initiation in term vs. preterm fed pigs. Microtubule‐associated protein light chain 3 II (LC3II) to total LC3 ratio in LD was decreased (P &lt; 0.01) by feeding regardless of GAB, indicating reduced autophagy‐lysosome system activation. There were no differences between groups in LD muscle RING‐finger protein‐1 (MuRF1) or atrogin‐1 abundance, indices of ubiquitin‐proteasome system activity. Similarly, no differences were observed between groups in phosphorylation of eukaryotic elongation factor 2 (eEF2) or eukaryotic initiation factor 2α (eIF2α), or in abundance of late endosomal/lysosomal adaptor, MAPK and MTOR activator 2 (LAMTOR2), Ras‐related GTP‐binding protein A (RagA), RagC, or DEP domain containing MTOR‐interacting protein (Deptor).ConclusionsPreterm piglets exhibit decreased body weight gain and feeding‐induced muscle protein synthesis compared to term piglets. These differences are due to blunted insulin response and reduced translation initiation signaling.Support or Funding InformationNIH HD085573, USDA CRIS 6250‐51000‐055, NIH HD072891, USDA NIFA 2013‐67015‐20438.

A harmine-derived beta-carboline displays anti-cancer effects in vitro by targeting protein synthesis

Growing evidence indicates that protein synthesis is deregulated in cancer onset and progression and targeting this process might be a selective way to combat cancers. While harmine is known to inhibit DYRK1A and intercalate into the DNA, tri-substitution was shown previously to modify its activity profile in favor of protein synthesis inhibition. In this study, we thus evaluated the optimized derivative CM16 in vitro anti-cancer effects unfolding its protein synthesis inhibition activity. Indeed, the growth inhibitory profile of CM16 in the NCI 60-cancer-cell-line-panel correlated with those of other compounds described as protein synthesis inhibitors. Accordingly, CM16 decreased in a time- and concentration-dependent manner the translation of neosynthesized proteins in vitro while it did not affect mRNA transcription. CM16 rapidly penetrated into the cell in the perinuclear region of the endoplasmic reticulum where it appears to target translation initiation as highlighted by ribosomal disorganization. More precisely, we found that the mRNA expression levels of the initiation factors EIF1AX, EIF3E and EIF3H differ when comparing resistant or sensitive cell models to CM16. Additionally, CM16 induced eIF2α phosphorylation. Those effects could explain, at least partly, the CM16 cytostatic anti-cancer effects observed in vitro while neither cell cycle arrest nor DNA intercalation could be demonstrated. Therefore, targeting protein synthesis initiation with CM16 could represent a new promising alternative to current cancer therapies due to the specific alterations of the translation machinery in cancer cells as recently evidenced with respect to EIF1AX and eIF3 complex, the potential targets identified in this present study.

Abstract A17: Probing a translational switch involved in tumor angiogenesis

Abstract As a tumor proliferates, it rapidly outgrows its blood supply, creating an oxygen-starved environment. Low oxygenation (hypoxia) suppresses the canonical biochemical pathway through which the translation of messenger RNAs (mRNAs) into proteins by the ribosome is typically initiated. Despite shutting down this general mechanism for initiating protein synthesis, the tumor can continue to initiate translation of a subset of mRNAs that are involved in angiogenesis and tumor survival. To explain this observation, it has been proposed that, in addition to the eukaryotic mRNA-specific 7-methyl guanosine cap at the 5' end, these pro-angiogenic mRNAs, of which Fibroblast Growth Factor-9 (FGF9) and Hypoxia Inducible Factor 1α (HIF1α) are prototypical members, contain highly stable structures in their 5' untranslated regions. These structures, also referred to as Cap Independent Translation Enhancers (CITEs), form part of a switch that activates a non-canonical pathway for initiating translation, presumably by binding directly to eukaryotic initiation factor 4G (eIF4G), thereby recruiting ribosomes in a manner distinct from the canonical 5' cap-dependent mechanism. To test this hypothesis, here we report the binding affinities of pro-angiogenic mRNAs, as well as control mRNAs lacking CITEs, to eIF4G as a measure of their ability to initiate translation in the hypoxic environment of a tumor. Understanding the dynamics of eIF4G-CITE interactions can inform the design and development of novel angiogenesis inhibitors that function by targeting these interactions. Citation Format: Maya Ramachandran, Somdeb Mitra, Ruben L. Gonzalez, Jr. Probing a translational switch involved in tumor angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A17.

The Intersubunit Bridge B1b of the Bacterial Ribosome Facilitates Initiation of Protein Synthesis and Maintenance of Translational Fidelity

In bacteria, ribosomal subunits are connected via 12 intersubunit bridges involving RNA–RNA, RNA–protein, and protein–protein interactions. The only protein–protein bridge in the ribosome is ribosomal intersubunit bridge 1b (B1b), which is mainly formed by the bacterial protein L31 (bL31) and connects the head domain of 30S subunit and the central protuberance of the 50S subunit. It is known to be the most dynamic intersubunit bridge. Here, we have evaluated the role of bL31 and thereby the bridge B1b in the working cycle of the ribosome. First, bL31-deficient ribosomes are severely compromised in their ability to ensure translational fidelity particularly in reading frame maintenance in vivo. Second, in the absence of bL31, the rate of initiation is significantly reduced both in vivo and in vitro. Third, polysome profile and subunit reassociation assays demonstrate that bL31 is important for stabilizing subunit joining in vivo and in vitro. Together, our results demonstrate that bL31 is important for determining translational fidelity and stabilizing subunit association. We conclude that the only protein–protein intersubunit bridge of the bacterial ribosome facilitates translation initiation and is essential for maintaining the reading frame of mRNA translation.

Measurements of translation initiation from all 64 codons in E. coli.

Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the ‘start codons’ for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.