Eukaryotic Translation Elongation Factor 1A (eEF1A) Domain I from S. cerevisiae Is Required but Not Sufficient for Inter-Species Complementation

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae.     

Phosphatidylethanolamine in Trypanosoma brucei is organized in two separate pools and is synthesized exclusively by the Kennedy pathway

Phosphatidylethanolamine is a major phospholipid class of all eukaryotic cells. It can be synthesized via the CDP-ethanolamine branch of the Kennedy pathway, by decarboxylation of phosphatidylserine, or by base exchange with phosphatidylserine. The contributions of these pathways to total phosphatidylethanolamine synthesis have remained unclear. Although Trypanosoma brucei, the causative agent of human and animal trypanosomiasis, has served as a model organism to elucidate the entire reaction sequence for glycosylphosphatidylinositol biosynthesis, the pathways for the synthesis of the major phospholipid classes have received little attention. We now show that disruption of the CDP-ethanolamine branch of the Kennedy pathway using RNA interference results in dramatic changes in phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. By targeting individual enzymes of the pathway, we demonstrate that de novo phosphatidylethanolamine synthesis in T. brucei procyclic forms is strictly dependent on the CDP-ethanolamine route. Interestingly, the last step in the Kennedy pathway can be mediated by two separate activities leading to two distinct pools of phosphatidylethanolamine, consisting of predominantly alk-1-enyl-acyl- or diacyl-type molecular species. In addition, we show that phosphatidylserine in T. brucei procyclic forms is synthesized exclusively by base exchange with phosphatidylethanolamine.     

The hypusine-containing translation factor eIF5A

Abstract

In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A (eIF5A) and its bacterial ortholog elongation factor P (EF-P) were initially characterized based on their ability to stimulate methionyl-puromycin (Met-Pmn) synthesis, a model assay for protein synthesis; however, the function of these factors in cellular protein synthesis has been difficult to resolve. Interestingly, a conserved lysine residue in eIF5A is post-translationally modified to hypusine and the corresponding lysine residue in EF-P from at least some bacteria is modified by the addition of a β-lysine moiety. In this review, we provide a summary of recent data that have identified a novel role for the translation factor eIF5A and its hypusine modification in the elongation phase of protein synthesis and more specifically in stimulating the production of proteins containing runs of consecutive proline residues.     

The elongation, termination, and recycling phases of translation in eukaryotes

This work summarizes our current understanding of the elongation and termination/recycling phases of eukaryotic protein synthesis. We focus here on recent advances in the field. In addition to an overview of translation elongation, we discuss unique aspects of eukaryotic translation elongation including eEF1 recycling, eEF2 modification, and eEF3 and eIF5A function. Likewise, we highlight the function of the eukaryotic release factors eRF1 and eRF3 in translation termination, and the functions of ABCE1/Rli1, the Dom34:Hbs1 complex, and Ligatin (eIF2D) in ribosome recycling. Finally, we present some of the key questions in translation elongation, termination, and recycling that remain to be answered.     

Inhibition of translation by cytotrienin A--a member of the ansamycin family

The ansamycins are a diverse and often physiologically active group of compounds that include geldanamycin and rifamycin, inhibitors of heat shock protein 90 and prokaryotic DNA-dependent RNA synthesis, respectively. Cytotrienin A is an ansamycin-type small molecule with potent antiproliferative and proapoptotic properties. Here, we report that this compound inhibits eukaryotic protein synthesis by targeting translation elongation and interfering with eukaryotic elongation factor 1A function. We also find that cytotrienin A prevents HUVEC tube formation and diminishes microvessel formation in the chorioallantoic membrane assay. These results provide a molecular understanding into cytotrienin A's previously reported properties as an anticancer apoptosis-inducing drug.     

The translation elongation factor 1A in tumorigenesis,signal transduction and apoptosis: Review article

Summary. An increasing number of evidences suggest the involvement of the eukaryotic elongation factor 1A, a core component of the protein synthesis machinery, at the onset of cell transformation. In fact, eEF1A is shown to be up-regulated in cell death; moreover, it seems to be involved in the regulation of ubiquitin-mediated protein degradation. In addition, eEF1A undergoes several post-translational modifications, mainly phosphorylation and methylation, that generally influence the activity of the protein. This article summarizes the present knowledges on the several extra-translational roles of eEF1A also in order to understand as the protein synthesis regulatory mechanisms could offer tools for cancer intervention.     

Cultured Aedes albopictus mosquito cells accumulate elongation factor-1 alpha (EF-1 alpha) during serum starvation

We examined survival, growth and protein synthesis in mosquito cells that had been maintained for up to 21 days in serum-free medium. On polyacrylamide gels, protein bands from "starved" cells remained discrete, and despite low levels of incorporation, radiolabeled bands were detectable, suggesting that low levels of protein synthesis were sustained. A prominent band that accumulated in serum-starved cells was digested with trypsin and analyzed by tandem mass spectrometry, which identified the protein as eukaryotic elongation factor (EF)-1 alpha EF-1 alpha is well-conserved among species, and differential accumulation of EF-1 alpha in serum-starved cells was verified by western blotting using a primary antibody to the homologous protein from Trypanosoma brucei. Aside from its importance in the elongation step of protein synthesis, EF-1 alpha has been shown to have a number of non-canonical functions, including interaction with viral RNA and a potential role in apoptosis. We anticipate that the prolonged viability of mosquito cells in serum-free medium may provide a system to explore whether EF-1 alpha accumulation is an adaptive response compatible with resumption of growth in the event that nutrients are replenished, or whether the excess EF-1 alpha represents an irreversible commitment to an apoptotic pathway.     

Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats

In the rat, denervation and hindlimb unloading are two commonly employed models used to study skeletal muscle atrophy. In these models, muscle atrophy is generally produced by a decrease in protein synthesis and an increase in protein degradation. The decrease in protein synthesis has been suggested to occur by an inhibition at the level of protein translation. To better characterize the regulation of protein translation, we investigated the changes that occur in various translation initiation and elongation factors. We demonstrated that both hindlimb unloading and denervation produce alterations in the phosphorylation and/or total amount of the 70-kDa ribosomal S6 kinase, eukaryotic initiation factor 2 alpha-subunit, and eukaryotic elongation factor 2. Our findings indicate that the regulation of these protein translation factors differs between the models of atrophy studied and between the muscles evaluated (e.g., soleus vs. extensor digitorum longus).     

Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3

The translation elongation machinery in fungi differs from other eukaryotes in its dependence upon eukaryotic elongation factor 3 (eEF3). eEF3 is essential in vivo and required for each cycle of the translation elongation process in vitro. Models predict eEF3 affects the delivery of cognate aminoacyl-tRNA, a function performed by eEF1A, by removing deacylated tRNA from the ribosomal Exit site. To dissect eEF3 function and its link to the A-site activities of eEF1A, we have identified a temperature-sensitive allele of the YEF3 gene. The F650S substitution, located between the two ATP binding cassettes, reduces both ribosome-dependent and intrinsic ATPase activities. In vivo this mutation increases sensitivity to aminoglycosidic drugs, causes a 50% reduction of total protein synthesis at permissive temperatures, slows run-off of polyribosomes, and reduces binding to eEF1A. Reciprocally, excess eEF3 confers synthetic slow growth, increased drug sensitivity, and reduced translation in an allele specific fashion with an E122K mutation in the GTP binding domain of eEF1A. In addition, this mutant form of eEF1A shows reduced binding of eEF3. Thus, optimal in vivo interactions between eEF3 and eEF1A are critical for protein synthesis.     

A Structural Domain Mediates Attachment of Ethanolamine Phosphoglycerol to Eukaryotic Elongation Factor 1A in Trypanosoma brucei

Ethanolamine phosphoglycerol (EPG) represents a protein modification that so far has only been found in eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is covalently attached to two conserved glutamate residues located in domains II and III of eEF1A. In contrast, Trypanosoma brucei eEF1A contains a single EPG attached to Glu362 in domain III. The sequence and/or structural requirements for covalent linkage of EPG to eEF1A have not been determined for any organism. Using a combination of biosynthetic labelling of parasites with tritiated ethanolamine and mass spectrometry analyses, we demonstrate that replacement of Glu362 in T. brucei eEF1A by site-directed mutagenesis prevents EPG attachment, whereas single or multiple amino acid substitutions around the attachment site are not critical. In addition, by expressing a series of eEF1A deletion mutants in T. brucei procyclic forms, we demonstrate that a peptide consisting of 80 amino acids of domain III of eEF1A is sufficient for EPG attachment to occur. Furthermore, EPG addition also occurs if domain III of eEF1A is fused to a soluble reporter protein. To our knowledge, this is the first report addressing amino acid sequence, or structure, requirements for EPG modification of eEF1A in any organism. Using T. brucei as a model organism, we show that amino acid substitutions around the modification site are not critical for EPG attachment and that a truncated version of domain III of eEF1A is sufficient to mediate EPG addition.     

Colonic Mucosal Proteome Signature Reveals Reduced Energy Metabolism and Protein Synthesis but Activated Autophagy during Anorexia‐Induced Malnutrition in Mice

Anorexia nervosa is an eating disorder often associated with intestinal disorders. To explore the underlying mechanisms of these disorders, the colonic proteome was evaluated during activity‐based anorexia. Female C57Bl/6 mice were randomized into three groups: Control, Limited Food Access (LFA) and Activity‐Based Anorexia (ABA). LFA and ABA mice had a progressive limited access to food but only ABA mice had access to an activity wheel. On colonic mucosal protein extracts, a 2D PAGE‐based comparative proteomic analysis was then performed and differentially expressed proteins were identified by LC‐ESI‐MS/MS. Twenty‐seven nonredundant proteins that were differentially expressed between Control, LFA, and ABA groups were identified. ABA mice exhibited alteration of several mitochondrial proteins involved in energy metabolism such as dihydrolipoyl dehydrogenase and 3‐mercaptopyruvate sulfurtransferase. In addition, a downregulation of mammalian target of rapamycin (mTOR) pathway was observed leading, on the one hand, to the inhibition of protein synthesis, evaluated by puromycin incorporation and mediated by the increased phosphorylation of eukaryotic elongation factor 2, and on the other hand, to the activation of autophagy, assessed by the increase of the marker of autophagy, form LC3‐phosphatidylethanolamine conjugate/Cytosolic form of Microtubule‐associated protein 1A/1B light chain 3 (LC3II/LC3I) ratio. Colonic mucosal proteome is altered during ABA suggesting a downregulation of energy metabolism. A decrease of protein synthesis and an activation of autophagy were also observed mediated by mTOR pathway.     

Structural Complementation of the Catalytic Domain of Pseudomonas Exotoxin A

The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions—supplied exogenously as genetic elements or purified protein fragments—had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.     

Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Balpha

The crystal structure of a complex between the protein biosynthesis elongation factor eEF1A (formerly EF-1alpha) and the catalytic C terminus of its exchange factor, eEF1Balpha (formerly EF-1beta), was determined to 1.67 A resolution. One end of the nucleotide exchange factor is buried between the switch 1 and 2 regions of eEF1A and destroys the binding site for the Mg(2+) ion associated with the nucleotide. The second end of eEF1Balpha interacts with domain 2 of eEF1A in the region hypothesized to be involved in the binding of the CCA-aminoacyl end of the tRNA. The competition between eEF1Balpha and aminoacylated tRNA may be a central element in channeling the reactants in eukaryotic protein synthesis. The recognition of eEF1A by eEF1Balpha is very different from that observed in the prokaryotic EF-Tu:EF-Ts complex. Recognition of the switch 2 region in nucleotide exchange is, however, common to the elongation factor complexes and those of Ras:Sos and Arf1:Sec7.     

Eukaryotic release factor 1 phosphorylation by CK2 protein kinase is dynamic but has little effect on the efficiency of translation termination in Saccharomyces cerevisiae

Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Delta mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.     

Global analysis of protein palmitoylation in African trypanosomes

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.     

The solution structure of the guanine nucleotide exchange domain of human elongation factor 1beta reveals a striking resemblance to that of EF-Ts from Escherichia coli.

BACKGROUND: In eukaryotic protein synthesis, the multi-subunit elongation factor 1 (EF-1) plays an important role in ensuring the fidelity and regulating the rate of translation. EF-1alpha, which transports the aminoacyl tRNA to the ribosome, is a member of the G-protein superfamily. EF-1beta regulates the activity of EF-1alpha by catalyzing the exchange of GDP for GTP and thereby regenerating the active form of EF-1alpha. The structure of the bacterial analog of EF-1alpha, EF-Tu has been solved in complex with its GDP exchange factor, EF-Ts. These structures indicate a mechanism for GDP-GTP exchange in prokaryotes. Although there is good sequence conservation between EF-1alpha and EF-Tu, there is essentially no sequence similarity between EF-1beta and EF-Ts. We wished to explore whether the prokaryotic exchange mechanism could shed any light on the mechanism of eukaryotic translation elongation. RESULTS: Here, we report the structure of the guanine-nucleotide exchange factor (GEF) domain of human EF-1beta (hEF-1beta, residues 135-224); hEF-1beta[135-224], determined by nuclear magnetic resonance spectroscopy. Sequence conservation analysis of the GEF domains of EF-1 subunits beta and delta from widely divergent organisms indicates that the most highly conserved residues are in two loop regions. Intriguingly, hEF-1beta[135-224] shares structural homology with the GEF domain of EF-Ts despite their different primary sequences. CONCLUSIONS: On the basis of both the structural homology between EF-Ts and hEF-1beta[135-224] and the sequence conservation analysis, we propose that the mechanism of guanine-nucleotide exchange in protein synthesis has been conserved in prokaryotes and eukaryotes. In particular, Tyr181 of hEF-1beta[135-224] appears to be analogous to Phe81 of Escherichia coli EF-Ts.     

Substrate preference and phosphatidylinositol monophosphate inhibition of the catalytic domain of the Per-Arnt-Sim domain kinase PASKIN

The Per-Arnt-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast, regulates glycogen synthesis and protein translation in mammals, and might be involved in insulin regulation in the pancreas. According to the current model, binding of a putative ligand to the PAS domain disinhibits the kinase domain, leading to PASKIN autophosphorylation and increased kinase activity. To date, only synthetic but no endogenous PASKIN ligands have been reported. In the present study, we identified a number of novel PASKIN kinase targets, including ribosomal protein S6. Together with our previous identification of eukaryotic elongation factor 1A1, this suggests a role for PASKIN in the regulation of mammalian protein translation. When searching for endogenous PASKIN ligands, we found that various phospholipids can bind PASKIN and stimulate its autophosphorylation. Interestingly, the strongest binding and autophosphorylation was achieved with monophosphorylated phosphatidylinositols. However, stimulated PASKIN autophosphorylation did not correlate with ribosomal protein S6 and eukaryotic elongation factor 1A1 target phosphorylation. Although autophosphorylation was enhanced by monophosphorylated phosphatidylinositols, di- and tri-phosphorylated phosphatidylinositols inhibited autophosphorylation. By contrast, target phosphorylation was always inhibited, with the highest efficiency for di- and tri-phosphorylated phosphatidylinositols. Because phosphatidylinositol monophosphates were found to interact with the kinase rather than with the PAS domain, these data suggest a multiligand regulation of PASKIN activity, including a still unknown PAS domain binding/activating ligand and kinase domain binding modulatory phosphatidylinositol phosphates.     

Eukaryotic elongation factor-2 (eEF2): its regulation and peptide chain elongation

Regulation at the level of translation in eukaryotes is feasible because of the longer lifetime of eukaryotic mRNAs in the cell. The elongation stage of mRNA translation requires a substantial amount of energy and also eukaryotic elongation factors (eEFs). The important component of eEFs, i.e. eEF2 promotes the GTP-dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome. Mostly the eEF2 is regulated by phosphorylation and dephosphorylation by a specific kinase known as eEF2 kinase, which itself is up-regulated by various mechanisms in the eukaryotic cell. The activity of this kinase is dependent on calcium ions and calmodulin. Recently it has been shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and mTOR signalling pathway. There are also various stimuli that control the peptide chain elongation in eukaryotic cell; some stimuli inhibit and some activate eEF2. These reports provide the mechanisms by which cells likely serve to slow down protein synthesis and conserve energy under nutrient deprived conditions via regulation of eEF2. The regulation via eEF2 has also been seen in mammary tissue of lactating cows, suggesting that eEF2 may be a limiting factor in milk protein synthesis. Regulation at this level provides the molecular understanding about the control of protein translocation reactions in eukaryotes, which is critical for numerous biological phenomenons. Further the elongation factors could be potential targets for regulation of protein synthesis like milk protein synthesis and hence probably its foreseeable application to synthetic biology.     

eEF1A: Thinking Outside the Ribosome

Eukaryotic translation elongation factor 1A (eEF1A) is one of the most abundant protein synthesis factors. eEF1A is responsible for the delivery of all aminoacyl-tRNAs to the ribosome, aside from initiator and selenocysteine tRNAs. In addition to its roles in polypeptide chain elongation, unique cellular and viral activities have been attributed to eEF1A in eukaryotes from yeast to plants and mammals. From preliminary, speculative associations to well characterized biochemical and biological interactions, it is clear that eEF1A, of all the translation factors, has been ascribed the most functions outside of protein synthesis. A mechanistic understanding of these non-canonical functions of eEF1A will shed light on many important biological questions, including viral-host interaction, subcellular organization, and the integration of key cellular pathways.     

eEF1A1 Overexpression Enhances Tumor Progression and Indicates Poor Prognosis in Hepatocellular Carcinoma

Liver is a major contributor of protein production physiologically. The aberrant state of protein synthesis leads to tumor progression. Eukaryotic elongation factor 1 alpha 1 (eEF1A1) is a major member of the eukaryotic elongation factor family that regulates protein synthesis. Although eEF1A1 plays an essential role in controlling the cell fate, its clinical significance in tumor development and progression has not been reported. Here, we aimed to uncover the expression and prognostic significance of eEF1A1 in hepatocellular carcinoma (HCC). Our data indicated that eEF1A1 expression was elevated in HCC cell lines and clinical samples at both the mRNA and protein levels. Immunohistochemistry revealed that eEF1A1 expression was upregulated in HCC samples compared with corresponding non-tumorous tissues. In 50 HCC cases with portal vein embolus, higher eEF1A1 immunoreactivity was detected in tumor metastases compared with the primary lesions. Kaplan–Meier analysis indicated that increased eEF1A1 expression was closely associated with unfavorable post-surgical overall and disease-free survival in 453 HCC patients. Moreover, multivariate analysis indicated eEF1A1 as an independent predictor for overall and disease-free survival. Collectively, our study suggests eEF1A1 as a novel prognostic biomarker and potential therapeutic target for HCC patients.