The eukaryotic elongation factor 1 gamma (eEF1γ) is a multi-domain protein, which consist of a glutathione transferase (GST)-like N-terminus domain. In association with α, β and δ subunits, eEF1γ forms part of the eukaryotic elongation factor complex, which is mainly involved in protein biosynthesis. The N-terminus GST domain of eEF1γ interacts with the β subunit. eEF1γ subunit is over-expressed in human carcinoma. The role of human eEF1γ (heEF1γ) is poorly understood. A successful purification of recombinant heEF1γ is the first step towards determining unknown properties of the protein, including putative GST-like activities and the structure of the protein. This paper describes the over-expression, purification and characterisation of recombinant full-length, and the N- and C-terminus domains of heEF1γ. All three recombinant heEF1γ constructs over-expressed in the soluble Escherichia coli cell fraction and were purified to homogeneity. Secondary structure analysis indicates that the heEF1γ constructs have high α-helical structural character. The full-length and N-terminus domain are dimeric, while the C-terminus is monomeric. Both full-length and N-terminus domain interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD=70.0 (±5.7) μM and with reduced glutathione (GSH). Glutathione sulfonate displaced ANS bound to hydrophobic binding sites in the recombinant N-terminus domain. Using the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, the N-domain showed some enzyme activity (0.03μmolmin(-1) mg(-1) protein), while the full-length heEF1γ did not catalyse the GSH-CDNB conjugation. Consequently, we hypothesize the presence of a presumed GST-like active site structure in the heEF1γ, which comprises a glutathione binding site and a hydrophobic substrate binding site.

Gbx2 encodes a DNA-binding transcription factor that plays pivotal roles during embryogenesis. Gain-and loss-of-function studies in several vertebrate species have demonstrated a requirement for Gbx2 in development of the anterior hindbrain, spinal cord, inner ear, heart, and neural crest cells. However, the target genes through which GBX2 exerts its effects remain obscure. Using chromatin immunoprecipitation coupled with direct sequencing (ChIP-Seq) analysis in a human prostate cancer cell line, we identified cis-regulatory elements bound by GBX2 to provide insight into its direct downstream targets. The analysis revealed more than 286 highly significant candidate target genes, falling into various functional groups, of which 51% are expressed in the nervous system. Several of the top candidate genes include EEF1A1, ROBO1, PLXNA4, SLIT3, NRP1, and NOTCH2, as well as genes associated with the Usher syndrome, PCDH15 and USH2A, and are plausible candidates contributing to the developmental defects in Gbx2(-/-) mice. We show through gel shift analyses that sequences within the promoter or introns of EEF1A1, ROBO1, PCDH15, USH2A and NOTCH2, are directly bound by GBX2. Consistent with these in vitro results, analyses of Gbx2(-/-) embryos indicate that Gbx2 function is required for migration of Robo1-expressing neural crest cells out of the hindbrain. Furthermore, we show that GBX2 activates transcriptional activity through the promoter of EEF1A1, suggesting that GBX2 could also regulate gene expression indirectly via EEF1A. Taken together, our studies show that GBX2 plays a dynamic role in development and diseases.

Recombinant factor VIII (FVIII), used for haemophilia A therapy, is one of the most challenging among the therapeutic proteins produced in heterologous expression systems. Deletion variant of FVIII, in which the entire domain B is replaced by a short linker peptide, was approved for medical use. Efficacy and safety of this FVIII deletion variant are similar to full-length FVIII preparations while the level of production in CHO cells is substantially higher. Typical levels of productivity for CHO cell lines producing deletion variant FVIII-BDD SQ, described elsewhere, are 0.5-2 IU/ml, corresponding to the concentration of FVIII of about 0.2 μg/ml. Using standard vectors based on the cytomegalovirus promoter (CMV) and the dihydrofolate reductase cDNA we have previously obtained the cell line secreting 0.5 IU/ml of FVIII-BDD, which roughly corresponds to the previously published data.

Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (SaEf-Tu) and Mycoplasma pneumoniae (MpnEf-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (MhpEf-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant MpnEf-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A + T rich genome may influence how positively-charged residues accumulate in SLiMs.

Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells.

Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3' end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk. Furthermore, the Seb1 interaction surface with Pol II largely overlaps with that of the elongation factor Spt5. Notably, Seb1 co-transcriptional recruitment is dependent on Spt5 dephosphorylation by the conserved PP1 phosphatase Dis2, which also dephosphorylates threonine 4 within the Pol II heptad repeated C-terminal domain. We propose that Dis2 orchestrates the transition from elongation to termination phase during the transcription cycle by mediating elongation to termination factor exchange and dephosphorylation of Pol II C-terminal domain.

Eukaryotic elongation factor 2 kinase (eEF2K) inhibitors may aid in the development of new therapeutic agents to combat cancer. Purified human eEF2K was obtained from an Escherichia coli expression system and a luminescence-based high-throughput screening (HTS) assay was developed using MH-1 peptide as the substrate. The luminescent readouts correlated with the amount of adenosine triphosphate remaining in the kinase reaction. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Nine initial hits showing inhibitory activities on eEF2K were identified from 56,000 synthetic compounds during the HTS campaign, of which, five were chosen to test their effects in cancer cell lines.

Elongation factor thermo-unstable (EF-Tu), an abundant multifunctional protein, is pivotal during protein synthesis and is an important antigen. Few studies have addressed the role of this protein in Brucella species, and the epitopes of this protein have not been reported. Here, we describe a monoclonal antibody (McAb), BD6, for EF-Tu in Brucella melitensis. Using western blotting involving a series of partially overlapping recombinant EF-Tu truncation peptides, a novel linear B-cell epitope, 110QTREHIL116 (EF), was identified. Alanine-scanning mutagenesis revealed that residues Q110, T111, R112, I115, and L116 were core residues involved in recognition. Sequence alignment suggested that the epitope peptide was conserved among bacterial species but differed by one amino acid residue (I115) from the host sequence. The epitope peptide was recognized by sera from B. melitensis-infected mice, and while recombinant epitope peptide induced a strong humoral immune response, the corresponding mouse peptide, QTREHLL, did not. These results suggested that I115 may be the key residue for the host immune system to distinguish between bacterial and self epitope EF sequences. Indirect immunofluorescence and western blotting assays showed that epitope peptide could be used in Saccharomyces cerevisiae, human embryonic kidney cell (HEK-293), and chicken fibroblast cell (DF1) expression systems and immunoprecipitation assay. Together, our results suggested that the McAb BD6 is a useful tool for further investigation of the potential functions of the EF-Tu protein in pathogen-host interactions, and that the epitope tag may be useful for application as a novel affinity tag to identify other bacterial pathogens, especially convenient for the identification of intracellular bacteria.

Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection.

Circular RNAs (circRNAs) are a large class of transcripts in the mammalian genome. Although the translation of circRNAs was reported, additional coding circRNAs and the functions of their translated products remain elusive. Here, we demonstrate that an endogenous circRNA generated from a long noncoding RNA encodes regulatory peptides. Through ribosome nascent-chain complex-bound RNA sequencing (RNC-seq), we discover several peptides potentially encoded by circRNAs. We identify an 87-amino-acid peptide encoded by the circular form of the long intergenic non-protein-coding RNA p53-induced transcript (LINC-PINT) that suppresses glioblastoma cell proliferation in vitro and in vivo. This peptide directly interacts with polymerase associated factor complex (PAF1c) and inhibits the transcriptional elongation of multiple oncogenes. The expression of this peptide and its corresponding circRNA are decreased in glioblastoma compared with the levels in normal tissues. Our results establish the existence of peptides encoded by circRNAs and demonstrate their potential functions in glioblastoma tumorigenesis.

Eukaryotic elongation factor 2 (eEF2) is an abundant and essential component of the translation machinery. The biogenesis of this 93 kDa multi-domain protein is assisted by the chaperonin TRiC/CCT. Here, we show in yeast cells that the highly conserved protein Hgh1 (FAM203 in humans) is a chaperone that cooperates with TRiC in eEF2 folding. In the absence of Hgh1, a substantial fraction of newly synthesized eEF2 is degraded or aggregates. We solved the crystal structure of Hgh1 and analyzed the interaction of wild-type and mutant Hgh1 with eEF2. These experiments revealed that Hgh1 is an armadillo repeat protein that binds to the dynamic central domain III of eEF2 via a bipartite interface. Hgh1 binding recruits TRiC to the C-terminal eEF2 module and prevents unproductive interactions of domain III, allowing efficient folding of the N-terminal GTPase module. eEF2 folding is completed upon dissociation of TRiC and Hgh1.

Mycoplasmas persist in the host for a long time, suggesting that they possess mechanisms for immune evasion. Factor H is a negative regulator of the complement system, which binds to host cells to avoid unexpected complement activation. In this study, we revealed that many mycoplasmas, such as Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma gallisepticum, Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma flocculare, and Mycoplasma bovis could hijack factor H such that they present themselves as a host tissue and thus escape from complement attack. Furthermore, the mechanism of recruiting factor H was identified in M. hyopneumoniae. M. hyopneumoniae binds factor H via factor H binding proteins, such as elongation factor thermo unstable (EF-Tu), P146, pyruvate dehydrogenase (acetyl-transferring) E1 component subunit alpha (PdhA), P46, Pyruvate dehydrogenase E1 component subunit beta (PdhB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and three different hypothetical proteins. The binding of factor H by EF-Tu further contributes to decreased C3 deposition on the M. hyopneumoniae surface and ultimately blocks further complement activation. In fact, binding of factor H occurs in a multifactorial manner; factor H is not only exploited by M. hyopneumoniae via its regulator activity to help mycoplasmas escape from complement killing, but also increases M. hyopneumoniae adhesion to swine tracheal epithelial cells, partially through EF-Tu. Meanwhile, the high sequence identity among EF-Tu proteins in the above-mentioned mycoplasmas implied the universality of the mechanism. This is the first report that mycoplasmas can escape complement killing by binding to factor H.

The elongation factor Tu has been identified as one of the most immunoreactive proteins that was recognized by human sera of GBS (group B streptococcus) positive patients. In this paper, we present the polypeptide-specific epitopes of the bacterial protein that are recognized by human antibodies: 28LTAAITTVLARRLP41 (peptide no. 3) and 294GQVLAKPGSINPHTKF309 (peptide no. 21). To determine the shortest amino acid sequence recognized by antibodies, truncation peptide libraries were prepared using the PEPSCAN method. The analysis of immunoreactivity of peptides with sera of GBS positive and negative women revealed that the most immunoreactive sequence was 306HTKF309. Moreover, we observed that this sequence also showed the highest specificity which was based on ratio of reactivity with sera of GBS positive relative to sera of GBS negative patients. Epitope was synthetized on Wang resin with the Fmoc strategy. Our results open the possibility to use 306HTKF309 peptide in diagnostic assays to determine Streptococcus agalactiae infection in humans.

Although correlations between RNA polymerase II (RNAPII) transcription stress, R-loops, and genome instability have been established, the mechanisms underlying these connections remain poorly understood. Here, we used a mutant version of the transcription elongation factor TFIIS (TFIISmut), aiming to specifically induce increased levels of RNAPII pausing, arrest, and/or backtracking in human cells. Indeed, TFIISmut expression results in slower elongation rates, relative depletion of polymerases from the end of genes, and increased levels of stopped RNAPII; it affects mRNA splicing and termination as well. Remarkably, TFIISmut expression also dramatically increases R-loops, which may form at the anterior end of backtracked RNAPII and trigger genome instability, including DNA strand breaks. These results shed light on the relationship between transcription stress and R-loops and suggest that different classes of R-loops may exist, potentially with distinct consequences for genome stability.

Expression of the fructose transporter gene SLC2A5 and histone acetylation in the transcribed region are induced by differentiation associated-signals such as glucocorticoids and p44/42 mitogen-activated protein kinase (MAPK) inhibition in small intestinal Caco-2 cells.

Translation elongation factor eIF5A binds to ribosomes to promote peptide bonds between problematic amino acids for the reaction like prolines. eIF5A is highly conserved and essential in eukaryotes, which usually contain two similar but differentially expressed paralogue genes. The human eIF5A-1 isoform is abundant and implicated in some cancer types; the eIF5A-2 isoform is absent in most cells but becomes overexpressed in many metastatic cancers. Several reports have connected eIF5A and mitochondria because it co-purifies with the organelle or its inhibition reduces respiration and mitochondrial enzyme levels. However, the mechanisms of eIF5A mitochondrial function, and whether eIF5A expression is regulated by the mitochondrial metabolism, are unknown. We analysed the expression of yeast eIF5A isoforms Tif51A and Tif51B under several metabolic conditions and in mutants. The depletion of Tif51A, but not Tif51B, compromised yeast growth under respiration and reduced oxygen consumption. Tif51A expression followed dual positive regulation: by high glucose through TORC1 signalling, like other translation factors, to promote growth and by low glucose or non-fermentative carbon sources through Snf1 and heme-dependent transcription factor Hap1 to promote respiration. Upon iron depletion, Tif51A was down-regulated and Tif51B up-regulated. Both were Hap1-dependent. Our results demonstrate eIF5A expression regulation by cellular metabolic status.

Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease.

AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed in situ with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism.

House dust mite (HDM) hypersensitivity increasingly affects millions of individuals worldwide. Although numerous major allergens produced by HDM species have been characterized, some of the less potent allergens remain to be studied. The present study aimed to obtain the recombinant allergen of Translation Elongation Factor 2 (TEF 2) from the HDM Dermatophagoides farinae by synthesizing, and then expressing the recombinant TEF 2 to identify its immunogenicity. In the present study, the D. farinae TEF 2 (Der f TEF 2) was synthesized, expressed and purified. The molecular characteristics of Der f TEF 2 were analyzed using bioinformatics approaches. The recombinant protein was purified via affinity chromatography, and the allergenicity was assessed using immunoblotting, ELISAs and skin prick tests. The gene for TEF 2 consists of 2,535 bp and encodes an 844‑amino acid protein. A positive response to recombinant Der f TEF 2 was detected in 16.2% of 37 patients with HDM allergies using skin prick tests. In addition, the immunoblotting indicated that the protein showed a high ability to bind serum IgE from patients allergic to HDMs, and that the recombinant TEF 2 was highly immunogenic. Bioinformatics analysis predicted 17 peptides as B cell epitopes (amino acids 29‑35, 55‑64, 92‑99, 173‑200, 259‑272, 311‑318, 360‑365, 388‑395, 422‑428, 496‑502, 512‑518, 567‑572, 580‑586, 602‑617, 785‑790, 811‑817 and 827‑836) and 14 peptides as T cell epitopes (amino acids 1‑15, 65‑79, 120‑134, 144‑159, 236‑250, 275‑289, 404‑418, 426‑440, 463‑477, 510‑524, 644‑658, 684‑698, 716‑730 and 816‑830). The software DNAStar predicted the secondary structure of TEF 2, and showed that 27 α‑helices and five β‑sheets were found in the protein. In conclusion, the present study cloned and expressed the Der f TEF 2 gene, and the recombinant protein exhibited immunogenicity, providing a theoretical bases, and references, for the diagnosis and treatment of allergic disease.

Elongation factor 1 A (EF1A), an essential regulator for protein synthesis, has been reported to participate in abiotic stress responses and environmental adaption in plants. However, the role of EF1A in abiotic stress response was barely studied in Medicago truncatula. Here, we identified elongation factor (EF) genes of M. truncatula and studied the salt stress response function of MtEF1A1 (MTR_6g021805).