EEF1D facilitates milk lipid synthesis by regulation of PI3K-Akt signaling in mammals.

Mammal's milk is an abundantly foremost source of proteins, lipids, and micronutrients for human nutrition and health. Understanding the molecular mechanisms underlying synthesis of milk components provides practical benefits to improve the milk quality via systematic breeding program in mammals. Through RNAi with EEF1D in primary bovine mammary epithelial cells, we phenotypically observed aberrant formation of cytoplasmic lipid droplets and significantly decreased milk triglyceride level by 37.7%, and exploited the mechanisms by which EEF1D regulated milk lipid synthesis via insulin (PI3K-Akt), AMPK, and PPAR pathways. In the EEF1D CRISPR/Cas9 knockout mice, incompletely developed mammary glands at 9th day postpartum with small or unformed lumens, and significantly decreased triglyceride concentration in milk by 23.4% were observed, as well as the same gene expression alterations in the three pathways. For dairy cattle, we identified a critical regulatory mutation modifying EEF1D transcription activity, which interpreted 7% of the genetic variances of milk lipid yield and percentage. Our findings highlight the significance of EEF1D in mammary gland development and milk lipid synthesis in mammals.

Revisiting the Schistosoma japonicum life cycle transcriptome for new insights into lung schistosomula development.

Schistosome parasites are complex trematode blood flukes responsible for the disease schistosomiasis; a global health concern prevalent in many tropical and sub-tropical countries. While established transcriptomic databases are accessed ad hoc to facilitate studies characterising specific genes or gene families, a more comprehensive systematic updating of gene annotation and survey of the literature to aid in annotation and context is rarely addressed. We have reanalysed an online transcriptomic dataset originally published in 2009, where seven life cycle stages of Schistosoma japonicum were examined. Using the online pathway analysis tool Reactome, we have revisited key data from the original study. A key focus of this study was to improve the interpretation of the gene expression profile of the developmental lung-stage schistosomula, since it is one of the principle targets for worm elimination. Highly enriched transcripts, associated with lung schistosomula, were related to a number of important biological pathways including host immune evasion, energy metabolism and parasitic development. Revisiting large transcriptomic databases should be considered in the context of substantial new literature. This approach could aid in the improved understanding of the molecular basis of parasite biology. This may lead to the identification of new targets for diagnosis and therapies for schistosomes, and other helminths.

Expression pattern of EEF1A2 in brain tumors: Histological analysis and functional role as a promoter of EMT.

AIMS: Glioblastomas are highly aggressive brain tumors with a very poor survival rate. EEF1A2, the proto-oncogenic isoform of the EEF1A translation factor family, has been found to be overexpressed and promoting tumorigenesis in multiple cancers. Interestingly, recent studies reported reduced expression of this protein in brain tumors, drawing our attention to find the functional role and mechanism of this protein in brain tumor progression. MAIN METHODS: Using representative cell line as models, the role of EEF1A2 in cell proliferation, migration and invasion were assessed using MTS assay, scratch wound-healing assay, transwell migration and invasion assay, respectively. Activation of key signaling pathways was assessed using western blots and real-time PCR. Finally, using immunohistochemistry we checked the protein levels of EEF1A2 in CNS tumors. KEY FINDINGS: EEF1A2 was found to increase the proliferative, migratory and invasive properties of cell lines of both glial and neuronal origin. PI3K activation directly correlated with EEF1A2 levels. Protein levels of key EMT markers viz. Twist, Snail, and Slug were increased upon ectopic EEF1A2 expression. Furthermore, EEF1A2 was found to affect the expression levels of key inflammatory cytokines, growth factors and matrix metalloproteases. IHC analysis showed that EEF1A2 is upregulated in tumor tissues compared to normal tissue. SIGNIFICANCE: EEF1A2 acts as an oncogene in both neuronal and glial cells and triggers an EMT program via PI3K pathway. However, it shows enhanced expression in neuronal cells of the brain than the glial cells, which could explain the previously reported anomaly.

eEF1A2 exacerbated insulin resistance in male skeletal muscle via PKCß and ER stress.

Recent studies raise the possibility that eukaryotic translation elongation factor 1 alpha (eEF1A) may play a role in metabolism. One isoform, eEF1A2, is specifically expressed in skeletal muscle, heart and brain. It regulates translation elongation and signal transduction. Nonetheless, eEF1A2's function in skeletal muscle glucose metabolism remains unclear. In the present study, suppression subtractive hybridisation showed a decrease in Eef1a2 transcripts in the skeletal muscle of diabetic Mongolian gerbils. This was confirmed at mRNA and protein levels in hyperglycaemic gerbils, and in db/db and high-fat diet-fed mice. Further, this downregulation was independent of Eef1a2 promoter methylation. Interestingly, adeno-associated virus-mediated eEF1A2 overexpression in skeletal muscle aggravated fasting hyperglycaemia, hyperinsulinaemia and glucose intolerance in male diabetic gerbils but not in female gerbil models. The overexpression of eEF1A2 in skeletal muscle also resulted in promoted serum glucose levels and insulin resistance in male db/db mice. Up- and downregulation of eEF1A2 by lentiviral vector transfection confirmed its inhibitory effect on insulin-stimulated glucose uptake and signalling transduction in C2C12 myotubes with palmitate (PA)-induced insulin resistance. Furthermore, eEF1A2 bound PKCß and increased its activation in the cytoplasm, whereas suppression of PKCß by an inhibitor attenuated eEF1A2-mediated impairment of insulin sensitivity in insulin-resistant myotubes. Endoplasmic reticulum (ER) stress was elevated by eEF1A2, whereas suppression of ER stress or JNK partially restored insulin sensitivity in PA-treated myotubes. Additionally, eEF1A2 inhibited lipogenesis and lipid utilisation in insulin-resistant skeletal muscle. Collectively, we demonstrated that eEF1A2 exacerbates insulin resistance in male murine skeletal muscle via PKCß and ER stress.

Two cases of early-onset myoclonic seizures with continuous parietal delta activity caused by EEF1A2 mutations.

BACKGROUND: Mutations in the elongation factor 1 alpha 2 (EEF1A2) gene have recently been shown to cause severe intellectual disability with early-onset epilepsy. The specific manifestations of mutations in this gene remain unknown. CASE REPORT: We report two cases of severe intellectual disability accompanied by early-onset epilepsy with continuous delta activity evident on electroencephalography. Both cases presented with developmental delay and repetitive myoclonic seizures in early infancy. Both cases showed continuous high-voltage delta activity over both parietal areas when awake, as revealed by interictal electroencephalograms. After the emergence of continuous delta activity, development stagnated. One case showed some development after relief of the seizures and epileptic activity, but drug resistant seizures recurred, and the development again became stagnant. In both cases, a de novo recurrent heterozygous mutation in EEF1A2 [c.364G>A (p.E122K)] was identified by whole-exome sequencing. CONCLUSION: This report provides clinical data on epileptic encephalopathy in patients with EEF1A2 mutation. Continuous high-voltage delta activity seen over both parietal areas may be a unique manifestation of EEF1A2 mutation. Epileptic activity may aggravate the effect of the mutation on brain development.

Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis.

Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity.

Critical role of eukaryotic elongation factor 1 alpha 1 (EEF1A1) in avian reovirus sigma-C-induced apoptosis and inhibition of viral growth.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome. It is well established that the ARV sigma-C protein induces apoptosis in host cells. However, the underlying molecular mechanism of this induction is still unclear. We report here the identification of eukaryotic elongation factor 1 alpha 1 (EEF1A1) as the interacting partner of σC. We found that σC-induced apoptosis in DF-1 cells could be completely abolished by knockdown of EEF1A1 by siRNA. Furthermore, knockdown of EEF1A1 markedly reduced ARV-induced apoptosis associated with decreased caspase-9 and -3 activation and cytochrome C release, leading to increased ARV growth in host cells. Thus, EEF1A1 plays a critical role in σC-induced apoptosis and inhibition of viral growth.

A non-canonical function of eukaryotic elongation factor 1A1: regulation of interleukin-6 expression.

Interleukin-6 is one of the most prominent triggers of inflammatory processes. We have shown recently that heteroarylketones (HAKs) interfere with stimulated interleukin-6 expression in astrocytes by suppression of STAT3 phosphorylation at serine 727. Surprisingly, this effect is not based on the inhibition of STAT3-relevant kinases. Therefore, we here used the structurally modified HAK compound biotin-HAK-3 in a reverse chemical approach to identify the relevant molecular target in UV-mediated cross-linking experiments. Employing streptavidin-specific 2D-immunoblotting followed by mass spectrometry we identified nine proteins putatively interacting with biotin-HAK-3. After co-immunoprecipitation, co-immunofluorescence, surface plasmon resonance analyses and RNAi-mediated knock-down, the eukaryotic elongation factor 1A1 (eEF1A1) was verified as the relevant target of HAK bioactivity. eEF1A1 forms complexes with STAT3 and PKCδ, which are crucial for STAT3(S727) phosphorylation and for NF-κB/STAT3-enhanced interleukin-6 expression. Furthermore, the intracellular HAK accumulation is strongly dependent on eEF1A1 expression. Taken together, the results reveal a novel molecular mechanism for a non-canonical role of eEF1A1 in signal transduction via direct modulation of kinase-dependent phosphorylation events.

EEF1A2 inactivates p53 by way of PI3K/AKT/mTOR-dependent stabilization of MDM4 in hepatocellular carcinoma.

UNLABELLED: Mouse Double Minute homolog 4 (MDM4) gene up-regulation often occurs in human hepatocellular carcinoma (HCC), but the molecular mechanisms responsible for its induction remain poorly understood. Here we investigated the role of the phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (PI3K/AKT/mTOR) axis in the regulation of MDM4 levels in HCC. The activity of MDM4 and the PI3K/AKT/mTOR pathway was modulated in human HCC cell lines by way of silencing and overexpression experiments. Expression of main pathway components was analyzed in an AKT mouse model and human HCCs. MDM4 inhibition resulted in growth restraint of HCC cell lines both in vitro and in vivo. Inhibition of the PI3K-AKT and/or mTOR pathways lowered MDM4 protein levels in HCC cells and reactivated p53-dependent transcription. Deubiquitination by ubiquitin-specific protease 2a and AKT-mediated phosphorylation protected MDM4 from proteasomal degradation and increased its protein stability. The eukaryotic elongation factor 1A2 (EEF1A2) was identified as an upstream inducer of PI3K supporting MDM4 stabilization. Also, we detected MDM4 protein up-regulation in an AKT mouse model and a strong correlation between the expression of EEF1A2, activated/phosphorylated AKT, and MDM4 in human HCC (each rho > 0.8, P < 0.001). Noticeably, a strong activation of this cascade was associated with shorter patient survival. CONCLUSION: The EEF1A2/PI3K/AKT/mTOR axis promotes the protumorigenic stabilization of the MDM4 protooncogene in human HCC by way of a posttranscriptional mechanism. The activation level of the EEF1A2/PI3K/AKT/mTOR/MDM4 axis significantly influences the survival probability of HCC patients in vivo and may thus represent a promising molecular target.

Cytotoxic glucosyltransferases of Legionella pneumophila.

Legionella is a gram-negative bacterium and the causative pathogen of legionellosis-a severe pneumonia in humans. A large number of Legionella effectors interfere with numerous host cell functions, including intracellular vacuole trafficking and maturation, phospholipid metabolism, protein ubiquitination, pro-/anti-apoptotic balances or inflammatory responses. Moreover, eukaryotic protein synthesis is affected by L. pneumophila glucosyltransferases Lgt1, Lgt2, and Lgt3. Structurally, these enzymes are similar to large clostridial cytotoxins, use UDP-glucose as a co-substrate and modify a conserved serine residue (Ser-53) in elongation factor 1A (eEF1A). The ternary complex consisting of eEF1A, GTP, and aminoacylated-tRNA seems to be the substrate for Lgts. Studies with Saccharomyces cerevisiae corroborated that eEF1A is the major target responsible for Lgt-induced cytotoxic activity. In addition to Lgt proteins, Legionella produces other effector glycosyltransferase, including the modularly composed protein SetA, which displays tropism for early endosomal compartments, subverts host cell vesicle trafficking and demonstrates toxic activities toward yeast and mammalian cells. Here, our current knowledge about both groups of L. pneumophila glycosylating effectors is reviewed.

Eukaryotic translation elongation factor 1-alpha 1 inhibits p53 and p73 dependent apoptosis and chemotherapy sensitivity.

The p53 family of transcription factors is a key regulator of cell proliferation and death. In this report we identify the eukaryotic translation elongation factor 1-alpha 1 (eEF1A1) to be a novel p53 and p73 interacting protein. Previous studies have demonstrated that eEF1A1 has translation-independent roles in cancer. We report that overexpression of eEF1A1 specifically inhibits p53-, p73- and chemotherapy-induced apoptosis resulting in chemoresistance. Short-interfering RNA-mediated silencing of eEF1A1 increases chemosensitivity in cell lines bearing wild type p53, but not in p53 null cells. Furthermore, silencing of eEF1A1 partially rescues the chemoresistance observed in response to p53 or p73 knockdown, suggesting that eEF1A1 is a negative regulator of the pro-apoptotic function of p53 and p73. Thus, in the context of p53-family signaling, eEF1A1 has anti-apoptotic properties. These findings identify a novel mechanism of regulation of the p53 family of proteins by eEF1A1 providing additional insight into potential targets to sensitize tumors to chemotherapy.

eEF1A2 promotes cell migration, invasion and metastasis in pancreatic cancer by upregulating MMP-9 expression through Akt activation.

eEF1A2 is a protein translation factor involved in protein synthesis that is overexpressed in various cancers, with important functions in tumor genesis and progression. We have previously showed that the ectopic expression of eEF1A2 is correlated with lymph node metastasis and perineural invasion in pancreatic cancer. In this study, we investigated the functional role of eEF1A2 in the regulation of cell migration, invasion, and metastasis in pancreatic cancer. Furthermore, we investigated the potential molecular mechanisms involved. By evaluating the invasive ability of a panel of pancreatic cancer cell lines with different metastatic potentials, eEF1A2 expression in cells was positively associated with their invasive ability. The knockdown of eEF1A2 by siRNA decreased the migration and invasion of PANC-1 cells. By contrast, the ectopic expression of exogenous eEF1A2 significantly promoted the migration and invasion of SW1990 cells. Stable eEF1A2 overexpression in a nude mouse model of peritoneal metastasis likewise dramatically enhanced the intraperitoneal metastatic ability of SW1990 cells. In addition, eEF1A2 overexpression could upregulate MMP-9 expression and activity. A significant positive correlation between the overexpression of both eEF1A2 and MMP-9 was observed in pancreatic cancer tissues. The inhibition of MMP-9 activity reduced the promoting effect of eEF1A2 on cell migration and invasion. Furthermore, eEF1A2-mediated cell migration and invasion, as well as MMP-9 expression and upregulation, were largely dependent on the eEF1A2-induced Akt activation. The findings suggested the potentially important role of eEF1A2 in pancreatic cancer migration, invasion, and metastasis. Thus, the results provide evidence of eEF1A2 as a potential therapeutic target in the treatment of aggressive pancreatic cancer.

The yeast rapid tRNA decay pathway competes with elongation factor 1A for substrate tRNAs and acts on tRNAs lacking one or more of several modifications.

The structural and functional integrity of tRNA is crucial for translation. In the yeast Saccharomyces cerevisiae, certain aberrant pre-tRNA species are subject to nuclear surveillance, leading to 3' exonucleolytic degradation, and certain mature tRNA species are subject to rapid tRNA decay (RTD) if they are appropriately hypomodified or bear specific destabilizing mutations, leading to 5'-3' exonucleolytic degradation by Rat1 and Xrn1. Thus, trm8-Δ trm4-Δ strains are temperature sensitive due to lack of m(7)G(46) and m(5)C and the consequent RTD of tRNA(Val(AAC)), and tan1-Δ trm44-Δ strains are temperature sensitive due to lack of ac(4)C(12) and Um(44) and the consequent RTD of tRNA(Ser(CGA)) and tRNA(Ser(UGA)). It is unknown how the RTD pathway interacts with translation and other cellular processes, and how generally this pathway acts on hypomodified tRNAs. We provide evidence here that elongation factor 1A (EF-1A) competes with the RTD pathway for substrate tRNAs, since its overexpression suppresses the tRNA degradation and the growth defect of strains subject to RTD, whereas reduced levels of EF-1A have the opposite effect. We also provide evidence that RTD acts on a variety of tRNAs lacking one or more different modifications, since trm1-Δ trm4-Δ mutants are subject to RTD of tRNA(Ser(CGA)) and tRNA(Ser(UGA)) due to lack of m(2,2)G(26) and m(5)C, and since trm8-Δ, tan1-Δ, and trm1-Δ single mutants are each subject to RTD. These results demonstrate that RTD interacts with the translation machinery and acts widely on hypomodified tRNAs.

The many roles of the eukaryotic elongation factor 1 complex.

The vast majority of proteins are believed to have one specific function. Throughout the course of evolution, however, some proteins have acquired additional functions to meet the demands of a complex cellular milieu. In some cases, changes in RNA or protein processing allow the cell to make the most of what is already encoded in the genome to produce slightly different forms. The eukaryotic elongation factor 1 (eEF1) complex subunits, however, have acquired such moonlighting functions without alternative forms. In this article, we discuss the canonical functions of the components of the eEF1 complex in translation elongation as well as the secondary interactions they have with other cellular factors outside of the translational apparatus. The eEF1 complex itself changes in composition as the complexity of eukaryotic organisms increases. Members of the complex are also subject to phosphorylation, a potential modulator of both canonical and non-canonical functions. Although alternative functions of the eEF1A subunit have been widely reported, recent studies are shedding light on additional functions of the eEF1B subunits. A thorough understanding of these alternate functions of eEF1 is essential for appreciating their biological relevance.

Eukaryotic translation elongation factor 1A induces anoikis by triggering cell detachment.

Anoikis, apoptosis because of loss of cell anchorage, is crucial for tissue homeostasis. Fibronectin not only provides a scaffold for cell anchorage but also harbors a cryptic antiadhesive site capable of inducing ß1-integrin inactivation. In this study, this cryptic antiadhesive site is implicated in spontaneous induction of anoikis. Nontransformed fibroblasts (NIH3T3) adhering to a fibronectin substratum underwent anoikis during serum starvation culture. This anoikis was caused by proteolytic exposure of the cryptic antiadhesive site in fibronectin by matrix metalloproteinase. Eukaryotic elongation factor 1A (eEF1A) was identified as a membrane receptor for the exposed antiadhesive site. Serum starvation raised the membrane residence of eEF1A, and siRNA-based disruption of this increase rendered cells anoikis-resistant. By contrast, cells became more susceptible to anoikis in parallel with increased membrane residence of eEF1A by enforced expression. These results demonstrate that eEF1A acts as a membrane receptor for the cryptic antiadhesive site of fibronectin, which contributes to cell regulation, including anoikis, through negative regulation of cell anchorage.

Guanine nucleotides regulate sphingosine kinase 1 activation by eukaryotic elongation factor 1A and provide a mechanism for eEF1A-associated oncogenesis.

Sphingosine kinase 1 (SK1) catalyses the formation of bioactive phospholipid sphingosine 1-phosphate (S1P). Elevated cellular SK1 activity and S1P levels enhance cell proliferation and survival, and are strongly implicated in tumourigenesis. Regulation of SK1 activity can occur through various mechanisms, including phosphorylation and protein-protein interactions. We have previously shown that eukaryotic elongation factor 1A (eEF1A) interacts with and directly activates SK1, but the mechanisms regulating this were undefined. Notably, eEF1A has GTPase activity and can exist in GTP- or GDP-bound forms, which are associated with distinct structural conformations of the protein. Here, we show that the guanine nucleotide-bound state of eEF1A regulates its ability to activate SK1, with eEF1A.GDP, but not eEF1A.GTP, enhancing SK1 activity in vitro. Furthermore, we show that enhancing cellular eEF1A.GDP levels through expression of a guanine nucleotide dissociation inhibitor of eEF1A, translationally controlled tumour protein (TCTP), increased SK1 activity in cells. We also examined a truncated isoform of eEF1A1, termed prostate tumour inducer-1 (PTI-1), which can induce neoplastic cell transformation through undefined mechanisms. PTI-1 lacks the G protein domain of eEF1A1 and is therefore unable to undergo the GTP-binding-induced conformational change. Notably, we found that PTI-1 can directly activate SK1 and that this seems to be essential for neoplastic transformation induced by PTI-1, as chemical SK1 inhibitors or overexpression of a dominant-negative SK1 blocked this process. Thus, this study defines the mechanism regulating eEF1A-mediated SK1 activation, and also establishes SK1 as being integral for PTI-1-induced oncogenesis.

Leishmania exosomes deliver preemptive strikes to create an environment permissive for early infection.

Herein, we review evidence supporting a role for Leishmania exosomes during early infection. We suggest a model in which Leishmania secreted microvesicles released into the extracellular milieu deliver effector cargo to host target cells. This cargo mediates immunosuppression and functionally primes host cells for Leishmania invasion. Leishmania ssp. release microvesicles and the amount of vesicle release and the specific protein cargo of the vesicles is sensitive to changes in environmental conditions that mimic infection. Leishmania exosomes influence the phenotype of treated immune cells. For example, wild-type (WT) exosomes attenuate interferon-γ-induced pro-inflammatory cytokine production (TNF-α) by Leishmania-infected monocytes while conversely enhancing production of the anti-inflammatory cytokine IL-10. The Leishmania proteins GP63 and elongation factor-1α (EF-1α) are found in secreted vesicles and are likely important effectors responsible for these changes in phenotype. GP63 and EF-1α access host cell cytosol and activate multiple host protein-tyrosine phosphatases (PTPs). Activation of these PTPs negatively regulates interferon-γ signaling and this prevents effective expression of the macrophage microbicidal arsenal, including TNF-α and nitric oxide. In addition to changing macrophage phenotype, WT vesicles dampen the immune response of monocyte-derived dendritic cells and CD4+ T lymphocytes. This capacity is lost when the protein cargo of the vesicles is modified, specifically when the amount of GP63 and EF-1α in the vesicles is reduced. It appears that exosome delivery of effector proteins results in activation of host PTPs and the negative regulatory effects of the latter creates a pro-parasitic environment. The data suggest that Leishmania exosomes secreted upon initial infection are capable of delivering effector cargo to naïve target cells wherein the cargo primes host cells for infection by interfering with host cell signaling pathways.

Omnipotent role of archaeal elongation factor 1 alpha (EF1α in translational elongation and termination, and quality control of protein synthesis.

The molecular mechanisms of translation termination and mRNA surveillance in archaea remain unclear. In eukaryotes, eRF3 and HBS1, which are homologous to the tRNA carrier GTPase EF1α, respectively bind eRF1 and Pelota to decipher stop codons or to facilitate mRNA surveillance. However, genome-wide searches of archaea have failed to detect any orthologs to both GTPases. Here, we report the crystal structure of aRF1 from an archaeon, Aeropyrum pernix, and present strong evidence that the authentic archaeal EF1α acts as a carrier GTPase for aRF1 and for aPelota. The binding interface residues between aRF1 and aEF1α predicted from aRF1·aEF1α·GTP ternary structure model were confirmed by in vivo functional assays. The aRF1/eRF1 structural domain with GGQ motif, which corresponds to the CCA arm of tRNA, contacts with all three structural domains of aEF1α showing striking tRNA mimicry of aRF1/eRF1 and its GTPase-mediated catalysis for stop codon decoding. The multiple binding capacity of archaeal EF1α explains the absence of GTPase orthologs for eRF3 and HBS1 in archaea species and suggests that universal molecular mechanisms underlie translational elongation and termination, and mRNA surveillance pathways.

Cathepsin D and eukaryotic translation elongation factor 1 as promising markers of cellular senescence.

Induction of premature senescence may be a promising strategy for cancer treatment. However, biomarkers for senescent cancer cells are lacking. To identify such biomarkers, we performed comparative proteomic analysis of MCF7 human breast cancer cells undergoing cellular senescence in response to ionizing radiation (IR). IR-induced senescence was associated with up-regulation of cathepsin D (CD) and down-regulation of eukaryotic translation elongation factor 1beta2 (eEF1B2), as confirmed by Western blot. The other elongation factor, eukaryotic translation elongation factor 1alpha1 (eEF1A1), was also down-regulated. IR-induced senescence was associated with similar changes of CD and eEF1 (eEF1A1 and eEF1B2) levels in the HCT116 colon cancer cell line and the H460 lung cancer cell line. Up-regulation of CD and down-regulation of eEF1 seemed to be specific to senescence, as they were observed during cellular senescence induced by hydrogen peroxide or anticancer drugs (camptothecin, etoposide, or 50 ng doxorubicin) but not during apoptosis induced by Taxol or 10 microg doxorubicin or autophagy induced by tamoxifen. The same alterations in CD and eEF1A1 levels were observed during replicative senescence and Ras oncogene-induced senescence. Transient cell cycle arrest did not alter levels of eEF1 or CD. Chemical inhibition of CD (pepstatin A) and small interfering RNA-mediated knockdown of CD and eEF1 revealed that these factors participate in cell proliferation. Finally, the senescence-associated alteration in CD and eEF1 levels observed in cell lines was also observed in IR-exposed xenografted tumors. These findings show that CD and eEF1 are promising markers for the detection of cellular senescence induced by a variety of treatments.

Regulation and functional role of eEF1A2 in pancreatic carcinoma.

Pancreatic cancer typically has an unfavourable prognosis due to late diagnosis and a lack of therapeutic options. Thus, it is important to better understand its pathological mechanism and to develop more effective treatments for the disease. Human chromosome 20q13 has long been suspected to harbour oncogenes involved in pancreatic cancer and other tumours. In this study, we found that eEF1A2, a gene located in 20q13, was significantly upregulated in pancreatic cancer. Little or no expression of eEF1A2 was detected in normal human pancreatic and chronic pancreatitis tissues, whereas increased eEF1A2 expression occurred in 83% of the pancreatic cancers we studied. Furthermore, using in vitro and in vivo model systems, we found that overexpression of eEF1A2 promoted cell growth, survival, and invasion in pancreatic cancer. Our data thus suggest that eEF1A2 might play an important role in pancreatic carcinogenesis, possibly by acting as a tumour oncogene.