TOPK promotes the growth of esophageal cancer in vitro and in vivo by enhancing YB1/eEF1A1 signal pathway.

T-LAK-originated protein kinase (TOPK), a dual specificity serine/threonine kinase, is up-regulated and related to poor prognosis in many types of cancers. Y-box binding protein 1 (YB1) is a DNA/RNA binding protein and serves important roles in multiple cellular processes. Here, we reported that TOPK and YB1 were both highly expressed in esophageal cancer (EC) and correlated with poor prognosis. TOPK knockout effectively suppressed EC cell proliferation and these effects were reversible by rescuing YB1 expression. Notably, TOPK phosphorylated YB1 at Thr 89 (T89) and Ser 209 (S209) amino acid residues, then the phosphorylated YB1 bound with the promoter of the eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) to activate its transcription. Consequently, the AKT/mTOR signal pathway was activated by up-regulated eEF1A1 protein. Importantly, TOPK inhibitor HI-TOPK-032 suppressed the EC cell proliferation and tumor growth by TOPK/YB1/eEF1A1 signal pathway in vitro and in vivo. Taken together, our study reveals that TOPK and YB1 are essential for the growth of EC, and TOPK inhibitors may be applied to retard cell proliferation in EC. This study highlights the promising therapeutic potential of TOPK as a target for treatment of EC.

Anisomycin inhibits Coxsackievirus B replication by promoting the lysosomal degradation of eEF1A1.

Group B Coxsackieviruses (CVB) are non-enveloped small RNA viruses in the genus Enterovirus, family Picornaviridae. CVB infection causes diverse conditions from common cold to myocarditis, encephalitis, and pancreatitis. No specific antiviral is available for the treatment of CVB infection. Anisomycin, a pyrrolidine-containing antibiotic and translation inhibitor, was reported to inhibit the replication of some picornaviruses. However, it is unknown if anisomycin can act as an antiviral against CVB infection. Here we observed that anisomycin showed potent inhibition on CVB type 3 (CVB3) infection with negligible cytotoxicity when applied at the early stage of virus infection. Mice infected with CVB3 showed markedly alleviated myocarditis with reduced viral replication. We found that CVB3 infection significantly increased the transcription of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1). CVB3 replication was suppressed by EEF1A1 knockdown, while elevated by EEF1A1 overexpression. Similar to the effect of CVB3 infection, EEF1A1 transcription was increased in response to anisomycin treatment. However, eEF1A1 protein level was decreased with anisomycin treatment in a dose-dependent manner in CVB3-infected cells. Moreover, anisomycin promoted eEF1A1 degradation, which was inhibited by the treatment of chloroquine but not MG132. We demonstrated that eEF1A1 interacted with the heat shock cognate protein 70 (HSP70), and eEF1A1 degradation was inhibited by LAMP2A knockdown, implicating that eEF1A1 is degraded through chaperone-mediated autophagy. Taken together, we demonstrated that anisomycin, which inhibits CVB replication through promoting the lysosomal degradation of eEF1A1, could be a potential antiviral candidate for the treatment of CVB infection.

miR-494-5p mediates the antioxidant activity of EPA by targeting the mitochondrial elongation factor 1 gene MIEF1 in HepG2 cells.

Eicosapentaenoic acid (EPA) shows antioxidant activity, which may be attributed to its regulatory effect on microRNA expression. Our preliminary study indicated that EPA upregulated miR-494-5p, which was possibly involved in the regulation of cellular stress responses. The current study aimed to address whether miR-494-5p was targeted by EPA to regulate cellular oxidative stress and its possible functional mechanism. The results showed that miR-494-5p mediated the antioxidant effect of EPA and miR-494-5p reduction deteriorated EPA-induced increase in the cellular antioxidant capacity of HepG2 cells. Moreover, the mitochondrial elongation factor 1 (MIEF1) gene was a target gene of miR-494-5p. Both miR-494-5p overexpression and MIEF1 knockdown significantly enhanced cellular antioxidant capacity, as indicated by a reduction in the reactive oxygen species level and an increase in the total cellular antioxidant capacity, along with enhancing antioxidant enzymes. Thus, miR-494-5p and MIEF1 had opposite effects on cellular antioxidant capacity. Furthermore, their regulatory effects on oxidative stress may have been attributed to modulation of mitochondrial function, biogenesis and homeostasis. Taken together, the findings indicated that miR-494-5p mediated EPA activity and promoted cellular antioxidant capacity by inhibiting the expression of MIEF1, which further modulated mitochondrial structure and activity. This study may provide novel insights into the post-translational regulation of antioxidation reactions, which involves the coordinated control of mitochondria.

Interfering with the expression of EEF1D gene enhances the sensitivity of ovarian cancer cells to cisplatin.

BACKGROUND: Eukaryotic translation elongation factors 1 δ (EEF1D), has garnered much attention with regards to their role in the drug resistance of cancers. In this paper, we investigated the effects and mechanisms of increasing the sensitivity of ovarian cancer cells to cisplatin or cis-dichlorodiammine platinum (DDP) by knockdown and knockout of EEF1D gene in cellular and animal models. METHODS: The EEF1D gene was knocked-down or -out by siRNA or CRISPR/Cas9 respectively in human ovarian cancer cell SKOV3, DDP-resistant subline SKOV3/DDP, and EEF1D gene in human primary ovarian cancer cell from 5 ovarian cancer patients with progressive disease/stable disease (PD/SD) was transiently knocked down by siRNA interference. The mice model bearing xenografted tumor was established with subcutaneous inoculation of SKOV3/DDP. RESULTS: The results show that reducing or removing EEF1D gene expression significantly increased the sensitivity of human ovarian cancer cells to DDP in inhibiting viability and inducing apoptosis in vitro and in vivo, and also boosted DDP to inhibit xenografted tumor growth. Interfering with EEF1D gene expression in mice xenografted tumor significantly affected the levels of OPTN, p-Akt, Bcl-2, Bax, cleaved caspase-3 and ERCC1 compared to DDP treated mice alone, and had less effect on PI3K, Akt and caspase-3. CONCLUSIONS: The knocking down or out EEF1D gene expression could enhance the sensitivity of ovarian cancer cells to DDP partially, which may be achieved via inactivating the PI3K/AKT signaling pathway, thus inducing cell apoptosis and decreasing repairment of DNA damage. Our study provides a novel therapeutic strategy for the treatment of ovarian cancer.

Expression of Hybrid Peptide EF-1 in Pichia pastoris, Its Purification, and Antimicrobial Characterization.

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.

Eimeria tenella Elongation Factor-1α (EF-1α) Coadministered with Chicken IL-7 (chIL-7) DNA Vaccine Emulsified in Montanide Gel 01 Adjuvant Enhanced the Immune Response to E. acervulina Infection in Broiler Chickens.

The current study was undertaken to assess the vaccine efficacy of Eimeria tenella EF-1α/chicken IL-7 (chIL-7) DNA vaccine when administered with Montanide Gel 01 adjuvant against live Eimeria acervulina challenge in commercial broiler chickens. The criteria used for the evaluation of vaccine efficacy were weight gain, duodenal lesion scores, oocyst counts, humoral antibody response, and duodenal proinflammatory cytokine gene expression. Chickens vaccinated with EF-1α (100 µg)/chIL-7 (20 µg) in Gel 01 PR adjuvant showed body weight gain similar to the uninfected control and higher oocyst shedding, a lower gut lesion score, and higher proinflammatory cytokine gene expression than did the infected controls. Moreover, chickens vaccinated with chIL-7 (20 µg) in Gel 01 PR adjuvant shed fewer oocysts with reduced gut lesion scores and produced higher levels of anti-EF-1α serum antibody than did the infected control. Chickens vaccinated with EF-1α (50 µg)/chIL-7 (20 µg) in Gel 01 PR adjuvant showed higher weight gains than did the infected control and shed significantly fewer oocysts than the infected control. Furthermore, chickens vaccinated with EF-1α (100 µg) in Gel 01 PR adjuvant demonstrated the lowest anti-EF-1α serum antibody levels. This study demonstrated the beneficial effects of using EF-1α and/or host cytokine chIL-7 DNA vaccine together with Gel 01 PR adjuvant to improve T-cell-mediated effector function in broiler chickens challenged with live E. acervulina.

Factor de alargamiento 1α (EF-1α) de Eimeria tenella administrado conjuntamente con vacuna de ADN de interleucina 7 de pollo (chIL-7) emulsionada en adyuvante de Montanide Gel 01 aumentó la respuesta inmune a la infección por E. acervulina en pollos de engorde. Este estudio se realizó para evaluar la eficacia de la vacuna con ADN de Eimeria tenella EF-1α /IL-7 de pollo (chIL-7) cuando se administró con el adyuvante Montanide Gel 01 contra el desafío con Eimeria acervulina viva en pollos de engorde comerciales. Los criterios utilizados para evaluar la eficacia de la vacuna fueron el aumento de peso, las puntuaciones de lesiones duodenales, los recuentos de ooquistes, la respuesta humoral de anticuerpos y la expresión de genes de citoquinas proinflamatorias duodenales. Los pollos vacunados con EF-1α (100 µg)/chIL-7 (20 µg) en el adyuvante Gel 01 PR mostraron un aumento de peso corporal similar al control no infectado y una mayor excreción de ooquistes, una puntuación más baja en la lesión intestinal y una mayor expresión de genes de citoquinas proinflamatorias en comparación con los controles infectados. Además, los pollos vacunados con chIL-7 (20 µg) en adyuvante Gel 01 PR eliminaron menos ooquistes con puntuaciones reducidas de lesión intestinal y produjeron niveles más altos de anticuerpos séricos anti-EF-1α en comparación con el control infectado. Los pollos vacunados con EF-1α (50 µg)/chIL-7 (20 µg) en adyuvante Gel 01 PR mostraron mayores ganancias de peso que el control infectado y eliminaron significativamente menos ooquistes que el control infectado. Además, los pollos vacunados con EF-1α (100 µg) en adyuvante Gel 01 PR demostraron los niveles más bajos de anticuerpos séricos anti-EF-1α. Este estudio demostró los efectos beneficiosos del uso de la vacuna de ADN EF-1α y/o la citoquina del huésped chIL-7 junto con el adyuvante Gel 01 PR para mejorar la función efectora mediada por células T en pollos de engorde desafiados con E. acervulina viva.

Molecular and functional properties of an archaeal phenylalanyl-tRNA synthetase from the hyperthermophile Sulfolobus solfataricus.

An archaeal phenylalanyl-tRNA synthetase (FRS) has been purified from the hyperthermophile Sulfolobus solfataricus (Ss). This enzyme is a heterotetramer made of two different subunits whose molecular mass is 56 kDa and 64 kDa, respectively. As thought, SsFRS is essential for the in vitro poly(Phe) synthesis. Interestingly, the enzyme is able to aminoacylate only endogenous tRNA but it does not seem to be a strictly ATP-dependent synthetase. SsFRS interacts with the elongation factor 1alpha isolated from the same source; this caused a significant enhancement of the SstRNA aminoacylation efficiency, thus indicating that, as well as in eukarya, in this archaeon a tRNA channelling mechanism should occur. The overall results presented in this paper show that the archaeal SsFRS behaves as the analogous enzymes isolated from eukaryal sources rather than those from eubacterial organisms.

Effects of the active aldehyde group generated by RNA N-glycosidase in the sarcin/ricin domain of rat 28S ribosomal RNA on peptide elongation.

Effects of the active aldehyde group of ribose C1' at position 4324 of rat 28S rRNA, in the inactivated ribosome generated by RNA N-glycosidases (trichosanthin, A-chain of cinnamomin and ricin), on peptide elongation have been studied. The aldehyde group inhibits the activities of eEF1A-dependent aminoacyl-tRNA binding to the inactivated ribosome and eEF1A-dependent GTPase, but increases eEF2-dependent activity. At a high concentration of RNA N-glycosidase, the generated aldehyde group also inhibits aminoacyl-tRNA binding to the inactivated ribosome in the absence of elongation factor and translocation activity. When the aldehyde group is reduced into a hydroxyl group by sodium borohydride or blocked with an amino acid through nucleophilic addition, the activities of eEF1A-dependent aminoacyl-tRNA binding and eEF1A-dependent GTPase of the inactivated ribosome are partially restored, but the altered activities of eEF2-dependent GTPase, translocation and aminoacyl-tRNA binding in the absence of elongation factor are not normalized. Thus, reduction or blockage of the aldehyde group with sodium borohydride or amino acids might change the conformation of the S/R domain in rat 28S ribosomal RNA to meet the requirement for eEF1A-dependent reactions, but not eEF2-involved reactions.