• Journal of the Korean Magnetic Resonance Society
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• v.16 no.2
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• pp.162-171
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• 2012

SAV2228 has an OB (Oligomer-Binding)-motif which is frequently used for nucleic acid recognition. To characterize the activity of translation initiation factor-1 (IF-1) from Staphylococcus aureus, SAV2228 was expressed and purified in Escherichia coli. We acquired 3D NMR spectra showing well dispersed and homogeneous signals which allow us to assign 94.4% of all $^1HN$, $^{15}N$, $^{13}C{\alpha}$, $^{13}C{\beta}$ and $^{13}CO$ resonances. We could predict a secondary structure of SAV2228 using TALOS and CSI from NMR data. SAV2228 was consisted of one ${\alpha}$-helix and five ${\beta}$-sheets. The predicted secondary structure, ${\beta}-{\beta}-{\beta}-{\alpha}-{\beta}-{\beta}$, was similar to other bacterial IF-1, but it was not completely same to the eukaryotic one. Assigned NMR peaks and secondary structre prediction can be used for the study on interaction with nucleic acid in the future.

• Molecules and Cells
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• v.39 no.2
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• pp.129-135
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• 2016

Eukaryotic translation initiation factor 2 alpha ($eIF2{\alpha}$), which is a component of the eukaryotic translation initiation complex, functions in cell death and survival under various stress conditions. In this study, we investigated the roles of $eIF2{\alpha}$ phosphorylation in cell death using the breast cancer cell lines MCF-7 and MCF-7/ADR. MCF-7/ADR cells are MCF-7-driven cells that have acquired resistance to doxorubicin (ADR). Treatment of doxorubicin reduced the viability and induced apoptosis in both cell lines, although susceptibility to the drug was very different. Treatment with doxorubicin induced phosphorylation of $eIF2{\alpha}$ in MCF-7 cells but not in MCF-7/ADR cells. Basal expression levels of Growth Arrest and DNA Damage 34 (GADD34), a regulator of $eIF2{\alpha}$, were higher in MCF-7/ADR cells compared to MCF-7 cells. Indeed, treatment with salubrinal, an inhibitor of GADD34, resulted in the upregulation of $eIF2{\alpha}$ phosphorylation and enhanced doxorubicin-mediated apoptosis in MCF-7/ADR cells. However, MCF-7 cells did not show such synergic effects. These results suggest that dephosphorylation of $eIF2{\alpha}$ by GADD34 plays an important role in doxorubicin resistance in MCF-7/ADR cells.

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.353-359
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• 2008

Hepatitis C virus (HCV) is known as the causative agent of blood transmitted hepatitis. Two viral proteins, E2 and NS5A, are known to exert interferon resistance of HCV via PKR pathway. Here, we report a third protein, the RNA-dependent RNA polymerase (NS5B) of HCV, induced interferon resistance inhibiting p56 pathway. p56 was shown to interact with p48 subunit of eukaryotic initiation factor 3 (eIF3). This interaction inhibited formation of ternary complex in translation initiation. Using dual reporter assay system, we observed that the translation decreased when interferon alpha was added to the culture. But, in the presence of HCV NS5B, the translation partly recovered. NS5B and p48 subunit of eIF3 were shown to interact. This interaction seems to inhibit the interaction between p48 and p56. This is the first report that a virus exerts interferon resistance via p56 pathway.

• Toxicological Research
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• v.8 no.2
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• pp.343-357
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• 1992

Tumor necrosis factor-${\alpha}$(TNF), a polypeptide hormone secreted primarily by activated macrophages, was originally identified on the basis of its ability to cause hemorrhagic necrosis and tumor regression in vivo. Subsequently, TNF has been shown to be an important component of the host responses to infection and cancer and may mediate the wasting syndrome known as cachexia. These systemic actions of TNF are reflected in its diverse effects on target cells in vitro. TNF initiates its diverse cellular actions by binding to specific cell surface receptors. Although TNF receptors have been identified on most of animal cells, regulation of these receptors and the mechanisms which transduce TNF receptor binding into cellular responses are not well understood. Therefore, in the present study, the mechanisms how TNF receptors are being regulated and how TNF receptor binding is being transduced into cellular responses were investigated in rat liver plasma membranes (PM) and ME-180 human cervical carcinoma cell lines. $^{125}I$-TNF bound to high ($K_d=1.51{\pm}0.35nM$)affinity receptors in rat liver PM. Solubilization of PM with 1% Triton X-100 increased both high affinity (from $0.33{\pm}0.04\;to\;1.67{\pm}0.05$ pmoles/mg protein) and low affinity (from $1.92{\pm}0.16\;to\;7.57{\pm}0.50$ pmoles/mg protein) TNF binding without affecting the affinities for TNF, suggesting the presence of a large latent pool of TNF receptors. Affinity labeling of receptors whether from PM or solubilized PM resulted in cross-linking of $^{125}I$-TNF into $M_r$ 130 kDa, 90 kDa and 66kDa complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, $^{125}I$-TNF binding to control and TNF-pretreated membranes were assayed. Specific binding was increased by pretreatment with TNF (P<0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF. As a next step, the post-receptor events induced by TNF were examined. Although the signal transduction pathways for TNF have not been delineated clearly, the actions of many other hormones are mediated by the reversible phosphorylation of specific enzymes or target proteins. The present study demonstrated that TNF induces phosphorylation of 28 kDa protein (p28). Two dimensional soidum dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) resolved the 28kDa phosphoprotein into two isoforms having pIs of 6.2 and 6.1. The pIs and relative molecular weight of p28 were consistent with those of a previously characterized mRNA cap binding protein. mRNA cap binding proteins are a class of translation initiation factors that recognize the 7-methylguanosine cap structure found on the 5' end of eukaryotic mRNAs. In vitro, these proteins are defined by their specific elution from affinity columns composed of 7-methylguanosine 5'-triphosphate($m^7$GTP)-Sepharose. Affinity purification of mRNA cap binding proteins from control and TNF treated ME-180 cells proved that TNF rapidly stimulates phosphorylation of an mRNA cap binding protein. Phosphorylation occurred in several cell types that are important in vitro models of TNF action. The mRNA cap binding protein phosphorylated in response to TNF treatment was purifice, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor-4E(eIF-4E). These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the diverse cellular actions of TNF.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.6
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• pp.1012-1022
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• 2020

Objective: Caseins and fatty acids of milk are synthesized and secreted by the epithelial cells of the mammary gland. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, has been shown to promote mammary development. This study was conducted to determine the effect of ATRA on casein synthesis and fatty acid composition in MAC-T cells. Methods: MAC-T cells were allowed to differentiate for 4 d, treated with ATRA (0, 1.0, 1.5, and 2.0 μM), and incubated for 3 d. We analyzed the fatty acid composition, the mRNA expression of casein and fatty acid synthesis-related genes, and the phosphorylation of casein synthesis-related proteins of MAC-T cells by gas chromatography, quantitative polymerase chain reaction, and western blotting, respectively. Results: In MAC-T cells, ATRA increased the mRNA levels of α_S1-casein and β-casein, janus kinase 2 (JAK2) and E74-like factor 5 of the signal transducer and activator of transcription 5 β (STAT5-β) pathway, ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 of the mammalian target of rapamycin (mTOR) pathway, inhibited the mRNA expression of phosphoinositide 3-kinase and eukaryotic initiation factor 4E of the mTOR pathway, and promoted the phosphorylation of STAT5-β and S6K1 proteins. Additionally, ATRA increased the de novo synthesis of fatty acids, reduced the content of long-chain fatty acids, the ratio of monounsaturated fatty acids to saturated fatty acids (SFA), the ratio of polyunsaturated fatty acids (PUFA) to SFA, and the ratio of ω-6 to ω-3 PUFA. The mRNA levels of acetyl-CoA carboxylase 1, fatty acid synthase, lipoprotein lipase, stearoyl-CoA desaturase, peroxisome proliferator-activated receptor gamma, and sterol regulatory element-binding protein 1 (SREBP1) were enhanced by ATRA. Conclusion: ATRA promotes the synthesis of casein by regulating JAK2/STAT5 pathway and downstream mTOR signaling pathway, and it improves the fatty acid composition of MAC-T cells by regulating SREBP1-related genes.

• Molecules and Cells
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• v.25 no.2
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• pp.172-177
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• 2008

Eukaryotic translation initiation factor 5B (eIF5B) plays a role in recognition of the AUG codon in conjunction with translation factor eIF2, and promotes joining of the 60S ribosomal subunit. To see whether the eIF5B proteins of other organisms function in Saccharomyces cerevisiae, we cloned the corresponding genes from Oryza sativa, Arabidopsis thaliana, Aspergillus nidulans and Candida albican and expressed them under the control of the galactose-inducible GAL promoter in the $fun12{\Delta}$ strain of Saccharomyces cerevisiae. Expression of Candida albicans eIF5B complemented the slow-growth phenotype of the $fun12{\Delta}$ strain, but that of Aspergillus nidulance did not, despite the fact that its protein was expressed better than that of Candida albicans. The Arabidopsis thaliana protein was also not functional in Saccharomyces. These results reveal that the eIF5B in Candida albicans has a close functional relationship with that of Sacharomyces cerevisiae, as also shown by a phylogenetic analysis based on the amino acid sequences of the eIF5Bs.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.447-453
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• 2006

Histidine kinase plays important roles in signal transduction in plant. We characterized the function of Arabidopsis histidine kinase 3 (AHK3) and analyzed the expression patterns of genes and proteins in its mutant ahk3 by trans-zeatin (t-zeatin). The ahk3 exhibited decreased sensitivity to t-zeatin during callus formation, seedling growth, and leaf senescence. From proteomic analysis of ahk3, eukaryotic translation initiation factor 5A-2, auxin binding glutathione S-transferase, and NDPK1 were identified not to be induced by t-zeatin, when compared to the wild-type. In addition, the expression levels of ARR4 and ARR16 among A-type response regulators (ARRs) markedly decreased in ahk3 by t-zeatin treatment. These results suggest that AHK3 plays an important role in cytokinin signaling and the proteins identified from proteomic analysis and specific ARRs, ARR4 and ARR16 may be directly or indirectly associated in AHK3-mediated cytokinin signaling.

• Journal of Life Science
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• v.23 no.7
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• pp.941-946
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• 2013

Local protein synthesis at subsynaptic sites plays a key role in the regulation of the protein composition in local domains. In this study, we carried out immunocytochemistry of cultured rat hippocampal neurons in various developmental stages to investigate the expression of eIF4E and its binding protein, eIF4EBP1. Both proteins were distributed in dendrites. In addition, eIF4EBP1 was highly expressed in the nucleus throughout the development, whereas eIF4E was not expressed in the nucleus. Punctate expression of eIF4E and eIF4EBP1 was evident in DIV 3. The colocalization rates of eIF4E or eIF4EBP1 puncta with PSD95 were higher in the dendrogenic than in the mature stages. In contrast, the colocalization rates of eIF4E and eIF4EBP1 puncta were higher in the mature than in the dendrogenic stages. As eIF4E is inactive when it is bound to eIF4EBP1, these data indicate that most dendritic eIF4E's are active during development but that they are mostly under inhibition in mature neurons.

• Journal of Life Science
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• v.23 no.4
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• pp.595-601
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• 2013

The clusters of orthologous groups of proteins (COG) algorithm was applied to identify essential proteins in eukaryotes and to measure the degree of conservation. Sixty-three orthologous groups, which were conserved in 66 microbial genomes, enlarged to 104 eukaryotic orthologous groups (KOGs) and 71 KOGs were conserved at the nuclear genome of 7 eucaryotes. Fifty-four of 71 translation-related genes were conserved, highlighting the importance of proteins in modern organisms. Translation initiation factors (KOG0343, KOG3271) and prolyl-tRNA synthetase (KOG4163) showed high conservation based on the distance value analysis. The genes of Caenorhabditis elegans appear to harbor high genetic variation because the genome showed the highest variation at 71 conserved proteins among 7 genomes. The 71 conserved genes will be valuable in basic and applied research, for example, targeting for antibiotic development.

• Journal of Integrative Natural Science
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• v.12 no.4
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• pp.127-132
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• 2019

Programmed cell death 4 (PDCD4) is a novel tumor suppressor that function in the nucleus and the cytoplasm and appears to be involved in the regulation of transcription and translation. Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate when cells exposed to environmental stresses. Since PDCD4 has implicated in translation repression through direct interaction with eukaryotic translation initiation factor 4A (eIF4A), we here investigated if PDCD4 has a functional role in the process of SG assembly under oxidative stresses. Using immunofluorescence microscopy, we found that PDCD4 is localized to SGs under oxidative stresses. Next, we tested if knockdown of PDCD4 has an effect on the assembly of SG using PDCD4-specific siRNA. Interestingly, SG assembly was accelerated and this effect was caused by sensitization of phosphorylation of eIF2α and dephosphorylation of eIF4E binding protein (4E-BP). These results suggest that PDCD4 has an effect on SG dynamics and possibly involved in cap-dependent translation repression under stress conditions.