• Journal of Microbiology and Biotechnology
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• 제34권6호
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• pp.1348-1355
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• 2024

The eukaryotic translation initiation factor eIF5B is a bacterial IF2 ortholog that plays an important role in ribosome joining and stabilization of the initiator tRNA on the AUG start codon during the initiation of translation. We identified the fluorophenyl oxazole derivative 2,2-dibromo-1-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)ethanone quinolinol as an inhibitor of fungal protein synthesis using an in vitro translation assay in a fungal system. Mutants resistant to this compound were isolated in Saccharomyces cerevisiae and were demonstrated to contain amino acid substitutions in eIF5B that conferred the resistance. These results suggest that eIF5B is a target of potential antifungal compound and that mutation of eIF5B can confer resistance. Subsequent identification of 16 other mutants revealed that primary mutations clustered mainly on domain 2 of eIF5B and secondarily mainly on domain 4. Domain 2 has been implicated in the interaction with the small ribosomal subunit during initiation of translation. The tested translation inhibitor could act by weakening the functional contact between eIF5B and the ribosome complex. This data provides the basis for the development of a new family of antifungals.

• Molecules and Cells
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• 제24권3호
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• pp.445-451
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• 2007

Translation initiation factor 4E (eIF4E) is a key regulator of protein synthesis. Abnormal regulation of eIF4E is closely linked to oncogenic transformation. Several regulatory mechanisms affecting eIF4E are discussed, including transcriptional regulation, phosphorylation and binding of an inhibitor protein. However it is not clear how the level of eIF4E protein is regulated under basal conditions. Here we demonstrate that Diap1 (Drosophila Inhibitor of Apoptosis Protein), a cell death inhibitor, binds directly to eIF4E and poly-ubiquitinates it via its E3 ligase activity, promoting its proteasome-dependent degradation. Expression of Diap1 caused a reduction of Cyclin D1 protein level and inhibited the growth stimulation induced by overexpression of eIF4E. Taken together, our results suggest that the level of eIF4E protein is regulated by Diap1, and that IAPs may play a role in cap-dependent translation by regulating the level of eIF4E protein.

• Journal of Plant Biotechnology
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• 제38권1호
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• pp.87-93
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• 2011

SAMDC는 폴리아민 생합성 과정에서 주효소로 작용하며 항상성을 유지하기위해 정교하게 조절된다. 카네이션 SAMDC 유전자는 5'-leader sequence에 54개 아미노산으로 구성된 small uORF가 존재한다. Translation 과정을 조절하는 uORF의 작용기작을 연구하기 위하여 35S 프로모터에 SAMDC 유전자의 uORF 부위와 GUS 유전자를 재조합한 형질전환 담배 식물체를 이용하였다. 본 실험에서는 SAMDC uORF 염기서열 혹은 SAMDC uORF 단백질에 의해서 downstream GUS ORF의 translation이 억제되었다. 특히 translation 억제는 개시코돈이 point-mutation된 construct에서 효과적으로 이루어졌다. 따라서 이러한 결과는 ribosomal stalling이 translation 억제 과정에 관여한 것으로 사료된다. 개시 코돈과 종결코돈을 가진 SAMDC uORF의 아미노산 서열을 frame shift 시키면 GUS 활성이 증가하였는데 이는 translation inhibitor로서 작용할 때 아미노산 서열이 중요하다는 것을 의미하며, 결국은 SAMDC uORF의 단백질 구조가 중요하게 작용할 가능성을 제시한다. 또한 유식물과 담배 꽃 등의 in vivo 상에서도 GUS 발현을 조직화학적으로 분석했을 때 small uORF가 존재할 경우 GUS 염색이 크게 저하되었지만, 개시코돈이나 혹은 종결코돈이 제거되도록 point-mutation 시킨 construct가 도입된 형질전환식물체에서는 SAMDC uORF의 억제효과가 크게 완화 되었다. 또한 가장 중요한 관찰 결과로는 small uORF 염기서열로부터 in vitro 시스템에서 5.7 kDa의 단백질이 실제적으로 합성되었음을 관찰하였다. 폴리아민 처리 후 GUS 단백질이 억제된 결과는 uORF로부터 합성된 단백질이 폴리아민 뿐 만 아니라 translation 과정에 관여하는 다른 요소들과 상호작용을 이루어 조절될 수 있음을 암시한다.

• Applied Biological Chemistry
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• 제32권3호
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• pp.222-231
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• 1989

대두에 존재하는 단백질 분해 효소 저해제로는 Kunitz trypsin inhibitor (SKTI), Bowman-Birk proteinase inhibitor, 그리고 그의 isoinhibitor들이 알려져 있다. SKTI는 분자량이 20K이며 181개의 아미노산으로 구성되어 있다. SKTI의 분자 구조 및 발현 특이성을 밝히기 위하여, 콩에서 순수 분리한 SKTI를 항원으로 사용하여 항체를 제조하였다. 이 항체는 항원으로 사용한 SKTI에 대하여 특이적으로 반응할 뿐만 아니라 기내에서 합성된 그의 전구체와도 특이적으로 반응하였다. 기내에서의 단백질 합성은 미성숙 대두 종자에서 mRNA를 분리한 후, wheat germ extract를 이용하여 실시하였다. 합성된 단백질 중에서 대두에 존재하는 SKTI는 검출되지 않는 반면에 면역침전법에 의해 분자량이 24K인 전구체가 존재하고 있음을 확인하였다. 이와 같이 SKTI는 mRNA로부터 전구체가 합성된 후 post-translational modification에 의해 완전한 SKTI로 변환됨을 알 수 있었다. 또한 SKTI는 대두 종자의 성숙과 함께 발현이 되는 것으로 보아 조직 및 분화 특이성 발현형태를 나타내는 것을 알 수 있었다.

• Animal cells and systems
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• 제14권3호
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• pp.147-154
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• 2010

It is well-established that phosphoinositide 3-kinase (PI3-kinase) regulates myogenesis by inducing transcription of myogenin, a key muscle regulatory factor, at the initiation of myoblast differentiation. In this study, we investigated the role of PI3-kinase in cells that have committed to differentiation. PI3-kinase activity increases during myogenesis, and this increase is sustained during the myogenic process; however, its function after the induction of differentiation has not been investigated. We show that LY294002, a PI3-kinase inhibitor, blocked myoblast fusion even after myogenin expression initially increased. In contrast to the inhibitory effects of LY294002 on myogenin mRNA levels during the initiation of differentiation, LY294002 blocked the accumulation of myogenin protein without affecting its mRNA level after differentiation was induced. Treatment with cycloheximide, a translation inhibitor, or actinomycin D, a transcription inhibitor, indicated that the stability of myogenin protein is lower than that of its mRNA. LY294002 inhibited the activities of several important translation factors, including eukaryotic elongation factor-2(eEF2), by altering their phosphorylation status. In addition, LY294002 blocked the incorporation of [$^{35}S$]methionine into newly synthesized proteins. Since myogenin has a relatively short half-life, LY294002-mediated inhibition of post-transcriptional processes resulted in a rapid depletion of myogenin protein. In summary, these results suggest that PI3-kinase plays an important role in regulating the expression of myogenin through post-transcriptional mechanisms after differentiation has been induced.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1646-1649
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• 2006

Protein synthesis is the ultimate outcome of gene expression which, in turn, is regulated by several translation factors. We attempted to identify substances that can inhibit the translation process in vitro when the outcome protein is luciferase. To this end, we developed a sensitive cell-free protein synthesis assay using luciferase as the reporter. The synthesis of luciferase increased proportionately as mRNA was added to a $15-{\mu}l$reaction medium in concentrations raging from 5 ng to 500 ng. The maximum amount of luciferase was synthesized when the media were incubated at $25^{\circ}C$ for 40 min. The concentration of each compound that inhibited luciferase production by 50% ($IC_{50}$) was calculated. Hygromycin, puromycin, and cycloheximide yielded an $IC_{50}$ of 0.008, 0.8, and $0.7{\mu}g/ml$, respectively. A filtrate of Streptomyces spp. isolates inhibited protein synthesis up to S-fold when added to the in vitro translation assay mixture.

• The Plant Pathology Journal
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• 제28권2호
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• pp.197-201
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• 2012

Potyviruses express their RNA genomes through the production of polyproteins that are processed in host cells by three virus-encoded proteases. Soybean plants produce large amounts of protease inhibitors during seed development and in response to wounding that could affect the activities of these proteases. The in vitro activities of two of the proteases of Soybean mosaic virus (SMV) and Tobacco vein mottling virus (TVMV) were compared in the rabbit reticulocyte lysate in vitro translation system using synthetic RNA transcripts. Transcripts produced from SMV and TVMV cDNAs that included the P1 and helper component-protease (HC-Pro) coding regions directed synthesis of protein products that were only partially processed. Unprocessed poly-proteins were not detected from transcripts that included all of the P1, HC-Pro, P3 and portions of the cylindrical inclusion protein coding regions of either virus. Addition of soybean trypsin inhibitor to in vitro translation reactions increased the accumulation of the unprocessed polyprotein from TVMV transcripts, but did not alter the patterns of proteins produced from SMV. These experiments suggest that SMV-and TVMV-encoded proteases are differentially sensitive to protease inhibitors.

• 대한의생명과학회지
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• 제22권4호
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• pp.211-214
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• 2016

House dust mite is an essential allergen in the pathogenesis of allergic diseases. Abnormal regulation of neutrophil apoptosis is an important pathogenic process in allergic diseases. In the present study, we investigated the effects of house dust mites on spontaneous apoptosis of neutrophils and its associated mechanisms. Extract of Dermatophagoides pteronissinus (DP) inhibited neutrophil apoptosis in a time-dependent manner. Cycloheximide (CHX), an inhibitor of translation, increased apoptosis of DP-treated neutrophils as well as control cells. The pro-apoptotic effect of CHX was blocked by DP in neutrophils. In addition, DP increased the phosphorylation of Bad in a time-dependent manner, indicating that it exerted an inhibitory effect on the function of Bad. These results suggest that DP has anti-apoptotic effects of neutrophils and may regulate protein synthesis and activation of Bad. Moreover, these findings may shed light on elucidation of allergy pathogenesis due to house dust mites.

• 한국식품위생안전성학회지
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• 제11권4호
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• pp.227-234
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• 1996

This study was designed to characterize endotoxin-induced prostaglandin production in primary cultured rat vascular smooth muscle cells (VSMC). The time course for prostaglandin synthesis in lipopolysaccharide (LPS)-stimulated VSMC showed that the maximum production was reached in 12 hours. LPS induced prostaglandin H2 synthase (PGHS) activity in VSMC and the time course profile in the changes of PGHS activity paralleled that of total prostaglandin production. Differential treatment showed that 4 hours' exposure to LPS was enough for the maximum effect on the prostaglandin production and this effect was completely inhibited by the co-treatment of actinomycin D, a transcription inhibitor. These results suggest that LPS effect might be determined within 4 hours. Actinomycin D increased PGHS activity without affecting prostaglandin production if added 4 hours after LPS treatment. On the other hand, cyclogeximide, a translation inhibitor, augmented LPS-induced prostaglandin production if treated during first four hours, but it inhibited LPS-induced PGHS activity regardless of treatment schedule. These results suggest the existence of multiple regulating mechanisms in the LPS-induced prostaglandin synthesis.

• Molecules and Cells
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• 제20권2호
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• pp.167-172
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• 2005

Ceruloplasmin (Cp) is a copper protein with important functions in iron homeostasis and in inflammation. Cp mRNA expression is induced by interferon (IFN)-${\gamma}$ in U937 monocytic cells, but synthesis of Cp protein is halted after about 12 h by transcript-specific translational silencing. The silencing mechanism requires binding of a 4-component cytosolic inhibitor complex, IFN-gamma-activated inhibitor of translation (GAIT), to a defined structural element (GAIT element) in the Cp 3'-UTR. Translational silencing of Cp mRNA requires the essential proteins of mRNA circularization, suggesting that the translational inhibition requires end-to-end mRNA closure. These studies describe a new mechanism of translational control, and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.