• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1993년도 제2회 신약개발 연구발표회 초록집
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• pp.55-55
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• 1993

Farnesyl Protein transferase(FPT)는 발암유전자 ras의 단백질 산물인 p$^{21}$의 post-translational modification의 첫 단계인 ras-farnesylation에 관여하는 효소로 본 연구에서는 정제된 FPT와 E. coli에서의 발현 system을 이용하여 FPT의 구조와 기능을 밝히고 이를 FPT 방해제의 설계에 이용하고자 한다. Bovine testis에 존재하는 FPT를 30%-50%의 Ammonium sulfate로 fractionation하고, DEAE-Sephacel, Sephacryl S-300 column을 통과시킨 후 peptide(KKCVIM) affinity column을 이용하여 순수 정제하였다. 정제된 효소의 분자량은 gel-filtration에 의해 100KDa으로 추정되었고 SDS-PAGE 결과 49KDa과 46KDa의 두 subunit로 구성되었음이 확인되었다. 효소활성에는 $Mg^{2＋}$ 와 $Zn^{2＋}$가 필수적이며 최적 pH는 7.0이었다. Yeast의 FPT의 두 subunit 유전자는 Yeast genomic DNA를 template로 사용하고 각 subunit에 specific한 합성된 primer들과 vent polymerase를 이용하여 Polymerase chain reaction을 통하여 얻었다. 두 유전자를 pBluescriptII SK＋ vector를 변형시킨 두 vector, pBSK＋4와 pBChl＋4에 재조합 시킨 후 E.coli에 transformation시켜 발현시켰다. 현재 정제된 Bovine FPT와 E. coli에서 발현된 Yeast FPT의 chemical modification과 site-directed mutagenesis를 통하여 FPT의 active site와 substrate binding site에 관한 연구를 진행시키고 있다.

• 약학회지
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• 제32권6호
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• pp.420-427
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• 1988

Post-translational modification of protein amino acid residues is a well known metabolic phenomenon. One such side chain modification, protein methylation, occur ubiquitously in nature, in organism ranging from prokaryotic to eukaryotic and the biological significance of protein methylation has begun to emerge. The observation that cytochrome C methylation facilitates the binding of this hemoprotein to mitochondria could be placed as the one of the examples along this line. However, the detail biological meaning of cytochrome C methylation is remained to be clarified. In the aspect of such reason this research was done. The results of this experiment were; 1) pure Euglena gracilis cytochrome C552 was isolated, 2) methylarginine and methylmethionine were not found in cytochrome C552 sequence, 3) however, Unknown Peak at 20.78min of retention time was found, and 4) this Unknown Peak was found only from Euglena cytochrome C552, so far.

• BMB Reports
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• 제54권11호
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• pp.541-544
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• 2021

Protein glycosylation is a common post-translational modification found in all living organisms. This modification in bacterial pathogens plays a pivotal role in their infectious processes including pathogenicity, immune evasion, and host-pathogen interactions. Importantly, many key proteins of host immune systems are also glycosylated and bacterial pathogens can notably modulate glycosylation of these host proteins to facilitate pathogenesis through the induction of abnormal host protein activity and abundance. In recent years, interest in studying the regulation of host protein glycosylation caused by bacterial pathogens is increasing to fully understand bacterial pathogenesis. In this review, we focus on how bacterial pathogens regulate remodeling of host glycoproteins during infections to promote the pathogenesis.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.331-336
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• 2005

Post translational modifications (PTMs) discovery is an important problem in proteomic. In the past, people discover PTMs by Tandem Mass Spectrometer based on ‘bottom-up’ strategy. However, such strategy suffers from the problem of failing to discover all PTMs. Recently, due to the improvement in proteomic technology, Taylor et al. proposed a database software to discover PTMs with ‘topdown’ strategy by FTMS, which avoids the disadvantages of ‘bottom-up’ approach. However, their proposed algorithm runs in exponential time, requires a database of proteins, and needs prior knowledge about PTM sites. In this paper, a new algorithm is proposed which can work without a protein database and can identify modifications in polynomial time. Besides, no prior knowledge about PTM sites is needed.

• 생명과학회지
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• 제24권2호
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• pp.196-208
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• 2014

식물의 생활사 동안 물부족 스트레스는 식물의 생장과 발달에 해로운 영향을 끼치는 대표적인 스트레스이다. 유용 작물의 생산성을 증가시키기 위하여, 물부족 스트레스를 극복하는 일은 식물학 연구 분야에서 가장 중요한 이슈로 대두되어 왔다. 식물의 호르몬 중에서 앱시스산은 물부족 스트레스에 대해 식물이 저항성을 나타내는데 가장 중요한 호르몬으로서 역할을 수행하며, 씨앗의 발아, 기공의 개폐, 유묘의 성장과 같은 다양한 발달 과정에도 관여하고 있다. 그러므로, 앱시스산에 의해 매개되는 식물의 신호전달 기작을 명확히 이해하는 것은 물부족 스트레스에 대한 내성을 갖는 유용 식물을 생산해내기 위해 가장 효과적인 방법이 될 것이다. 한편, 인산화, 유비퀴틴화와 같은 번역 후 변형 과정은 식물이 다양한 환경적 스트레스 하에서 신속하게 적응을 하기 위해 가장 효율적인 기작으로 인식되어 왔는데, 이는 전사수준에서의 조절과 달리 이미 존재하는 신호전달 물질의 활성과 안정성을 직접적으로 빠르게 조절할 수 있기 때문이다. 본 총설에서는 앱시스산의 신호전달 과정과 관련된 최근 연구 동향을 업데이트하고자 하며, 특히 이러한 신호전달 과정을 앱시스산 수송, 인식, 신호전달 및 번역 후 변형 과정에 초점을 맞추어 알아보고자 한다. 또한 그러한 조절 기작이 농업분야에서 유용 작물을 생산하는데 어떻게 적용될 수 있는지에 대한 향후 전망에 대해서도 기술하고자 한다.

• Journal of Microbiology and Biotechnology
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• 제27권3호
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• pp.610-615
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• 2017

When Shigella infect host cells, various effecter molecules are delivered into the cytoplasm of the host cell through the type III secretion system (TTSS) to facilitate their invasion process and control the host immune responses. Among these effectors, the S. flexneri effector OspF dephosphorylates mitogen-activated protein kinases and translocates itself to the nucleus, thus preventing histone H3 modification to regulate expression of proinflammatory cytokines. Despite the critical role of OspF, the mechanism by which it localizes in the nucleus has remained to be elucidated. In the present study, we identified a potential small ubiquitin-related modifier (SUMO) modification site within OspF and we demonstrated that Shigella TTSS effector OspF is conjugated with SUMO in the host cell and this modification mediates the nuclear translocation of OspF. Our results show a bacterial virulence factor can exploit host post-translational machinery to execute its intracellular trafficking.

• Preventive Nutrition and Food Science
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• 제5권4호
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• pp.184-188
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• 2000

HMG-CoA reductase is th rate-limiting enzyme of cholesterol biosynthesis. As intracellular levels of cholesterol should be regulated elaborately in response to external stimuli an internal needs, the expression of the HMG-CoA reductase gene is regulated intricately at several different levels from transcription to post-translational modification. In this study, we investigated the regulatory mechanism of HMG-CoA reductase gene expression at the post-transcriptional/pre-translational levels in a baby hamster kidney cell line, C100. when 25-hydroxycholesterol was added to cells cultured in medium containing 5% delipidized fetal bovine serum and 25$\mu$M lovastatin, the levels of HMG-CoA reductase mRNA decreased rapidly, which seemed to be due to the increased degradation of reductase mRNA. These suppressive effects of 25-hydroxycholesterol on MG-CoA reductase mRNA levels were blocked by a translation inhibitor, cycloheximide. Similarly, actinomycin D and 5,6-dichloro-1-$\beta$-D-ribofuranosylbenzimidazole, transcription inhibitors, blocked the 25-hydroxycholesterol-mediated degradation of HMG-CoA reductase mRNA. These results indicate that new protein/RNA synthesis is required for the degradation of HMG-CoA reductase mRNA. In addition, data from the transfection experiments shows that cis-acting determinants, regulating the stability of reductase mRNA, were scattered in the sequence corresponding to 1766-4313 based on the sequence of Syrian hamster HMG-CoA reductase cDNA. Our data suggests that sterol-mediated destabilization of reductase mRNA might be one of the important regulatory mechanism of HMG-CoA reductase gene expression.

• Molecules and Cells
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• 제41권2호
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• pp.127-133
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• 2018

Chromatin remodeling factors are involved in many cellular processes such as transcription, replication, and DNA damage response by regulating chromatin structure. As one of chromatin remodeling factors, remodeling and spacing factor 1 (RSF1) is recruited at double strand break (DSB) sites and regulates ataxia telangiectasia mutated (ATM) -dependent checkpoint pathway upon DNA damage for the efficient repair. RSF1 is overexpressed in a variety of cancers, but regulation of RSF1 levels remains largely unknown. Here, we showed that protein levels of RSF1 chromatin remodeler are temporally upregulated in response to different DNA damage agents without changing the RSF1 mRNA level. In the absence of SNF2h, a binding partner of RSF1, the RSF1 protein level was significantly diminished. Intriguingly, the level of RSF1-3SA mutant lacking ATM-mediated phosphorylation sites significantly increased, and upregulation of RSF1 levels under DNA damage was not observed in cells overexpressing ATM kinase. Furthermore, failure in the regulation of RSF1 level caused a significant reduction in DNA repair, whereas reconstitution of RSF1, but not of RSF1-3SA mutants, restored DSB repair. Our findings reveal that temporal regulation of RSF1 levels at its post-translational modification by SNF2h and ATM is essential for efficient DNA repair.

• Mass Spectrometry Letters
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• 제13권4호
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• pp.139-145
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• 2022

Protein glycosylation is a common post-translational modification by non-template-based biosynthesis. In fungal biotechnology, which has great applications in pharmaceuticals and industries, the importance of research on fungal glycoproteins and glycans is accelerating. In particular, the importance of quantitative analysis of fungal glycans is emerging in research on the production of filamentous fungal proteins by genetic modification. Reliable mass spectrometry-based techniques for quantitative glycomics have evolved into chemical, enzymatic, and metabolic stable isotope labeling methods. In this study, we intend to expand quantitative glycomics by metabolic isotope labeling of glycans in Aspergillus niger, a filamentous fungus model, by the MILPIG method. We demonstrate that incubation of filamentous fungi in a culture medium with carbon-13 labeled glucose (1-¹³C₁) efficiently incorporates carbon-13 into N-linked glycans. In addition, for quantitative validation of this method, light and heavy glycans are mixed 1:1 to show the performance of quantitative analysis of various N-linked glycans simultaneously. We have successfully quantified fungal glycans by MILPIG and expect it to be widely applicable to glycan expression levels under various biological conditions in fungi.

• Archives of Pharmacal Research
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• 제25권6호
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• pp.759-769
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• 2002

Mutated forms of ras are found in many human tumors and the rate of incidence is significantly higher in colon and pancreatic cancers. The protein product from the ras oncogene is a small G-protein, $p21^{ras}{\;}(Ras)$ that is known to playa key role in the signal transduction cascade and cell differentiation and proliferation. Mutated Ras is unable to regulate itself and remains constantly activated, leading to uncontrolled cell growth. The function of Ras in signal transduction requires its location near the growth factor receptor at the cell membrane. However, Ras does not have a transmembrane domain. Ras requires farnesylation to increase its hydrophobicity and subsequent plasma membrane association for its transforming activity. This key post-translational modification is catalyzed by the enzyme Ras farnesyltransferase (FTase), which transfers a farnesyl group from farnesylpyrophosphate to the C-terminal cysteine of the Ras protein. The requirement has focused attention on FTase as a target for therapeutic intervention. Selective inhibition of FTase will prevent Ras protein from association with the plasma membrane, leading to a disruption of oncogenic Ras function.