• 약학회지
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• 제50권4호
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• pp.263-267
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• 2006

CD1d is an unique antigen presenting molecule which provides antigenic repertoires to NKT cells. To examine molecules required for CD1d antigen presentation, we determined an interaction between CD1d and several endoplasmic reticulum (ER) resident molecular chaperones by co-immunoprecipitation. Results indicated that calnexin and calreticulin seem to be bound to mouse CD1d, but TAP and tapasin do not bind. Further, we screened an yeat two hybrid system to identify proteins that help mouse CD1d transportation in the cytosol. We found that two proteins, heat shock protein a sub-unit $(Hsp90{\alpha})$ and protein kinase C and casein kinase substrate in neurons 3 (PACSIN-3), interact with CD1d. Future study will be focus on the role of these molecules during the CD1d antigen presentation.

• The Plant Pathology Journal
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• 제31권2호
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• pp.108-114
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• 2015

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about $6.39{\times}10^5$ transformants/$3{\mu}g$ pGADT7-Rec. The titer of the primary cDNA library was $2.5{\times}10^8cfu/mL$. The numbers for the cDNA library was $2.46{\times}10^5$. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.76-78
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• 2002

Heat shock proteins (HSPs) are induced in most living cells by mild heat treatment, ethanol, heavy metal ions and hypoxia. In yeast Saccharomyces cerevisiae, mild heat pretreatment strongly induces Hsp104 and thus provide acquired thermotolerance. The ability of hsp104 deleted mutant $({\triangle}hsp104)$ to acquire tolerance to extreme temperature is severely impaired. In providing thermotolerance, two ATP binding domains are indispensible, as demonstrated in ClpA and ClpB proteases of E. coli. The mechanisms by which Hsp104 protects cells from severe heat stress are not yet completely elucidated. We have investigated regulation of mitochondrial metabolic pathways controlled by the functional Hsp104 protein using $^{13}C_NMR$ spectroscopy and observed that the turnover rate of TCA cycle was enhanced in the absence of Hsp104. Production of ROS, which are toxic to kill cells radiply via oxidative stress, was also examined by fluorescence assay. Mitochondrial dysfunction was manifested in increased ROS levels and higher sensitivity for oxidative stress in the absence of Hsp104 protein expressed. Finally, we have identified mitochondrial complex I and Ferritin as binding protein(s) of Hsp104 by yeast two hybrid experiment. Based on these observations, we suggest that Hsp104 protein functions as a protector of oxidative stress via either keeping mitochondrial integrity, direct binding to mitochonrial components or regulating metal-catalyzed redox chemistry.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.286-291
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• 2002

Histone deacetylase I (HDAC1) works as one of the components in a nucleosome remodeling (NuRD) complex that consists of several proteins, including metastasis-associated protein 1 (MTA1). Since the protein-protein interaction of HDAC1 and MTA1 would appear to be important for both the integrity and functionality of the HDAC1 complex, the interruption of the HDAC1 and MTA1 interaction may be an efficient way to regulate the biological function of the HDAC1 complex. Based on this idea, a yeast two-hybrid system was constructed with HDAC1 and MTA1 expressing vectors in the DNA binding and activation domains, respectively. To verify the efficiency of the assay system, 3,500 microbial metabolite libraries were tested using the paper disc method, and KB0699 was found to inhibit the HDAC1 and MTA1 interaction without any toxicity to the wild-type yeast. Furthermore, KB0699 blocked the interaction of HDAC1 and MTA1 in an in vitro GST pull down assay and induced morphological changes in B16/BL6 melanoma cells, indicating the interruption of the HDAC1 complex function. Accordingly, these results demonstrated that the yeast assay strain developed in this study could be a valuable tool for the isolation of a HDAC1 complex disruptor.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2004년도 The 3rd Annual Conference for The Korean Society for Bioinformatics Association of Asian Societies for Bioinformatics 2004 Symposium
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• pp.265-271
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• 2004

The interaction network of protein -protein plays an important role to understand the various biological functions of cells. Currently, the high -throughput experimental techniques (two -dimensional gel electrophoresis, mass spectroscopy, yeast two -hybrid assay) provide us with the vast amount of data for protein-protein interaction at the proteome scale. In order to recognize the role of each protein in their network, the efficient bioinformatical and computational analysis methods are required. We propose a systematic and mathematical method which can analyze the protein -protein interaction network rigorously and enable us to capture the biological and physical essence of a topological character and stability of protein -protein network, and sensitivity of each protein along the biological pathway of their network. We set up a Laplacian matrix of spectral graph theory based on the protein-protein network of yeast proteome, and perform an eigenvalue analysis and apply a perturbation method on a Laplacian matrix, which result in recognizing the center of protein cluster, the identity of hub proteins around it and their relative sensitivities. Identifying the topology of protein -protein network via a Laplacian matrix, we can recognize the important relation between the biological pathway of yeast proteome and the formalism of master equation. The results of our systematic and mathematical analysis agree well with the experimental findings of yeast proteome. The biological function and meaning of each protein cluster can be explained easily. Our rigorous analysis method is robust for understanding various kinds of networks whether they are biological, social, economical...etc

• 한국미생물·생명공학회지
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• 제14권3호
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• pp.209-212
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• 1986

YEp 13 plasmid에 B. amyloliquefaciens의 $\alpha$-amylase gene을 cloning시켜서 얻은 hybrid plasmid를 E. coli C 600으로 형질전환시켜서 amylase 활성을 나타내는 균주를 선별하였다. 선별된 E. coli C 600균주를 plasmid추출하여 전기영동해 본 결과 plasmid가 매우 불안정하였으며, 그중 가장 단순한 plasmid band를 지니고 있으며 amylase활성이 강한 E. coli균주를 선별하였다. 선별된 균주의 균체내에 있는 2개의 plasmid DNA를 분리하여 각각의 plasmid를 pTG 17-1, pTG 17-2로 명명하였으며 S. cerevisiae MC 16에서 형질전환이 가능한 pTG 17-2 DNA를 제한효소 EcoRI과 Pst I으로 restriction한 결과 EcoRI으로 처리한 경우는 7.3, 4.8, 2.4 kb인 3개의 분획으로 나타났으며 Pst I으로 처리한 경우는 linear로 14.5kb임을 알았으며 이로써 pTG 17-2 plasmid의 size가 약 14kb임을 알았다. 또한 E.coli균체내에서의 ampicillin sensitive로써 이 plasmid의 ampicillin resistance site가 결실되었음을 알았고 효모의 형진전환체로 부터의 $\alpha$-amylase는 균체외로 분비되지 않았고 효모균체내의 $\alpha$-amylase는 Somogyi-Nelson방법과 Agar diffusion 방법으로 확인하였다.

• The Plant Pathology Journal
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• 제32권5호
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• pp.377-387
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• 2016

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, is one of the most important viruses of cultivated tomatoes worldwide, mainly causing yellowing and curling of leaves with stunting in plants. TYLCV causes severe problems in sub-tropical and tropical countries, as well as in Korea. However, the mechanism of TYLCV infection remains unclear, although the function of each viral component has been identified. TYLCV C4 codes for a small protein involved in various cellular functions, including symptom determination, gene silencing, viral movement, and induction of the plant defense response. In this study, through yeast-two hybrid screenings, we identified TYLCV C4-interacting host proteins from both healthy and symptom-exhibiting tomato tissues, to determine the role of TYLCV C4 proteins in the infection processes. Comparative analyses of 28 proteins from healthy tissues and 36 from infected tissues showing interactions with TYLCV C4 indicated that TYLCV C4 mainly interacts with host proteins involved in translation, ubiquitination, and plant defense, and most interacting proteins differed between the two tissues but belong to similar molecular functional categories. Four proteins-two ribosomal proteins, S-adenosyl-L-homocysteine hydrolase, and 14-3-3 family protein-were detected in both tissues. Furthermore, the identified proteins in symptom-exhibiting tissues showed greater involvement in plant defenses. Some are key regulators, such as receptor-like kinases and pathogenesis-related proteins, of plant defenses. Thus, TYLCV C4 may contribute to the suppression of host defense during TYLCV infection and be involved in ubiquitination for viral infection.

• 생명과학회지
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• 제26권6호
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• pp.698-704
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• 2016

세포내소기관과 단백질 복합체의 운반은 kinesin superfamily proteins (KIFs)에 의해 매개된다. 처음으로 밝혀진 kinesin인 kinesin 1은 motor단백질로서 세포 내에서 미세소관을 따라 이동하며, 다양한 세포내 소기관이나 단백질복합체를 운반한다. Kinesin 1은 장쇄(KHC, 또한 KIF5s로도 통칭) 2분자와 단쇄(KLCs) 2분자로 구성된 4합체(tetramer) 구조를 가진다. KIF5s의 운반체 결합영역을 포함하는 말단영역은 다수의 운반체와 결합하지만, 결합운반체에 관하여 아직 충분히 밝혀지지 않았다. 본 연구에서 KIF5A의 결합 단백질을 동정하기 위하여 효모 two-hybrid screening을 수행하였고 철 저장 및 해독 기능을 하는 단백질인 ferritin heavy chain (Frt-h)을 찾아내었다. Frt-h은 KIF5A의 아미노산 800번과 940번 사이의 부위와 결합하며, 다른 KIF5s와도 결합함을 효모 two-hybrid assay로 확인하였다. 또한 Frt-h의 coiled-coil 도메인이 KIF5A와의 결합에 필수영역임을 밝혔다. 한편, ferritin light chain (Frt-l) 또한 KIF5s와 결합함을 효모 two-hybrid assay로 확인하였다. 이러한 단백질간의 결합을 glutathione S-transferase (GST) pull-down assay를 통하여 검증하였다. 추가적으로 생쥐의 뇌 파쇄액을 항 KHC 항체로 면역침강을 행한 결과, KLC1뿐만 아니라 Frt-h와 Frt-l도 같이 침강하였다. 이러한 결과들은 세포 내에서 kinesin 1이 ferritin 복합체를 운반함을 시사한다.

• 미생물학회지
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• 제53권4호
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• pp.292-296
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• 2017

진핵생물에서 THO/TREX 복합체는 전사 신장, pre-mRNA 가공 및 mRNA의 핵에서 방출에 중요한 역할을 담당한다. 이 복합체는 진화적으로 잘 보존되어 있지만, 생명체에 따라 구성성분과 기능에 차이가 존재한다. 이 논문에서는 출아효모보다는 고등생물과 더 유사한 분열효모 Schizosaccharomyces pombe에서 THO/TREX 복합체의 한 구성요소인 spTex1가 생장과 mRNA의 방출에 필수적이지 않다는 것을 보였다. spTex1 유전자의 결실과 과발현 어느 것도 생장의 결함과 $poly(A)^+$ RNA가 핵 안에 축적되는 현상을 거의 보이지 않았다. 또한 spTex1-GFP 단백질은 주로 핵 안에 위치하였다. Yeast two-hybrid와 Co-immunoprecipitation 분석에서 S. pombe Tex1은 THO/TREX 복합체의 주요 구성인자인 spHpr1 (THOC1), spTho2 (THOC2)과 상호작용을 하였다. 이와 같은 결과들은 S. pombe의 Tex1도 THO/TREX 복합체의 구성인자이지만, 생장과 mRNA 방출에는 중요한 역할을 하지 않음을 의미한다.

• 생명과학회지
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• 제20권10호
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• pp.1451-1457
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• 2010

Serotonin (5-hydroxytryptamine (5-HT))는 신경계의 세포-세포 간의 신호전달의 주요한 신경전달물질이다. 세포막에 존재하는 serotonin transporter (SERT)는 연접간격에 존재하는 5-HT를 세포 내로 재흡수 하여 세포외부의 5-HT 농도를 조절하지만 그 기전은 아직 밝혀지지 않았다. 본 연구에서는 yeast two-hybrid system을 사용하여 SERT의 C-말단이 protein kinase C-$\varepsilon$ (PKC-$\varepsilon$)과 특이적으로 결합함을 알았다. PKC-$\varepsilon$는 PKC의 isotype으로 calcium 비의존적이며 phorbol ester/diacylglycerol 민감성 serine/threonine kinase이다. $Na^+/Cl^-$ 의존성 SLC6 gene family의 다른 수송체는 PKC-$\varepsilon$과 결합하지 않았다. Deletion mutant들을 사용하여 SERT는 PKC-$\varepsilon$의 C-말단부위와 결합함을 알았으며, 또한 이 단백질간의 결합을 GST pull-down assay로 확인하였다. PKC-$\varepsilon$는 in vitro에서 SERT의 N-말단의 펩티드를 인산화시켰다. 이러한 결과들은 PKC-$\varepsilon$에 의한 SERT의 인산화가 세포막에 존재하는 SERT의 활성을 조절하는 역할을 할 가능성을 시사한다.