Histone Lysine Methylation

곽상준,Kwak, Sahng-June Korean Society of Life Science 2007 생명과학회지 Vol.17 No.3

유핵세포의 게놈(genome)은 단백-DNA복합체인 염색질(chromatin)의 형태로 존재하는데, 생명현상을 유지하기 위해서는 생명체 또는 세포가 처한 상황에 맞게 염색질의 구조를 변화시키는 역동적인 조절기전이 필요하다. 염색질을 구성하는 기본단위는 히스톤 8량체 (histone octamer)를 포함하는 뉴클레오좀(nucleosome)이다. 히스톤 단백에는 여러 종류의 공유결합성 수식이 일어나는데, 그 중 하나가 라이신 잔기(lysine residue)에 일어나는 메틸화이다. 최근 수년간의 연구로 여러 개의 히스톤 라이신 메틸화효소(histone lysine methyltransferase, HKMT), 이에 결합하는 염색질단백 및 메틸화와 관련된 후생유전학적 현상이 밝혀졌으며, 특히 정밀한 연구방법을 동원한 다방면의 실험을 통하여 비록 자세한 기전과 전체적인 윤곽의 규명은 미흡하더라도 라이신 메틸화가 후생유전학적 변화를 초래하는 일부 과정이 규명 되었다. 또한 여러 종류의 라이신 탈메틸화효소가 최근에 발견됨에 따라, 아세틸화, 인산화등 다른 공유결합성 수식보다는 상대 적으로 안정되더라도, 히스톤 메 틸화로 유발되는 후생유전학적 변화가 불가역성이 아님을 알게 되었다. Our genome exists in the form of chromatin, and its structural organization should be precisely regulated with an appropriate dynamic nature for life. The basic unit of chromatin is a nucleosome, which consists of a histone octamer. These nucleosomal histones are subject to various covalent modifications, one of which is methylation on certain lysine residues. Recent studies in histone biology identified many histone Iysine methyltransferases (HKMTs) responsible for respective lysine residues and uncovered various kinds of involved chromatin associating proteins and many related epigenetic phenotypes. With the aid of highly precise experimental tools, multi-disciplinary approaches have widened our understanding of how lysine methylation functions in diverse epigenetic processes though detailed mechanisms remain elusive. Still being considered as a relatively more stable mark than other modifications, the recent discovery of lysine demethylases will confer more flexibility on epigenetic memory transmitted through histone lysine methylation. In this review, advances that have been recently observed in epigenetic phenotypes related with histone lysine methylation and the enzymes for depositing and removing the methyl mark are provided.

폐상피세포에서 Dexamethasone에 의한 NF-${\kappa}B$ Transactivation 억제기전에 관한 연구

이계영,김윤섭,고미혜,박재석,지영구,김건열,곽상준,Lee, Kye-Young,Kim, Yoon-Seop,Ko, Mi-Hye,Park, Jae-Seok,Jee, Young-Koo,Kim, Keun-Youl,Kwak, Sahng-June 대한결핵및호흡기학회 2000 Tuberculosis and Respiratory Diseases Vol.48 No.5

Glucocorticoid receptor (GR) functions as a suppressor of inflammation by inhibiting the expression of many cytokine genes activated by NF-${\kappa}B$. The goal of this study is to investigate the mechanism by which GR repress NF-${\kappa}B$ activation in lung epithelial cells. We used A549 and BEAS-2B lung epithelia! cell lines. Using Ig$G{\kappa}$-NF-${\kappa}B$ luciferase reporter gene construct, we found that dexamethasone significantly suppressed TNF-$\alpha$-induced NF-${\kappa}B$ activation and the overexpression of GR showed dose-dependent reduction of TNF-$\alpha$-induced NF-${\kappa}B$ activity in both cell lines. However, DNA binding of NF-${\kappa}B$ induced by TNF-$\alpha$ in electromobility shift assay was not inhibited by dexamethasone. Super shift assay with anti-p65 antibody demonstrated the existence of p65 in NF-${\kappa}B$ complex induced by $\alpha$ Western blot showed that $I{\kappa}B{\alpha}$ degradation induced by TNF-$\alpha$ was not affected by dexamethasone and $I{\kappa}B{\kappa}$ was not induced by dexamethasone, neither. To evaluate p65 specific transactivation, we adopted co-transfection study of Gal4-p65TA1 or TA2 fusion protein expression system together with 5xGal4-luciferase vector. Co-transfection of GR with Gal4-p65TA1 or TA2 repressed luciferase activity profoundly to the level of 10-20% of p65TA1- or TA2-induced transcriptional activity. And this transrepressional effect was abolished by co-transfection of CBP of SRC-1 expression vectors. These results suggest that GR-mediated transrepression of NF-${\kappa}B$ in lung epithelial cells is through competing for binding to limiting amounts of transcriptional coactivators, CBP or SRC-1.

Multiplex PCR-aided Differential Diagnosis of Taeniid Species

Hye-Jung Lee(이혜정),Min Seo(서민),Sahng-June Kwak(곽상준) 한국생명과학회 2010 생명과학회지 Vol.20 No.6

아시아조충과 무구조충의 편절은 형태학적으로 유사해 감별진단하기가 쉽지 않다. 하지만 아시아조충의 경우 감염자에서 낭미충증 등의 심각한 합병증을 일으킬 가능성을 배제할 수 없으므로 두 기생충의 정확한 진단이 필요하다. 최근 기생충학 분야에서 DNA 서열에 기초한 진단 방법이 널리 쓰이고 있다. 본 연구에서는 다중 중합 효소연쇄반응을 이용해 한국인에서 발견된 태니아 속 조충류의 감별진단을 시도해 보고자 했다. 중합효소연쇄반응을 위해 Ta4978F, Ts5058F, Tso7421F, Rev7915 4개의 시발체(프라이머)를 사용했으며, 그 결과 태니아의 종 동정이 정확하고 신속하게 이루어질 수 있었다. 중합효소연쇄반응 방법을 도입한다면 한국에서 인체 태니아 조충의 역학적 소견을 용이하게 재검토할 수 있으리라고 생각한다. Differential diagnosis of the taeniid proglottids between Taenia asiatica and T. sagniata is a daunting task due to their close morphological similarity. However, to correctly diagnose them on time is important in managing infected patients, as well as for reducing serious complications such as cysticercosis. Currently, DNA-based methods for the dissection of genomic information of parasites are being employed to make accurate and rapid diagnoses in the field of parasitology. In this study, multiplex PCR was established and exploited to identify exact species of taeniid adult worms recovered from Korean people. To discriminate one from the other other, primers-Ta4978F, Ts5058F, Tso7421F, and Rev7915- were used for the multiplex PCR, which provided swift and precise identification of the taeniid worms being observed. Also,having instituted PCR methodology, we ascertained that easiness would be achieved to reassess and re-evaluate Korean endemic data on human taeniid cestodes.

폐상피세포에서 Triptolide에 의한 NF-${\kappa}B$ 의존성 IL-8 유전자 전사활성 억제기전

지영구,김윤섭,윤세영,김용호,최은경,박재석,김건열,채기남,곽상준,이계영,Jee, Young-Koo,Kim, Yoon-Seup,Yun, Se-Young,Kim, Yong-Ho,Choi, Eun-Kyoung,Park, Jae-Seuk,Kim, Keu-Youl,Chea, Gi-Nam,Kwak, Sahng-June,Lee, Kye-Young 대한결핵및호흡기학회 2001 Tuberculosis and Respiratory Diseases Vol.50 No.1

연구배경 : 폐상피세포가 능동적으로 IL-8을 분비한다는 것은 주지의 사실이다. NF-${\kappa}B$는 IL-8 발현 조절에 있어서 가장 중요한 역할을 담당하는 전사인자이다. Triptolide는 최근 밝혀진 NF-${\kappa}B$ 억제제로서 중국한약제인 뇌공등 (Tripterygium Wilfordii)에서 추출된약제이다. 연자들은 새로운 NF-${\kappa}B$ 억제제인 triptolide가 폐상피세포에서 NF-${\kappa}B$ 의존성 IL-8 유전자의triptolide가 염증성 폐질환에서 새로운 치료제로서의 가능성을 확인하기 위하여 본 연구를 시행하였다. 방법 : 폐상피세포로서 A549 사용하였고 triptolide는 미국의 Pharamagenesis(Palo Alto, CA)사로부터 제공받았다. NF-${\kappa}B$ 활성유도물질로는 IL-$1{\beta}$(R&D)와 PMA(Sigma)를 이용하였다. IL-8 유전자의 발현은 RT-PCR과 ELISA를 이용하여 측정 하였다. NF-${\kappa}B$의존성 IL-8 유전자의 전사활성을 평가하기 위하여는 IL-8 NF-${\kappa}B$ luciferase construct를 안정적으로 유전자주입한 A549 IL-8 NF-${\kappa}B$ luciferase 세포주를 제조해 사용하였고 NF-${\kappa}B$ DNA 결합은 electromobility shift assay(EMSA)를 이용하였다. p65 전사활성을 assay하기 위해서는 Gal4-p65 fusion protein expression system을 유전자주입과 luciferase assay를 통하여 시행하였다. Transcriptional coactivator의 역할을 규명 하가 위하여서는 CBP(CREB-binding protein)와 SRC-1(steroid receptor coactivator-1) 발현 벡터를 유전자 주입하고 luciferase assay를 이용하여 확인하였다. 결과 : Luciferase assay로 triptolide가 PMA와 IL-$1{\beta}$자극에 의한 IL-8 NF-${\kappa}B$ 활성을 의미있게 감소시킴을 확인하였다. IL-8 ELISA와 RT-PCR로 triptolide가 PMA와 IL-$1{\beta}$ 자극에 의해 유도되는 IL-8 발현을 각각 단백질과 mRNA 수준에서 억제함을 관찰하였다. Triptolide가 PMA와 IL-$1{\beta}$에 의한 IL-8 NF-${\kappa}B$의 전사활성을 억제시킨 반면 EMSA와 $I{\kappa}B{\alpha}$ Western blot을 이용한 실험에서는 triptolide가 NF-${\kappa}B$ DNA 결합과 $I{\kappa}B{\alpha}$의 분해에 전혀 영향을 미치지 못함을 확인하였다. 이러한 전사활성 억제와 DNA 결합 간의 불일치의 원인으로서는 DNA 결합 이후에 발생하는 핵내 에서의 transactivation에 triptolide가 영향을 미치리라고 생각되어 p65 transactivation study를 Gal4-p65T A(p65의 transactivation domain) fusion protein 발현 시스템과 luciferase assay를 이용하여 시행한 결과 triptolide가 p65 transactivation을 억제함으로써 NF-${\kappa}B$를 억제함을 확인하였다. 그러나 CBP나 SRC-1과 같은 coactivator의 역할을 규명하기 위한 유전자주입 실험에서 triptolide에 의한 p65 transactivation 억제에 대해 CBP나 SRC-1의 과발현이 별다른 영향을 미치지 못하였다. 결론 : Triptolide는 폐상피세포에서 NF-${\kappa}B$ 의존성 IL-8 유전자의 전사활성을 억제하고 그 기전은 $I{\kappa}B{\alpha}$ 경로가 아닌 핵내에서의 p65 transactivation 억제에 의해 발생하며 이에는 CBP나 SRC-1과 같은 coactivator가 관여하지 않음을 확인하였다. Background : NF-${\kappa}B$ is the most important transcriptional factor in IL-8 gene expression. Triptolide is a new compound that recently has been shown to inhibit NF-${\kappa}B$ activation. The purpose of this study is to investigate how triptolide inhibits NF-${\kappa}B$-dependent IL-8 gene transcription in lung epithelial cells and to pilot the potential for the clinical application of triptolide in inflammatory lung diseases. Methods : A549 cells were used and triptolide was provided from Pharmagenesis Company (Palo Alto, CA). In order to examine NF-${\kappa}B$-dependent IL-8 transcriptional activity, we established stable A549 IL-8-NF-${\kappa}B$-luc. cells and performed luciferase assays. IL-8 gene expression was measured by RT-PCR and ELISA. A Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation and an electromobility shift assay was done to analyze NF-${\kappa}B$ DNA binding. p65 specific transactivation was analyzed by a cotransfection study using a Gal4-p65 fusion protein expression system. To investigate the involvement of transcriptional coactivators, we perfomed a transfection study with CBP and SRC-1 expression vectors. Results : We observed that triptolide significantly suppresses NF-${\kappa}B$-dependent IL-8 transcriptional activity induced by IL-$1{\beta}$ and PMA. RT-PCR showed that triptolide represses both IL-$1{\beta}$ and PMA-induced IL-8 mRNA expression and ELISA confirmed this triptolide-mediated IL-8 suppression at the protein level. However, triptolide did not affect $I{\kappa}B{\alpha}$ degradation and NF-$_{\kappa}B$ DNA binding. In a p65-specific transactivation study, triptolide significantly suppressed Gal4-p65T Al and Gal4-p65T A2 activity suggesting that triptolide inhibits NF-${\kappa}B$ activation by inhibiting p65 transactivation. However, this triptolide-mediated inhibition of p65 transactivation was not rescued by the overexpression of CBP or SRC-1, thereby excluding the role of transcriptional coactivators. Conclusions : Triptolide is a new compound that inhibits NF-${\kappa}B$-dependent IL-8 transcriptional activation by inhibiting p65 transactivation, but not by an $I{\kappa}B{\alpha}$-dependent mechanism. This suggests that triptolide may have a therapeutic potential for inflammatory lung diseases.

GRβ 과발현이 스테로이드에 의한 IL-8 합성 및 분비에 미치는 영향

서지희 ( Ji Hee Seo ),김윤섭 ( Youn Seop Kim ),이순일 ( Sun Il Lee ),박재석 ( Jae Seuk Park ),이계영 ( Kye Young Lee ),곽상준 ( Sahng June Kwak ),지영구 ( Young Koo Jee ) 대한천식알레르기학회 2005 천식 및 알레르기 Vol.25 No.2

트란스페린에 대한 단클론항체의 생산과 방사면역측정법의 개발

한현식,곽상준 충북대학교 의과대학 충북대학교 의학연구소 1997 忠北醫大學術誌 Vol.7 No.1

트란스페린은 주로 간에서 생산되는 금속결합 운반 당단백으로 주요한 역할은 헤모글로빈의 합성을 위하여 철을 운반하는 것이다. 근래 트란스페린의 철공급 외의 기능으로 감염에 대한 숙주 저항에 관한 견해들이 있다. 현재 트란스페린을 직접 측정하는 방법인 방사면역 확산법은 혈장의 농도를 측정하는 데에는 문제가 없으나 체액에 미량 존재하는 부위의 측정에는 민감도가 떨어진다. 본연구에서는 트란스페린에 대한 단클론항체를 만들어 이들의 특성을 분석하고, 이를 이용한 민감한 측정법을 개발하며, 나아가 트란스페린치와 숙주저항성과의 관련성을 보고자 하였다. 단글론항체를 3가지 얻었는데, 이들은 모두 IgG₁이었고 이항체는 트란스페린의 철결합 유무에 관계 없이 같은 정도로 결합하였다. 트란스페린의 항원결정기는 서로 같거나 아주 근접하여 위치하는 것으로 생각 된다. 트란스페린을 방사성옥소로 표식하고, 폴리스타이린관을 고상으로 이용한 면역측정법을 개발하였는데, 검색구간은 0.1 - 20 ㎍/㎖ 이었고, 정밀도 및 분석적 회수율이 높았다. 혈청은 1,000배로 희석하여 측정하고, 소변은 희석하지 않고 측정할 수 있었다. 급성 감염성 질환과 비염증성 질환에서 얻은 혈청과 소변의 트란스페린치를 측정한 결과 유의한 차이를 보이지는 않았다 Transferrin is a metal-binding transport glycoprotein mainly produced in the liver. In addition to the iron supplying function, host resistance to infection of transferrin has been recently proposed. Transferrin is currently measured by radial immunodiffusion, but its sensitivity is low, especially for assaying body fluid other than serum. For developing a more sensitive assay system, monoclonal antibodies to human transferrin were produced, all of which are of the IgG₁subclass. These monoclonal antibodies bound transferrin to the same degree regardless of iron binding. The epitopes of transferrin seemed to be nearly the same or very close in position, so that competitive binding immunoassay system was developed in which transferrin was labelled with I125 and polystyrene tube was used as a solid phase. The detectable rang of transferrin by the assay system was between 0.1 and 20㎍/ml. The reproducibility and the recovery were satisfactory Serum was assayed after diluting 1 to 1000, but urine was assayed without dilution. The transferrin levels of acute infectious subjects were not significantly different from those of non-inflammatory subjects in urine as well as serum.

대장균에서 재조합 Rat Guanine deaminase 유전자의 발현, 정제 및 분석

성연선,곽상준,박대성,김향원,이희영 단국대학교 1998 論文集 Vol.33 No.-

Guanine deaminase(EC, 3.5.4.3, ;Guanine aminohydrolase, GAH, GDA) catalyzes the deamination reaction of guanine to xanthine irreversibly. The cDNA encoding rat guanine deaminase had been isolated from a λZAPII rat brain expression library using antibody generated against purified rat guanine deaminase. Toe obtain recombinant GDA and analyze the property of catalytically conserved retion, here we expressed the recombinant GDA in E.coli and showed the retention of its catalytic activity similar to native rat GDA. To make the construct carrying coding region to GDA in pGEX4T prokaryotic expression vector, the region encompassing open reading frame of pBlue-GDA was PCR amplified and subcloned into pGEX4T prokaryotic expression vector with correct reading frame of fusion carrier glutathione S-transferase. After transformation to E.coli DH5a. The bacteria carrying pGEX-GDA was grwon in the condition of IPTG induction. The fusion protein GST-GDA was purified with GSH-sepharose affinity chromatography. The purified GST-GDA was subsequently digested with biotin-conjugated thrombin, and thrombin was removed by streptavidin agarose. Then sample were treated with GSH-sepharose affinity chromatography to remove the contaminant GST and GST-GDA. The purified recombinant GDA showed 70% of specific activity relative to that of purified rat GDA. The recombinant GDA showed identical molecular weight with rat GDA in SDS-PAGE.