C2C12 근육아세포에서 trichostatin A에 의한 NF-κB DNA 결합 활성과 근육발생에 미치는 영향

임운기,김경창,신혜자 한국생명과학회 2002 생명과학회지 Vol.12 No.1

골격근 세포의 분화는 근육특이 유전자들의 전사적 활성과 근육아세포에서 근육소관으로의 형태적 분화로 특징지어진다. 본 연구에서는 TSA가 근육형성의 일련의 과정에서 NF-kB DNA 결합 활성과 융합에 미치는 영향을 조사하였다. 대조군과 비교해서 TSA가 처리된 C2C12 myoblast는 융합하여 근육소관을 형성할 수 없었으며 NF-kB DNA 결합 활성은 억제되었다. 이런 현상들이 TSA에 의한 직접적인 것인지 알아보기 위해서 TSA가 처리되지 않고 분화를 유도하기 위해서 사용된 배지를 농축하여 C2C12 myoblast에 TSA와 함께 동시에 처리하였다. 그 결과 세포는 융합하여 근육소관을 형성하였으며 NF-kB DNA 결합 활성이 회복되었다. 이러한 결과는 TSA가 아마도 여러 관련 인자들을 통해 myoblast의 융합과 NF-kB DNA 결합 활성을 억제함으로 근육형성과정에 영향을 미침을 시사한다. The differentiation of skeletal muscle precursor cells in culture is marked by the transcriptional activation of muscle-specific genes and the morphological differentiation of myoblast into multinucleate myotube. In this study, we examined the effect of TSA (Trichostatin A) on WF-kB DNA binding activity and muscle cell fusion in the process of myogenesis. Under TSA treatment, C2C12 myoblast could not fuse to myotube and its NF-kB DNA binding activity was also blocked. To investigate whether these phenomenons were affected by TSA in direct or not, differentiation media (DM) used to differentiate cells without TSA was concentrated and added to C2C12 myoblast with TSA simultaneously. C2C12 myoblast was fused to myotube and NF-kB DNA binding activity was recovered. These results suggest that TSA affects on the differentiation of myoblast, perhaps through several factors, by inhibiting myoblst fusion and blocking NF-kB DNA binding activity.

미생물에 의한 폐기름 탄화수소의 분해

정재갑,임운기,신혜자 한국생명과학회 1999 생명과학회지 Vol.9 No.1

폐기름 유출지역에서 분리 동정된 미생물 Xl, X2, X3은 폐유나 그 주성분인 난분해성 물질들을 유일 탄소원으로 자랄 수 있었다. Naphthalene과 2-methyl naphthalene은 7일만에 약 80$\%$ 분해되었다. Hexane과 hexadecane은 거의 대부분 분해되며 60$\%$의 분해가 폐유에서 관찰되었다. 합성 계면활성제인 Triton X-100와 Tween 20은 세포의 성장과 분해에 오히려 저해함을 보였다. Xl, X2은 그람 음성을 X3은 그람 양성을 보이며 항생제 ampicillin에 저항성을 가진다. Xl의 30kb plasmid을 E.coli에 transform하여 유전공학적 활용 가능성을 보였다. Sediment samples from the waste-oil spilled sites were screened for microorganisms able to degrade the components of crude oil, and 3 strains that could degrade were obtained. The isolated 3 strains (Xl, X2 and X3) metabolized naphthalene and 2-methyl naphthalene about 80$\%$ as well as hexane and hexadecane about 60~70$\%$ as a sole carbon source in 7 days. The degradation of the waste oil was about 60$\%$. The addition of synthetic surfactant, Triton-X 100 or Tween 20 slightly inhibited the growth of the populations. Xl and X2 were gram negative and X3 was gram positive. Xl and X3 showed ampicillin resistancy. Xl strain having 30kb plasmid has been selected for genetic study. The plasmid was isolated and transformed into E. coli. showing the possibility of the genetically engineered degrader.

대장균 트립토판 중합효소 α 소단위체의 응집 형성에 미치는 잔기 173 치환체의 억제 효과

정재갑,박후휘,임운기 한국생명과학회 2022 생명과학회지 Vol.32 No.9

Aggregation of normally soluble proteins can cause disease-related problems. Tryptophan synthase α-subunit (αTS) in E. coli adopts one of most popular structural scaffolds, the TIM barrel fold. Previous mutagenesis of the αTS gene resulted in many aggregation-prone mutant proteins. Here, Y173F (Tyr at residue 173 to Phe) substitution, which imparts increased stability, was tested for its ability to suppress aggregation of aggregation-prone mutant proteins (Y4C, S33L, P28L, P28S, G44S, D46N, P96L, and P96S). Aggregation was suppressed in all eight severe aggregate-forming mutants (all differing in their mutation positions), by the Y173F replacement. P28L αTS, which was available in pure form, was further analyzed and showed reduced secondary structure content, lower stability, and a looser structure with more exposed hydrophobic surface compared to the wild type protein. A double mutant P28L/Y173F protein showed almost no indication of these changes compared to the wild type protein. We hypothesized that Tyr at position 173 in αTS is positioned at the hydrophobic core and may serve to suppress the aggregation of this protein caused by other residues. Important residue (s) could be working widely in the prevention/suppression of protein aggregation.

트립토판 합성효소의 이소조절성에 미치는 리간드

김일,신혜자,임운기,김한도,Kim, Il,Shin, Hye-Ja,Im, Woon-Ki,Kim, Han-Do 한국생명과학회 2004 생명과학회지 Vol.14 No.1

트립토판 합성효소의 이소조절성에 작용하는 리간드가 알려져 있다. $\beta$반응의 기질인 세린만이 있는 조건에서 일가 양이온과 glycerophosphate의 리간드효과를 잔기치환체를 사용하여 조사하였다. 이들 리간드가 이 효소의 이소조절성에 관여하고 있음을 보여주고 있다. Various ligands function as regulators in the allosteric control of tryptophan synthase. Effects of the monovalent cations and glycerophosphate on the mutant tryptophan synthases were examined in the presence of L-serine. The results showed that these compounds might play roles in the allosteric control of the proteins.

SPR 근거 DNA 칩을 이용한 페놀 화합물 특이 CapR 조절 단백질과 촉진유전자와의 상호작용 연구

박선미,박후휘,임운기,신혜자 한국생명과학회 2003 생명과학회지 Vol.13 No.1

Aromatic compounds are of major concern among environmental pollutants due to their toxicity and persistence. To monitor aromatic compounds in a real time with a better sensitivity, a new method of SPR (surface plasmon resonance) based on DNA chip (Biacore 3000) was developed here. It is thought that CapR regulatory protein as a complex with phenol, could bind to their corresponding promoter, Po. Biotinylated DNA oligomers for the promoter was synthesized by PCR and coupled onto streptoavidin-linked CM5-chip. CapR regulatory proteins were purified after cloning their genes in pET21a (+) vector and expressing proteins. The interaction was assessed by the system where the regulatory proteins flowed with or without phenol through the cells of DNA chip. CapR regulatory protein even in the presence of phenol had no response to its promoter, Po, suggesting that other factor(s) might be required for the activation of Po promoter. The present work reveals a promising possibility of the SPR-based DNA chip in monitoring specific environmental pollutants in a real time.

트립토판 합성효소의 β반응속도에 미치는 일가양이온의 영향

김일,신혜자,임운기,김한도,Kim, Il,Shin, Hye-Ja,Im, Woon-Ki,Kim, Han-Do 한국생명과학회 2004 생명과학회지 Vol.14 No.1

대장균 트립토판 합성효소의 $\beta$반응의 빠른 속도 반응에 일가 양이온의 영향을 D56N과 D56G 잔기치환체를 대상으로 조사하였다. 일가 양이온을 처리하였을 때 생성되는 중간반응 생성물의 생성과 분해 속도가 영향을 받았다. 56번 자리 잔기치환체도 야생형에 비해 반응의 중간산물의 생성과 분해에 있어 모두 느린 속도를 보여주고 있다. 이들 잔기 치환체에 미치는 일가 양이온의 영향 또한 달라졌다. 이 결과는 대장균의 효소에서도 56번잔기가 이소조절에 관여하고 있고, 이 과정에서 일가 양이온들이 이소조절 리간드역할을 수행하는 것을 보여준다. Effects of monovalent cations were examined on the fast $\beta$reaction of $\alpha$D56N and $\alpha$D56G mutant tryptophan synthase. Reaction rates for the production and degradation of intermediates in the reaction were changed in the presence of cathons. The mutant proteins showed different reaction rates from those of wild-type protein, and additional changes occurred in the presence of cations. The results showed that monovalent cations and $\alpha$D56 are important in allosteric properties of this protein.

트립토판 중합요소 알파 소단위체 $Pr28$longrightarrowLeu 잔기 치환체의 구조 변화

김은주,신혜자,임운기 한국생명과학회 2001 생명과학회지 Vol.11 No.1

A mutant tryptophan synthase $\alpha$-subunit, where Pro28 was replaced with Leu, tends to be expressed in recombinant E. coli. CD and fluorescence spectra of this protein indicate some changes in secondary and tertiary structure. Wild type protein was more or less affected by {TEX}$Ca^{2+}${/TEX} ion in regards of the fluorescent properties of its native, unfolded and intermediate forms, but the mutant protein was not at all. The dramatic structural changes may be related to the aggregation of this mutant protein.

대장균 트립토판 생성효소의 소단위체간 상호조절

조원진(Won Jin Cho),임운기(Woon Ki Lim) 한국생명과학회 2017 생명과학회지 Vol.27 No.12

대장균 트립토판 생성효소는 α2β2 복합체로 구성되며, 트립토판 생합성에서 최종 2 단계의 반응에 관여한다. 두 개의 소단위체는 분자 터널로 연결되어 있어, 기질 채널링이 일어난다. 활성 부위간 상호 조절하는 정교한 조절 기작에는 α-루프 L6(αL6), αL2, αL3이 관여한다. 본 연구에서는 이 자리의 잔기치환체를 써서 소단위체에 특이적으로 결합하는 리간드의 영향을 조사하여 소단위체간 상호 조절기작에 따른 구조 변화를 살펴보았다. αTS의 활성부위에 결합하는 D,L-α-glycerophosphate(GP)는 모든 잔기치환체를 야생형 수준으로 회복시켰다. βTS의 기질인 L-Ser는 다양한 효과를 나타낸다. 야생형과 NS104에서는 속도가 감소한 반면, GD51과 PH53에서는 거의 영향이 없었고, PT53와 DG56은 증가하였다. 이는 반응 중간 화학종의 분포의 변화와 연관될 가능성을 제시한다. GP와 L-Ser를 동시에 처리했을 때는 특이하게도 PH53는 가장 안정한 잔기치환체였다. 이는 Pro53가 소단위체간의 조절기작에서 중요한 역할을 하는 것을 시사한다. Escherichia coli tryptophan synthase (TS) contains α2β2, which catalyzes the final two steps in Trp biosynthesis. A molecular tunnel exists between the two active sites of α and β subunits in TS. Via intersubunit communication, TS increases catalytic efficiency, including substrate channeling. The β subunit of TS is composed of two domains, one of which, the COMM (communication) domain, plays an important role in intersubunit communication. The α subunit has a TIM barrel structure. This protein has functional regions at the C terminal of β pleated sheets and in its loop regions. Three regions of the α subunit (αL6 [α-loop L6], αL2, and αL3) are implicated in intersubunit communication. In the present study, conformational changes in αL6 were monitored by measuring the sensitivity of mutant proteins in these regions to trypsin. The addition of a α subunit-specific ligand, D,L-α-glycerophosphate (GP), partially restored the sensitivity of mutant proteins to trypsin. In contrast, the addition of the β subunit-specific ligand L-serine (Ser) resulted in varied sensitivity to trypsin, with an increase in PT53 (substitution of Pro with Thr at residue 53) and DG56, decrease in NS104 and wild type, and no change in GD51 and PH53. This finding may be related to several reaction intermediates formed under this condition. The addition of both GP and Ser led to a highly stable state of the complex. The present results are consistent with the current model. The method used herein may be useful for screening residues involved in intersubunit communication.

트립토판 중합효소 알파 소단위체의 in vitro 구조재형성시 Ca^2+과 Mg^2+이온의 단백질 응집체 형성 촉진 효과

천광호,김종원,신혜자,임운기 부산대학교 유전공학연구소 1999 분자생물학 연구보 Vol.15 No.-

The effect of cations on the formation of protein aggregates was examined by in vitro refolding of mutant tryptophan synthase α-subunit in which Pro 24 was replaced by Leu. NH^+_4, K^+ and NA^+ had no effect, but Mg^2+ and Ca^2+ stimulated the formation of protein aggregates in dose-dependent manner. It is suggested that Mg^2+ and Ca^2+ may be implicated in the formation of protein aggregates in vivo.

24번 잔기가 치환된 트립토판 중합효소 $\alpha$ 소단위체들의 구조풀림 성질

정지은,박후휘,신혜자,임운기 한국생명과학회 1999 생명과학회지 Vol.9 No.6

대장균 트립토판 중합요소 (tryptophan synthase) $\alpha$ 소단위체의 24번 잔기인 트레오닌이 메티오닌, 알라닌 또는 세린으로 치환되고 잔기 139에 트립토판이 있는 단백질를 정제하여, 여러가지 요소 농도에 대한 트립토판 형광값의 변화를 측정하여 변성곡선을 얻었다. 이들 단백질들은 모두 F139W 단백질에 비해 구조형성 성질에 큰 변화를 일으켰으며, 잔기 24번은 구조형성에 중요한 것으로 추정된다. The doubly altered mutant tryptophan synthase $\alpha$-subunits, in which Thr 24 was replaced by Ser, Leu or Lys in addition to F139W substitution, were purified. Urea-induced unfolding equilibrium curves of these proteins, monitored by fluorescence intensity of tryptophan, show that the alterations of residue 24 resulted in marked changes in folding properites, suggesting the importance of this residue in folding of this protein.