Streptomyces coelicolor (Muller)의 과산화수소 대응 반응에 관련된 단백질 양상의 분석

정혜정,노정혜 한국미생물학회 1993 미생물학회지 Vol.31 No.2

Streptomyces coelicolor (Muller) 의 세포에 $100 \mu$M 의 과산화수소를 1시간 동안 처리하여 과산화수소 스트레스중에 생성되는 단백질을 L-[$^{35}S$] methionine 을 이용하여 순간 표지하였다. 단백질을 추출하여 2차원 겔 전기영동으로 분석한 결과 지수 성장기의 세포가 가지는 총세포 단백질 중 약 100개의 단백질의 합성이 과산화수소에 의해 증가되는 것을 관찰하였다. 이들을 Pin(peroxide inducible) 단백질이라고 명명하고 과상화수소 처리 후 발현이 증가되는 시간에 따라 세 그룹으로 나누었다. 약 60 개의 Pin 단백질은 과산화수소 처리후 20분 이내에 발현이 증가하여 1시간동안 지속적으로 다량 합성되었다. 정체 성장기의 세포에서는 62개의 단백질의 합성이 과산화수소에 의해 증가되었으며, 이 중 21 개의 단백질은 지수성장기의 Pin 단백질과 일치하였다. 과산화수소에 대한 저항성이 증가한 세가지 돌연변이체의 단백질을 지수 성장기에서 추출하여 2 차원 겔 전기영동으로 분석한 결과, 각각의 경우 15, 17, 15개의 Pin 단백질을 야생형보다 항상적으로 다량합성하는 것을 관찰하였다. 이 pin 단백질 중 9개 (D74.7 a, E76.0c, E23.3, F50.7, F47.2a, F25.5, G39.6b, G24.0, H39.6a) 는 두가지 돌연변이체 모두에게 증가되었다. 따라서 이 단백질들은 S. coelicolor 가 과산화수소 스트레스에 대응하는데 있어 중요한 역할을 담당한다고 추정된다. Streptomyces coeUc%r (Muller) cells were treated with $100 \mu$M hydrogen peroxide for I hour and proteins synthesized during hydrogen peroxide stress were labeled with L-[$^{35}S$]-methionine. Total cellular proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. In exponential growth phase, synthesis of about 100 proteins was increased by hydrogen peroxide treatment. These proteins were named as Pin (£eroxide-inducib]e) proteins and classified into 4 subgroups according to their induction time after hydrogen peroxide treatment. About 60 of them were found to be induced within 20 minutes and maintained throughout I hour of treatment. In stationary growth phase. synthesis of 62 proteins was increased by hydrogen peroxide and 21 of them were the same Pins found in exponential growth phase. Proteins from the mutants which are resistant to hydrogen peroxide were obtained in exponential growth phase and compared with those from the wild type on two-dimensional gel. The three mutants, N7, N9. and N24, were found to have higher constitutive leve]s of ]5, 17, and 15 Pin proteins respectively, than the wild type. 9 of these Pin proteins (D74.7a, E76.0c, E23.3. F50.7, F47.2a. F25.5, G39.6b, G24.0, H39.6a) increased in two of the three mutants and 3 proteins (F39.7, H6I.7. 120.8) increased in all of the three mutants. These proteins might play important roles in the response of S. coelic%r to hydrogen peroxide.

인체 위선암에서 변이 p53 단백질 발현과 임상병리학적 소견 및 예후와의 관계

양영일,오상훈,최영길,김재홍 인제대학교 1998 仁濟醫學 Vol.19 No.2

본 연구는 위암의 발생 및 진행과정에 관여할 것으로 생각되는 대표적인 종양 억제 유전자인 p53 단백질의 발현이 위암 환자의 임상병리학적 소견과 예후에 어떤 영향을 미치는지 알아보고자, 암조직의 비율이 높은 파라핀포매 위암 조직 절편 44예를 대상으로 하여 면역조직 화학 염색으로 p53 단백질 발현을 검사하였다. 대상 환자의 생존율을 구하였고 각각의 임상 및 병리 소견과의 관계를 비교하여 다음과 같은 결과를 얻었다. 1) 변이 p53 단백질 발현은 44예 중 20예(45.5%)에서 관찰되었다. 이 중 조기 위암 15예중 6예(40.0%), 진행 위암 29예중 14예(45.3%)로 의미있는 차이를 보이지 않았고 환자의 연령이 60세 이상인 경우에 p53 유전자의 발현율이 높았으나 통계적 의의는 없었다(P=0.076). 그리고 성별, 종양의 크기, 종양의 위치, 위벽의 침윤심도, 조직학적인 분화도 및 H. pylori 감염 유무에 따른 발현율의 차이도 없었다. 2) 영역 림프절 전이가 있는 경우(P=0.046), 림프관 침습이 있는 경우(P=0.019) 및 병기가 진행할수록(P=0.049) 의의있게 p53 단백질의 발현율이 높았다. 3) 전체 환자의 평균 생존기간은 60.1±33.1개월이었고 5년 생존율은 56.8% 였다. p53 단백질 발현이 관찰되었던 환자들의 평균 생존기간은 53.9±34.2개월이었고 5년 생존율은 42.8%로 의의있게 불량한 예후를 보였다(p=0.049). 4) 예후 인자의 다변량 분석의 결과, 영역 림프절 전이와 위벽의 침윤심도가 의의있는 예후 인자였으며 변이 p53 단백질 발현은 독립적인 예후 인자가 아니었다. 본 연구에서는 조기 위암과 진행 위암 환자에 있어서 p53 유전자 변이가 비슷한 빈도로 관찰되었고, 위벽의 침윤심도에 따라서도 골고루 분포되어 있는 점으로 보아서 위암의 진행 과정에서 초기 및 진행 단계에 p53 유전자의 변이가 관여하며, 특히 p53 유전자의 변이는 림프절전이 및 종양의 진행 정도에도 영향을 미치는 것을 알 수 있었다. 또한 변이 p5,3 단백질 발현이 있을 때 의의있게 불량한 예후를 보였지만 독립적인 예후 인자로 활용하기에는 앞으로 더 깊은 연구가 이루어져야 하겠다. Striking advances in molecular analysis of human gastrointestinal cancer indicate that malignant transformations of normal epithelial cell require multiple processes associated with an accumulation of multiple gene abnormalities affecting DNA repair genes, oncogenes, and tumor suppressor genes. The objective of this study was to determine the availability of mutant p53 expression as a marker of prognosis at gastric carcinoma and its relationship to clinicopathological characteristics. The expression of mutant p53 protein was studied immunohistochemically in 44 gastric carcinomas. The correlation of mutant p53 protein expression in tumor tissue, with clinicopathologic variables and the stage of disease were analyzed. Results showed that 45.5% of the gastric carcinomas expressed elevated levels of mutant p53 protein. The clinicopathologic variables including age, gender, size and location of tumor, depth of invasion, histopathologic differentiation, and Helicobacter pylori infection were not related with mutant p53 protein expression. Otherwise p53 expression positively correlated with regional lymph node metastasis(P=0.046), lymphatic invasion(P=0.019) and advanced stage(P = 0.049). The overall 5-year surval rate was 56.6%. Survival analysis revealed significant impact of mutant p53 expression on survival, in which the 5-year survival rate was 42.8% for patients with p53 positive tumor and 70.8% with negative p53 tumor.(p=0.049) In multivariate analysis adjusted for the other clinicopathological variables, the most significant independent prognostic factors were regional lymph node metastasis and depth of invasion, however, expression of mutant p53 protein was not independent prognostic factor. These findings suggest that mutation of p53 protein occurs in early stage of malignant transformation and may contribute to the progression of the tumor. Based on this study, investigations with a larger number of patients are needed to establish the role of p53 as an independent prognostic indicator in gastric carcinoma.

Generation and Biological Characterization of a Neutralization-Resistant Mutant of Newcastle Disease Virus

Park, Mi-Ja,Kye, Soo-Jeong,Kim, Ji-Ye,Kim, Saeromi,Seul, Hee-Jung,Park, Choi-Kyu,Choi, Kang-Seuk The Korean Society for Microbiology 2012 Journal of Bacteriology and Virology Vol.42 No.4

A neutralization-resistant mutant of Newcastle disease virus (NDV) Kr005 strain belonging to class II genotype VII was generated using a neutralizing monoclonal antibody and its biological effects were assessed. The mutant showed single amino acid substitution (E to K) at position 347 of the hemagglutinin-neuraminidase (HN) protein (E347K mutant). The E347K mutant exhibited marked rounding of the cells and few syncytia in infected chicken embryofibroblast (CEF) cells. The hemadsorption and neuraminidase activities of the E347K mutant of the wild-type virus were 118% and 166%, respectively. The mutant produced a rapid elution pattern whereas the wild type had a slow elution pattern. Growth kinetics studies showed that the E347K mutant produced an 80-times higher yield of extracellular virus in CEF cells compared with the wild-type virus. The time-course virus titer showed a marked increase in mutant-infected cells from 6 h to 12 h post infection (pi), which was consistent with the titer pattern time-course for NA activity. The E347K mutant virus showed a slight decrease in virulence compared to the wild-type virus, but there was no change in pathotype when measured by in vivo pathogenicity testing. These results suggest that an E347K mutation in HN protein might be associated with increased NA activity and subsequent enhancement of virus release from infected cells without change in viral pathotype.

Synechocystis sp. PCC6803을 이용한 Photosystem I- mutants의 색소 및 틸라코이드막 단백질 분석

전은경,장남기 한국잔디학회 1997 한국잔디학회지 Vol.11 No.1

Pigments and thylakoid membrane proteins were investigated in wild type and PS I- mutants from Synechocystis sp. PCC6803 Comparing morphological features, B2 was less fluorescent than the other strains. The contents of chlorophyll a were propotional to the FNR activity in thylakoid membrane. The FNR activity of mutants was lower than that of wild type. In the result of pigments analysis, mutants had smaller cholophyll a than that of wild type. The major carotenoid was found to he $\beta$-caroene, but aeaxanthin was barely detected in thylakoid membrane of mutants. The polypeptide, 14.8kD was detected by electrophoresis in mutants. It was considered to be the modification of 15.4kD in wild type. Membrane polypeptides of 17.6 and 19.7kD were not detected in mutants. In the result of western blotting, subunit I was detected in all strains, but subunit II was barely detected in mutants. Subunit II was not detected in B2 at all. In view of the results so far achieved, the changes of contents of chlorophyll and zeaxanthin were affected by the defficiency or modification of functional domain in subunit I. Also the modification in subunit I affected the subunit II- binding site in PS I. As the result, efficiency of photosynthesis was decreased. Key words: Synechoystis sp. PCC6803, PS I - mutant, Photosynthetic efficiency, Pigment,Thylakoid membrane proteins, Subunit I, II.

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression

Chung, Jeong Min,Lee, Sangmin,Jung, Hyun Suk Elsevier 2017 Protein expression and purification Vol.133 No.-

<P><B>Abstract</B></P> <P>Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The expression and purification condition for truncated actin binding proteins was optimized from <I>Escherichia coli</I> strain. </LI> <LI> The treatment of sarkosyl-detergent increases the solubility of bacterial recombinant proteins. </LI> <LI> Minimizing concentration of sarkosyl was suggested for enhancing the production of soluble proteins. </LI> </UL> </P>

RiSLnet: Rapid Identification of Smart Mutant Libraries using Protein Structure Network. Application to thermal stability enhancement

김진영,( Roopali Upadhyay ),홍은영,이선구,서주현,김병기 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0

A key point of protein stability engineering is to identify specific target residues that do not harm original activity of the protein. Here, we propose a method, called RiSLnet (Rapid identification of Smart mutant Library using residue network), to identify such residues by combining network analysis for protein residue interactions, identification of conserved residues, and evaluation of relative solvent accessibility. Application to lysine decarboxylase (CadA) confirmed its accuracy and effectiveness. RiSLnet successfully identified beneficial mutants with decent (~60%) accuracy and significantly reduced the number of candidate residues (~99%) for mutation. Finally, a combinatorial mutant designed by RiSLnet and in silico saturation mutagenesis resulted in an increase of half-life (T1/2) to 114.9 min at 58°C, which is approximately two-fold higher than that of the wild-type.

콩 돌연변이 계통의 단백질 특성

Moo Hee Yang,Joe W. Burton 韓國作物學會 1994 Korean journal of crop science Vol.39 No.3

콩단백질의 황 아미노산함량은 가축 영양학상 중요한 위치를 차지하기 때문에 신계통이 가져야만 할 필수조건일지도 모른다. 콩 계통간에 저장단백질의 유전적변이가 존재한다면 이는 기존의 육종방법을 통하여 콩의 종자단백질 구성성분을 유전적으로 변경하여 품질을 개량할 수 있는 가능성을 시사하고 있다. 본 연구는 여러 문헌에 보고된 콩종자 저장단백질의 돌연변이 계통들을 선별하여 콩단백질의 품질을 향상시키기 위한 육종 재료로서의 가능성을 평가하기 위하여 실행되었다. 수집된 돌연변이 계통들은 저장단백질의 또 다른 특성을 나타내었다. 그 돌연변이 계통들 중에서 Keburi(P.I.417016), Keburi(P.I.506817), P.I.154608-1 등은 황 아미노산 함량이 상대적으로 다른 돌연변이 계통보다 높은 1.9, 2.1, 1.8%를 나타내었으며, 이는 7S 단백질인 α ', α ,β 단백질 함량이 상대적으로 낮기 때문인 것으로 나타났다. 그러므로 그 돌연변이 계통들 중에서 Keburi(P.I.417016), Keburi(P.I.506817), P.I.54608-1 등은 황 아미노산 함량을 향상시키기 위한 중요한 육종재료로, 그 외 돌연변이 계통들은 다른 용도의 육종 재료로 이용할 수 있을 것으로 추측된다. The sulfur amino acid composition in soybean (Glycine max L.) seeds may be an essential characteristic of new cultivars for some animal diets. Variation in seed storage protein among genotypes might make it possible to improve the quality of seed protein by genetically altering seed storage protein composition through plant breeding. This study was carried out to determine if mutant strains have potential for improving seed protein quality in soybean. Ten mutant strains had a distinct characteristic of seed storage protein subunits. Among the mutant strains, the sulfur amino acid compositions(methionine plus cystein) of Keburi(P.I.417016), Keburi(P.I.506817), and P.I.54608-1 were relatively higher than those of the others and were 1.9, 2.1, and 1.8%, repectively, which might be due to low levels of α , α ', and β subunits of 7S protein. Therefore, it is concluded that the mutant strains, Keburi(P.I.417016), Keburi(P.I.506817), and P.I.54608-1 appear to be potential materials for a breeding program for improving sulfur amino acid composition, and the others also seem to be possible breeding materials for other uses.

Transduced Tat-DJ-1 protein inhibits cytokines-induced pancreatic RINm5F cell death

( Hyo Sang Jo ),( Hyeon Ji Yeo ),( Hyun Ju Cha ),( Sang Jin Kim ),( Su Bin Cho ),( Jung Hwan Park ),( Chi Hern Lee ),( Eun Ji Yeo ),( Yeon Joo Choi ),( Won Sik Eum ),( Soo Young Choi ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.5

Loss of pancreatic β-cells by oxidative stress or cytokines is associated with diabetes mellitus (DM). DJ-1 is known to as a multifunctional protein, which plays an important role in cell survival. We prepared cell permeable wild type (WT) and mutant type (M26I) Tat-DJ-1 proteins to investigate the effects of DJ-1 against combined cytokines (IL-1β, IFN-γ and TNF-α)-induced RINm5F cell death. Both Tat-DJ-1 proteins were transduced into RINm5F cells. WT Tat-DJ-1 proteins significantly protected against cell death from cytokines by reducing intracellular toxicities. Also, WT Tat-DJ-1 proteins markedly regulated cytokines-induced pro- and anti-apoptosis proteins. However, M26I Tat-DJ-1 protein showed relatively low protective effects, as compared to WT Tat-DJ-1 protein. Our experiments demonstrated that WT Tat-DJ-1 protein protects against cytokine-induced RINm5F cell death by suppressing intracellular toxicities and regulating apoptosisrelated protein expression. Thus, WT Tat-DJ-1 protein could potentially serve as a therapeutic agent for DM and cytokine related diseases. [BMB Reports 2016; 49(5): 297-302]

Transduced Tat-DJ-1 protein inhibits cytokines-induced pancreatic RINm5F cell death

( Hyo Sang Jo ),( Hyeon Ji Yeo ),( Hyun Ju Cha ),( Sang Jin Kim ),( Su Bin Cho ),( Jung Hwan Park ),( Chi Hern Lee ),( Eun Ji Yeo ),( Yeon Joo Choi ),( Won Sik Eum ),( Soo Young Choi ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.6

Loss of pancreatic β-cells by oxidative stress or cytokines is associated with diabetes mellitus (DM). DJ-1 is known to as a multifunctional protein, which plays an important role in cell survival. We prepared cell permeable wild type (WT) and mutant type (M26I) Tat-DJ-1 proteins to investigate the effects of DJ-1 against combined cytokines (IL-1β, IFN-Υ and TNF-α)-induced RINm5F cell death. Both Tat-DJ-1 proteins were transduced into RINm5F cells. WT Tat-DJ-1 proteins significantly protected against cell death from cytokines by reducing intracellular toxicities. Also, WT Tat-DJ-1 proteins markedly regulated cytokines-induced pro- and anti-apoptosis proteins. However, M26I Tat-DJ-1 protein showed relatively low protective effects, as compared to WT Tat-DJ-1 protein. Our experiments demonstrated that WT Tat-DJ-1 protein protects against cytokine-induced RINm5F cell death by suppressing intracellular toxicities and regulating apoptosisrelated protein expression. Thus, WT Tat-DJ-1 protein could potentially serve as a therapeutic agent for DM and cytokine related diseases. [BMB Reports 2016; 49(5): 297-302]

OsCIPK31, a CBL-Interacting Protein Kinase Is Involved in Germination and Seedling Growth under Abiotic Stress Conditions in Rice Plants

Hai-long Piao,Yuan-hu Xuan,Su Hyun Park,제병일,Soon Ju Park,Sung Han Park,김철민,Jin Huang,Guo Kui Wang,김민정,강상모,이인중,Taek-Ryoun Kwon,Yong Hwan Kim,Un-sang Yeo,이기환,손대영,한창덕 한국분자세포생물학회 2010 Molecules and cells Vol.30 No.1

Calcineurin B-like protein-interacting protein kinases (CIPKs) are a group of typical Ser/Thr protein kinases that mediate calcium signals. Extensive studies using Arabi-dopsis plants have demonstrated that many calcium sig-natures that activate CIPKs originate from abiotic stresses. However, there are few reports on the functional demon-stration of CIPKs in other plants, especially in grasses. In this study, we used a loss-of-function mutation to charac-terize the function of the rice CIPK gene OsCIPK31. Expo-sure to high concentrations of NaCl or mannitol effected a rapid and transient enhancement of OsCIPK31 expression. These findings were observed only in the light. However, longer exposure to most stresses resulted in down-regulation of OsCIPK31 expression in both the presence and absence of light. To determine the physiological roles of OsCIPK31 in rice plants, the sensitivity of oscipk31::Ds, which is a transposon Ds insertion mutant, to abiotic stresses was examined during germination and seedling stages. oscipk31::Ds mutants exhibited hypersensitive phenotypes to ABA, salt, mannitol, and glucose. Com-pared with wild-type rice plants, mutants exhibited re-tarded germination and slow seedling growth. In addition, oscipk31::Ds seedlings exhibited enhanced expression of several stress-responsive genes after exposure to these abiotic stresses. However, the expression of ABA meta-bolic genes and the endogenous levels of ABA were not altered significantly in the oscipk31::Ds mutant. This study demonstrated that rice plants use OsCIPK31 to modulate responses to abiotic stresses during the seed germination and seedling stages and to modulate the expression of stress-responsive genes.