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Methylase를 사용한 Escherichia coli에서 Triplex 존재에 관한 연구
임향숙,김성조,강성만,Rhim, Hyangshuk,Kim, Sungjo,Kang, Seongman 한국미생물학회 1998 미생물학회지 Vol.34 No.4
Escherichia coli 세포에서 intramolecular triplex가 형성될 수 있는지를 조사하기 위하여 genetic assay를 도입하였다. Two dimensional gel electrophoresis 방법을 사용하여 $(G)_9AATTC(G)_9$ 혹은 $(GAA)_9TTC(GAA)_8$ sequences가 in vitro에서 intramolecular triplex 구조를 형성하는 것을 확인하였다. Genetic assay를 위하여 temperature-sensitive EcoRI methylase 유전자를 포함한 플라스미드와 triplex를 형성할 수 있는 $(G)_9AATTC(G)_9$ 혹은 $(GAA)_9TTC(GAA)_8$ sequences를 포함한 플라스미드로 E. coli를 cotransform 하였다. EcoRI methylase가 발현되는 permissive temperature에서 pur pyr sequences를 포함하는 플라스미드는 EcoRI methylase 작용에 kinetically 저항성을 보여주었다. 이러한 결과는 pur pyr sequence들이 EcoRI methylase가 작용하기 어려운 triplex 구조를 E. coli 세포에서 형성한다는 것을 증거한다. We have introduced a genetic assay to study the existence of intramolecular triplexes in Escherichia coli. A plasmid containing the gene that encodes a temperature-sensitive EcoRI methylase was cotransformed with different plasmids containing inserts, $(G)_9AATTC(G)_9$ and $(GAA)_9TTC(GAA)_8$, that are able to form intramolecular triplexes in vitro. Inhibition of methylation in vivo was found for $(G)_9AATTC(G)_9$ and $(GAA)_9TTC(GAA)_8$, suggesting that the pur pyr sequences adopt unusual strucures in E. coli. In addition, experiments using two dimensional gel electrophoresis confirmed that intramolecular triplexes are formed for the pur pyr sequences under negative supercoiling. These results demonstrate the existence of intramolecular triplexes in E. coli.
Lee, Sun-Joo,Choi, Dongwon,Rhim, Hyangshuk,Kang, Seongman Biochemical Society 2005 Biochemical journal Vol.389 No.2
<P>We reported previously that the human RNF2 (RING finger protein 2) protein is an E3 ubiquitin ligase that interacts with the human ubiquitin-conjugating enzyme Hip-2/hE2-25K. In the present study, we show that RNF2 interacts with S6' ATPase, a subunit of the proteasomal 19 S regulatory complex. S6' interacts with RNF2 through its N-terminal RING domain, and RNF2 interacts with S6' through its C-terminal region. Interestingly, the RNF2-S6' interaction increases the ATP hydrolysis activity of the S6' protein. Moreover, S6' ATPase activity is highly increased in the presence of ubiquitinated proteins. The present study suggests that the E3 ubiquitin ligase RNF2 might have a dual function: facilitating the ubiquitination of its target substrates and recruiting the substrates to the proteasome. Furthermore, ATP hydrolysis in the E3/proteasome complex might act as an important signal for the protein degradation pathway.</P>
PHB2 interacts with RNF2 and represses CP2c-stimulated transcription.
Lee, Sun-Joo,Choi, Dongwon,Rhim, Hyangshuk,Choo, Hyo-Jung,Ko, Young-Gyu,Kim, Chul Guen,Kang, Seongman Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2008 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.319 No.1
<P>RNF2, a polycomb group protein, is an important component of PRC complex regulating transcriptional activity. Recently, several RNF2 interacting proteins have been identified. Thus, RNF2 might have multiple activities, depending on its interacting partner proteins. In the present study, using the yeast two-hybrid system, we have found that RNF2 interacts with the PHB2 protein. Luciferase reporter assays showed that RNF2 represses the CP2c-stimulated luciferase activity in a PHB2 dose-dependent manner. Further experiments with RNF2 deletion mutants indicated that RNF2(1-158) is sufficient for both the physical association and functional co-operation with the PHB2 protein. Co-immunoprecipitation experiments revealed that PHB2 and CP2c bind to the N- and C-terminals of RNF2, respectively. Luciferase reporter assays using alpha-globin promoter with CP2-binding elements hinted that RNF2 and PHB2 are involved in the CP2-stimulated expression of the alpha-globin gene. Our study suggests a novel mechanism by which RNF2 and PHB2 modulate the CP2-mediated transcriptional pathway.</P>
Moon, Jeong-Mi,Kim, Goo-Young,Rhim, Hyangshuk Kluwer Academic Publishers 2012 Biotechnology letters Vol.34 No.10
<P>Mammalian expression vectors are used to overexpress genes of interest in mammalian cells. High temperature requirement protein A1 (HtrA1), used as a specific target, was expressed from the pHA-M-HtrA1 plasmid in HEK293T cells, inducing cell death. Expression of HtrA1 was driven by the pHA-M-HtrA1 mammalian expression vector in E. coli resulting in growth suppression of E. coli in an HtrA1 serine protease-dependent manner. By using various combinations of promoters, target genes and N-terminal tags, the T7 promoter and N-terminal HA tag in the mammalian expression vector were shown to be responsible for expression of target genes in E. coli. Thus the pHA-M-HtrA1 plasmid can be used as a novel, rapid pre-test system for expression and cytotoxicity of the specific target gene in E. coli before assessing its functions in mammalian cells.</P>
Yun, Si-Eun,Nam, Min-Kyung,Rhim, Hyangshuk Elsevier 2018 Biochimica et biophysica acta, General subjects Vol.1862 No.7
<P><B>Abstract</B></P> <P><B>Background</B></P> <P>Regulating apoptosis is a common and essential therapeutic strategy for cancer and neurodegenerative disorders. Based on basic studies of apoptotic mechanisms, various researches have attempted to overcome the pathogenesis of such diseases by activating or inhibiting apoptosis. Generally, the biochemical characteristics of the target molecules should be evaluated along with understanding of their mechanisms of action during drug development. Among apoptotic regulators, XIAP serves as a potent negative regulator to block apoptosis through the inhibition of caspase (CASP)-9 and -3/7. Although XIAP is an attractive target with such apoptotic-modulating property, biochemical and biophysical studies of XIAP are still challenging.</P> <P><B>Methods</B></P> <P>In this study, the CASP-9 and -3/7 inhibitors XIAP, 242Δ and Δ230 were prepared using the pGEX expression system and biochemically characterized.</P> <P><B>Results</B></P> <P>These inhibitors were expressed in <I>Escherichia coli</I> at a concentration of ≥20 mg/L culture under a native condition with 0.01 mM IPTG induction. Notably, using a simple and rapid affinity purification technique, these CASP-9 and -3/7 inhibitors have been purified, yielding ≥5 mg/L culture at approximately 90% purity.</P> <P><B>Conclusions</B></P> <P>We have determined that HtrA2 specifically binds to the BIR2 and BIR3 of XIAP at a 1:1 molecular ratio. Moreover, in vitro cell-free CASP-9 and -3/7 activation-apoptosis assays have demonstrated that these purified XIAP proteins dramatically inhibit CASP-9 and -3/7 action.</P> <P><B>General significance</B></P> <P>Our system is suitable for biochemical studies, such as quantitation of the number of molecules acting on the apoptosis regulation, and provides a basis and insights that can be applied to the development of therapeutic agents for neurodegenerative disorders and cancer.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Development of a simple and rapid method for bacterial expression and purification of XIAP proteins, wild type, 242Δ, and Δ230 </LI> <LI> Structural verification of the purified XIAP proteins using GST pull-down with HtrA2 </LI> <LI> Functional verification of the purified XIAP proteins using cell-free caspase activation-apoptosis assays </LI> </UL> </P>
Choi, Soo Young,Park, Jinseu,Kang, Young Hee,Lee, Yoon,Lee, Hangyu,Rhim, Hyangshuk The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.4
The human immunodeficiency virus type 1 (HIV-1), transactivator of transcription (Tat), is one of the viral gene products that is essential for HIV-1 replication. The HIV-1 Tat protein regulates transcription from an HIV-1 long terminal repeat (LTR) and affects the gene expression of cellular proteins during infection. In order to develop an expression system to overexpress and simply purify HIV-1 Tat proteins, the HIV-1 Tat coding sequences that contain one or two exons were amplified using PCR and cloned into a pET vector, which contains a consecutive stretch of six histidine residues at the amino-terminus. The reconstituted vectors were overexpressed in the E. coli strain and the soluble recombinant proteins were purified to be homogeneity in a single step by Ni??-nitrilotriacetic acid Sepharose chromatography under nondenaturing conditions. Recombinant HIV-1 LTR promoter in a dose-dependent manner when introduced into mammalian cells. In addition, treatment of human endothelial cells with purified Tat proteins resulted in a significant increase in the level of vascular cell adhesion molecule-1 (VCAM-1) expression. These results indicate that the recombinant HIV-1 Tat proteins are active in transactivating viral and cellular described in this study will facilitate in characterizing the biological functions of the Tat proteins.