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Immunoaffinity purification of SRT-tagged human creatine kinase by peptide elution
Lee, Jae-Rin,Hahn, Hwa-Sun,Yu, Jae-Ran,Kim, Seong-Tae,Yang, Jun-Mo,Hahn, Myong-Joon Elsevier 2005 Journal of biotechnology Vol.117 No.3
<P><B>Abstract</B></P><P>The mouse monoclonal antibody (Mab), SRT10, recognizes a linear epitope of 10 amino acids (ThrPheIleGlyAlaIleAlaThrAspThr). When these epitope-tagged fusion proteins are expressed in mammalian cells, the Mab can detect the tagged proteins by immunoblotting, immunocytochemistry and immunoprecipitation. Here, we describe an efficient method for the purification of SRT-tagged recombinant human creatine kinase (CK) transiently expressed in mammalian cells. This method utilizes the expression of the N-terminal- or C-terminal-tagged CK in transiently transfected HEK293 cells followed by binding to anti-SRT-agarose affinity resin and competitive elution with SRT peptide. Recombinant CK was purified near homogeneity as judged by SDS-PAGE.</P>
Kim, Dong-Eun,Hahn, Myong-Joon,Park, Kyung-Hee 동의대학교 산업기술개발연구소 2006 産業技術硏究誌 Vol.20 No.-
Tyrosinase requires copper ion at the active site to oxidize phenols to catechols. Here, inhibitory action of a copper-chelating ammonium tetrathiotungstate (ATTT) was investigated. ATTT completely inactivated the mushroom tyrosinase activity in a dose-dependent manner. Progress-of-substrate reaction kinetics using the two-step kinetic pathway revealed that ATTT acts as a kinetically competitive inhibitor in vitro, and the enzyme-ATTT complex subsequently undergoes reversible conformational change, leading to complete inactivation of the tyrosinase activity. In the melanin producing cell lines ATTT exhibited more potent inhibitory effect on the tyrosinase than previously known inhibitors such as kojic acid and hydroquinone.
이재린,안진현,Chang-Dae Bae,양준모,Myong-Joon Hahn,Hwa-Sun Hahn,Young-Hoon Kim 생화학분자생물학회 2007 Experimental and molecular medicine Vol.39 No.4
Zinc finger protein 133 (ZNF133) is composed of a Krüppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1.
Kim, Young-Hoon,Lee, Jae-Rin,Hahn, Myong-Joon Elsevier 2018 Biochemical and biophysical research communication Vol.496 No.2
<P><B>Abstract</B></P> <P>Epithelial-stromal interaction 1 (<I>EPSTI1</I>) was first discovered as a gene induced in breast cancer epithelial cells by co-cultured stromal fibroblasts. There are many reports on the role of Epsti1 in cancer malignancy. Epsti1 is now well known in regulating cancer. Recently, the role of Epsti1 in the immune response has been reported; these reports suggest the role of Epsti1 in immune function, immune privilege, and autoimmune diseases. Furthermore, they show that Epsti1 is expressed in various types of immune cells. In this study, we observed that Epsti1 is highly expressed in macrophages exposed to IFNγ and lipopolysaccharide (LPS), which classically activates macrophages. Polarization of macrophage to classically activated (M1) or alternatively activated (M2) is important for mounting responses against various infections. The M1 and M2 types of macrophage have a distinct role in the immune system. However, the molecular mechanism of modulation of the macrophage type is not well defined. Our results showed that the M2 type macrophage phenotype is enhanced in Epsti1-deficient bone marrow-derived macrophages (BMDM). In addition, Epsti1 deficiency suppresses induction of pro-inflammatory genes in BMDMs via inhibition of Stat1 and p65 nuclear localization and phosphorylation. Surprisingly, <I>Epsti1</I>−/− mice show decreased numbers of M1 macrophages in the peritoneal cavity. These findings identify Epsti1 as a modulator of macrophage activation and polarization via the Stat1 and p65 pathways, and suggest a potentially important role of Epsti1 in immunotherapies against inflammatory diseases.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Epsti1 is highly expressed in macrophages exposed to IFNγ and lipopolysaccharide (LPS). </LI> <LI> The M2 type macrophage phenotype is enhanced in Epsti1-deficient bone marrow-derived macrophages. </LI> <LI> <I>Epsti1</I>−/− mice show decreased the number of M1 macrophage in the peritoneal cavity. </LI> </UL> </P>
DNA methyltransferase-3a interacts with p53 and represses p53-mediated gene expression.
Wang, Y Alan,Kamarova, Yeugeniya,Shen, Kate C,Jiang, Zhongliang,Hahn, Myong-Joon,Wang, Yaolin,Brooks, S C Landes Bioscience 2005 Cancer biology & therapy Vol.4 No.10
<P>Genome stability maintenance is regulated by both genetic and epigenetic mechanisms. DNA methylation is the predominant epigenetic mechanism in regulation of gene expression and in suppression of mobile DNA elements from random integration in the genome. The importance of DNA methylation in tumorigenesis has been demonstrated in cancer cells, which harbor global genomic DNA hypomethylation and regional hypermethylation at CpG islands of tumor suppressor genes. DNA methylation is mediated by a class of DNA methyltransferases (Dnmts) involved in de novo methylation of genomic DNA and in the maintenance of DNA methylation patterns during replication. Global genomic DNA demethylation induced by 5-Aza-deoxycytidine activates the p53 signaling pathway and induces apoptosis, suggesting that DNA methylation mediated by Dnmts is associated with p53 signaling in maintaining genome stability. In this report, we show that Dnmt3a interacts with p53 directly and represses p53-mediated transactivation of the p21 gene. It was found that trans-repression by Dnmt3a does not require the methyltransferase activity implying that transcriptional repression does not involve promoter silencing through DNA methylation by Dnmt3a. Finally, the activity of Dnmt3a in vivo was demonstrated when this enzyme was overexpressed in a breast cell line in which Dnmt3a repressed p21 upregulation following DNA damage. The results presented in this study provide new understanding of tumor promotion as mediated by Dnmt3a through its interaction with p53, and suppression of the p53-mediated transcription of tumor suppressor genes. Given that the expression of Dnmts is increased in certain cancers, it is likely that increased Dnmts could block the transactivation function of p53 following its induction by chemotherapeutic drugs resulting in chemoresistance. The use of a DNA methyltransferase inhibitor would therefore restore the p53 tumor suppression function and the utilization of such an inhibitor in combination with DNA damage agents might be an effective therapy for certain cancers.</P>