Methylation and demethylation of DNA and histones in chromatin: the most complicated epigenetic marker

윤홍덕 생화학분자생물학회 2017 Experimental and molecular medicine Vol.49 No.-

Pleurotus ostreatus에서 분비된 Laccase의 보결단 추정

윤홍덕,신광수,강사욱,하영칠,정가진,김규중 한국미생물학회 1991 미생물학회지 Vol.29 No.4

Extracellular laccase secreted from Pleurotus ostreatus was activated by $Cu^{2+}$ and $Cu^{+}$ . The enzyme was strongly inactivated by 8-hydroxyquinoline, potassium cyanide, sodium azide, sodium bisulfite and 2-mercaptoethanol. The two ionogenic groups, which have pKa values of 5.60-5.70 and 6.70-6.85 respectively, were found to relate with the active site of this enzyme. The oxidation reactions were brought about by initial single electron transfer process on the active site. The enzyme was found to be a metalloprotein which had about 3.9 cupric ions per molecule of protein as a prosthetic group. The enzyme showed a strong peak at 605 nm and a weak shoulder at 330 nm in UV-Visible absorption spectrum. Both signals disappeated upon treatment of the enzyme with 4 electron equivalent ascorbate. These results indicate that type I Cu peak and type III Cu shoulder are present in laccase.

Lentinus edodes 에서 분비되는 Laccase 의 특성

정인범,윤홍덕,맹진수,강사욱,하영칠,정가진,최형태,김재헌 한국미생물학회 1992 미생물학회지 Vol.30 No.4

Lentinus edodes 에서 분비된 laccase 를 DEAE Sephadex A-50, Con A-Sepharose, Sephadex G-150 크로마토그래피를 통해서 순수분리하였다. 효소는 분자량이 87 KDa 정도인 하나의 소단위체로 되어 있고, 12.0% 의 당을 함유하고 있었다. 그리고 N-말단 아미노산 서열은 Pleurotus oxtreatus 와 Cloriolus hirsutus 의 laccase 와 유사하였다. 효소의 최적 pH 는 4.8 이고 최적 온도는 $40^{\circ}C$ 이었다. 그리고 본 효소는 pH 7-9 와 $30^{\circ}C$이하에서 비교적 안정하였다. Syringaldazine 에 대한 $K_{M}$ 은 $0.4\mu$ M 이었고 $k_{cat}$ 은$ 77 sec^{-1}$ 이었다. This layer chromatography 에 의한 본 효소의 반응 산물들의 분리양상이 Pleurotus ostreatus 의 laccase 의 경우와 유사하였다. Extracellular laccase excreted from Lentinus edodes ATCC 48085 was purified through a series of DEAF, Sephadex A-50. Con A-Sepharosc and Sephadex G-150 chromatography. Extracellular enzyme. which consists of a single polypeptide, has a n~olecular mass of 87.000 daltons and contains 12.0'%, carbohydrate. The N-terminal amino acid sequence (I5 residues) of the puritied enzyme was similar to that of laccases of PIeurotus ostreatus and Coriolus hirsutus. The enzyme showed optimal activity at near pH 4.8 and $40^{\circ}C$. The enzyme was stable at pH 7-9 and below $30^{\circ}C$. $K_{M}$ and $k_{cat}$ values for syringaldazine were estimated to be $0.4\mu\textrm{M}$ and 77 sec, respectively. The developed patterns of reaction products of thevenzyme on thin layer chromatography were similar to those of laccase of Pleurotus ostreatus.

pVHL-Mediated Transcriptional Repression of c-Myc by Recruitment of Histone Deacetylases

황인영,윤홍덕,조은정,Jae-Seok Roe,설자환,김화연 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.2

The biological functions of Myc are to regulate cell growth, apoptosis, cell differentiation and stem-cell self-renewal. Abnormal accumulation of c-Myc is able to induce excessive proliferation of normal cells. von Hippel-Lindau protein (pVHL) is a key regulator of hypoxia-inducible factor1 (HIF1), thus accumulation and hyperactivation of HIF1 is the most prominent feature of VHL-mutated renal cell carcinoma. Interestingly, the Myc pathway is reported to be activated in renal cell carcinoma even though the precise molecular mechanism still remains to be established. Here, we demonstrated that pVHL locates at the c-Myc promoter region through physical interaction with Myc. Furthermore, pVHL reinforces HDAC1/2 recruitment to the Myc promoter, which leads to the auto-suppression of Myc. Therefore, one possible mechanism of Myc auto-suppression by pVHL entails removing histone acetylation. Our study identifies a novel mechanism for pVHL-mediated negative regulation of c-Myc transcription.

Integrated miRNA and mRNA Expression Profiling in Response to Eriodictyol in Human Endothelial Cells

이승은,박혜림,윤홍덕,조정제,안현종,박증석,박용식 한국바이오칩학회 2017 BioChip Journal Vol.11 No.3

Eriodictyol, a flavonoid commonly found in citrus fruits, shows neurotrophic, antioxidant, anti- inflammatory, and anti-cancer effects under diverse pathophysiological conditions such as vascular diseases. In this study, we evaluated whether eriodictyol modulates miRNA and mRNA expression. Using microarray analysis, we examined miRNA and mRNA expression in human endothelial cells treated with 10 μM of eriodictyol for 24 h. Using numerous bioinformatic systems, we evaluated the signatures of the potential biological processes and signaling pathways. Our results provide insight into the underlying molecular mechanisms of action of eriodictyol and suggest that eriodictyol can be used for therapeutic intervention in vascular disease.

Integrated Analysis of miRNA and mRNA Expression Profiles in Human Endothelial Cells Exposed to Fisetin

이승은,박혜림,윤홍덕,김혜미,진영호,조정제,안현종,박증석,박용식 한국바이오칩학회 2017 BioChip Journal Vol.11 No.3

MicroRNAs (miRNAs) play vital regulatory roles in various biological processes including cell differentiation, proliferation and apoptosis. Several studies have examined the expression profiles of miRNAs in response to different drugs and chemicals and suggested that expression patterns were associated with the effects of drugs and chemicals. Fisetin, a polyphenol found in a wide range of plants, has been reported to exhibit numerous biological activities, such as antioxidant, anti-inflammatory, antiangiogenic, and antiproliferative properties. In the present study, we evaluated whether fisetin regulates miRNA and mRNA expression. Using microarray analysis, we measured global miRNA and mRNA expression in human endothelial cells treated with 10 μM of fisetin for 24 h. Using several bioinformatic systems, we determined the signatures of potential biological processes and signaling pathways such as cell migration, apoptotic signaling pathway, cell motility, and cardiovascular system development. Our findings provide insight into the molecular mechanisms of action of fisetin and can be used to improve therapeutic intervention in vascular diseases.

Integrated Analysis of Changed microRNA Expression in Crotonaldehyde-exposed Human Endothelial Cells

박혜림,이승은,손건우,윤홍덕,박용식 한국바이오칩학회 2016 BioChip Journal Vol.10 No.2

Crotonaldehyde (CRA), a reactive α,β-unsaturated aldehyde, is produced by natural and synthetic processes such as incomplete combustion of many compounds and can be found in cigarette smoke. CRA induces the formation of DNA adducts and suppresses the expression of glutathione, resulting in endothelial dysfunction, inflammation and vascular diseases. MicroRNAs (miRNAs) are small, non-coding RNAs that negatively modulate gene expression by targeting mRNAs for translational repression or degradation. They are also important factors involved in cell growth, apoptosis, and proliferation in cardiovascular disease. In this study, to examine the effect of CRA on the expression profiles of miRNAs in human umbilical vein endothelial cells (HUVECs), we performed pair-wise correlation analyses and identified 162 miRNAs with altered expression upon treatment of HUVECs with 10 μM CRA. In addition, we discovered a significant anti-correlation between 55 miRNAs and 11 mRNA. Differentially expressed miRNAs were further validated by Gene Ontology (GO) enrichment analysis. Our results suggest that modified expression of miRNAs caused by CRA treatment is related to endothelial dysfunction and might contribute to further understanding the molecular mechanisms of vascular diseases.

Histone acylation marks respond to metabolic perturbations and enable cellular adaptation

조찬희,박석재,Oh Sungjoon,Choi Jinmi,김은경,윤홍덕,조은정 생화학분자생물학회 2020 Experimental and molecular medicine Vol.52 No.-

Acetylation is the most studied histone acyl modification and has been recognized as a fundamental player in metabolic gene regulation, whereas other short-chain acyl modifications have only been recently identified, and little is known about their dynamics or molecular functions at the intersection of metabolism and epigenetic gene regulation. In this study, we aimed to understand the link between nonacetyl histone acyl modification, metabolic transcriptional regulation, and cellular adaptation. Using antibodies specific for butyrylated, propionylated, and crotonylated H3K23, we analyzed dynamic changes of H3K23 acylation upon various metabolic challenges. Here, we show that H3K23 modifications were highly responsive and reversibly regulated by nutrient availability. These modifications were commonly downregulated by the depletion of glucose and recovered based on glucose or fatty acid availability. Depletion of metabolic enzymes, namely, ATP citrate lyase, carnitine acetyltransferase, and acetyl-CoA synthetase, which are involved in Ac-CoA synthesis, resulted in global loss of H3K23 butyrylation, crotonylation, propionylation, and acetylation, with a profound impact on gene expression and cellular metabolic states. Our data indicate that Ac-CoA/CoA and central metabolic inputs are important for the maintenance of histone acylation. Additionally, genome-wide analysis revealed that acyl modifications are associated with gene activation. Our study shows that histone acylation acts as an immediate and reversible metabolic sensor enabling cellular adaptation to metabolic stress by reprogramming gene expression.

Menin represses JunD transcriptional activity in protein kinase C -mediated Nur77 expression

김형수,이지은,김부연,조은정,김성태,윤홍덕 생화학분자생물학회 2005 Experimental and molecular medicine Vol.37 No.5

TCR signaling leading to thymocyte apoptosis is mediated through the expression of the Nur77 family of orphan nuclear receptors. It has been shown that the Nur77 promoter is activated by at least two signaling pathways, one mediated by calcium and the other by protein kinase C (PKC). MEF2D has been known to regulate Nur77 expression in a calcium- dependent manner. The mechanism by which calcium regulates MEF2D is through dissociation of calcium-sensitive MEF2 corepressors (Cabin1/ HDACs, HDAC4/5) and the association with calcineurin- activated transcription factor NF-AT and the coactivator p300. However, little is known about how PKC activates the Nur77 promoter. Herein, we report that PKC targets AP-1 like response element in the Nur77 promoter where JunD constitutively binds. PKC θ triggers mitogen-activated protein kinase- inediated phosphorylation of JunD, and increases transcriptional activity of JunD, cooperatively with p300. Menin is identified as the transcriptional corepressor for JunD via recruitmen t of mSin3- istone deacetylases. In fact, Menin represses PKC θ / p300-mediated transcriptional activity of JunD in T cell. Its dynamic regulation of histone modifiers with JunD is responsible for PKC θ -synergistic effect on Nur77 expression in T cell.

Down syndrome critical region 1 enhances the proteolytic cleavage of calcineurin

Ji-Eun Lee,장현철,조은정,윤홍덕 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.7

Down syndrome critical region 1 (DSCR1), an oxidative stress-response gene, interacts with calcineurin and represses its phosphatase activity. Recently it was shown that hydrogen peroxide inactivates calcineurin by proteolytic cleavage. Based on these facts, we investigated whether oxidative stress affects DSCR1- mediated inactivation of calcineurin. We determined that overexpression of DSCR1 leads to increased proteolytic cleavage of calcineurin. Convertsely, knockdown of DSCR1 abolished calcineurin cleavage upon treatment with hydrogen peroxide. The PXIIXT motif in the COOH-terminus of DSCR1 is responsible for both binding and cleavage of calcineurin. The knockdown of overexpressed DSCR1 in DS fibroblast cells also abrogated calcineurin proteolysis by hydrogen peroxide. These results suggest that DSCR1 has the ability to inactivate calcineurin by inducing proteolytic cleavage of calcineurin upon oxidative stress.