• Journal of Microbiology and Biotechnology
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• 제11권4호
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• pp.592-597
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• 2001

The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and H+. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase(E1), dihydrolipoamide acetyltransferase(E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, cloning and characterization of a gene for human E3BP have been carried out. A cDNA encoding the human E3BP was isolated by database search and cDNA library screening. The primary structure of E3BP has some similar characteristics with that of E2 in the lipoyl domain and the carboxyl-terminal domain, based on the nucleotide sequence and the deduced amino acid sequence. However, the conserved amino acid moiety including the histidine residue for acetyltransferase activity in E2 is not conserved in the case of human E3BP. The human E3BP was expressed and purified in E. coli. The molecular weight of the protein, excluding the mitochondrial target sequence, was about 50 kDa as determined by SDS-PAGE. Cloning of human E3BP and expression of the recombinant E3BP will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

• IMMUNE NETWORK
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• 제2권1호
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• pp.35-40
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• 2002

Background: CTLA-4 (Cytotoxic T Lymphocyte associated Antigen 4, CD152) has been known as a homologue of CD28, an accessory molecule providing a key costimulatory signal for successful antigen-driven activations of T lymphocyte. Most of biochemical and cell biological characteristics of the CD152 protein remain unknown while those of CD28 have been characterized in detail. Methods: In this study CD152 expression in both $CD4^+$ and $CD8^+$ PBLs was studied by using flow cytometry. And intracellular CD152 multiprotein complex was purified and used for generating antibodies recognizing proteins composing of intracellular CTLA-4 multi protein complex. Results: Level of surface expression of this molecule was peaked at 2 days of PHA stimulation in flow cytometric analysis. 40~45% of PHA blast cells were $CD152^+$ in both of two subsets at this stage and the level of expression were equivalent in both two subsets. Contrary to this surface expression, intracellular expression was peaked at day 3 and it was preferentially induced in $CD8^+$ cells and about 60% of $CD8^+$ cells were $CD152^+$ at this stage. High molecular weight (>350 kD) intacellular CD152 protein complex purified by using preparative electrophoresis were immunized into rabbits and then 3 different anti-P34PC4, anti-P34PC7 and anti-P34PC8 antibodies were obtained. Using these 3 antibodies two unknown antigens associated with intracellular CD152 multiprotein complex were found and their molecular weights were 54 kD and 75 kD, respectively. Among these, the former was present as 110 kD homodimer in non-reducing condition. Conclusion: It seemed that 34 kD intracellular CD152 molecule forms high molecular weight multiprotein complex at least with 2 proteins of 75 kD monomer and 110 kD homodimer.

• Biomolecules & Therapeutics
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• 제29권5호
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• pp.527-535
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• 2021

Sclerostin (SOST), a regulator of bone formation in osteocytes, inhibits the canonical Wnt signaling by interacting with low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to prevent Wnt binding. Loss-of-function mutations of the SOST gene caused massive bone outgrowth and SOST-null mouse exhibited a high bone density phenotype. Therefore, SOST has been suggested as a promising therapeutic target for osteoporosis. A few previous studies with X-ray crystallography identified the binding interfaces between LRP6 and SOST, but there are limitations in these studies as they used truncated SOST protein or SOST peptide. Here, we analyzed the conformational dynamics of SOST-LRP6 E1E2 complex using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We examined the effect of the C-terminal tail of SOST on LRP6 conformation upon complex formation. HDX-MS analysis suggested a new potential binding interface for the C-terminal region of SOST that was missing from the previous crystal structure of the SOST-LRP6 E1E2 complex.

• 한국동물학회지
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• 제39권4호
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• pp.393-399
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• 1996

전사개시전 복합체(preinitiation complex)에서 단백질간의 상호작용을 연구하시 위해 zero-Iength croessinking방법을 이용하였다. DNA template가 결합한 금속 지지체를 이용하여 in vitro에서 전사개시전 복합체를 형성시키고, 이렇게 만든 복합체를 1-ethyl-3-(3-dimethylaminopropyl) carbodlimide(EDC)로 croessinking시켰다. $\beta$-mercaptoethanol를 첨가하여 croessinking반응을 멈추게 한 다음, EDC로부터 전사개시전 복합체를 분리하였다. TBF,TFIIB,GAL4-AH등으로 구성된 전사개시전 복합체에 이러한 방법을 적용함으로써 TBF가 GAL4-AH,TFIIB와 각각 직접적으로 결합하고 있음을 규명하였다. 반면에 GAL4-AH와 TFIIB가 croessinking된 산물은 확인할 수 없었다. 이러한 결과들은 GAL4-AH,TFIIB,TBP,DNA로 구성된 전사개시전 복합체에서 GAL4-AH는 TFIIB와 안정적인 결합을 하고 있지 않음을 암시한다.

• 정보처리학회논문지:소프트웨어 및 데이터공학
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• 제2권12호
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• pp.899-902
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• 2013

본 논문에서는 두 개의 단백질 분자가 결합하여 복합체를 구성할 때, 두 분자 간의 접촉 영역을 발견하여 3차원 공간에서 가시화하는 방법을 제시한다. 두 분자 간의 접촉 영역은 기하학적인 측면에서 서로 상보성을 보이며, 상보적인 결합의 크기를 나타내기 위한 방법으로 접촉 영역의 면적을 계산하는 방법이 주로 사용되어 왔다. 접촉 영역의 면적을 수치화한 결과와 접촉 영역의 단순 표시는 서로 다른 접촉 영역에 대해 상대적인 결합 강도를 비교하기에는 적합하지만, 접촉 영역이 가진 기하학적인 특성을 분석하기에는 부적합하다. 본 논문에서는 접촉 영역에서 상보성을 표시하기 위해 상대 분자와의 거리 정보를 가시화하는 방법을 제시한다.

• Journal of the Korean Society for Industrial and Applied Mathematics
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• 제17권2호
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• pp.87-102
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• 2013

An n-string tangle is a three dimensional ball with n-strings which are properly embedded in the ball. In early 90's, C. Ernst and D. Sumners first used a tangle to describe a DNA-protein complex. In this model, DNA is represented by a string and protein is represented by a ball. Mu is a protein which binds to DNA at three sites and a DNA-Mu complex is called Mu-transpososome. Knowing the DNA topology within Mu-transpososome is very important to understand DNA transposition by Mu protein. In 2002, Pathania et al. determined that the DNA configuration within the Mu transpososome is three branched and five noded [12]. In 2007, Darcy et al. analyzed this by using mathematical tangle and concluded that the three branched and five noded DNA configuration is the only biologically reasonable solution [4]. In this paper, based on the result of Pathania et al. and Darcy et al., the author determines the DNA topology within the DNA-Mu complex after the whole Mu transposition process. Furthermore, a new experiment is designed which can support the Pathania et al.'s result. The result of this new experiment is predicted through mathematical knot thory.

• BMB Reports
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• 제56권11호
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• pp.618-623
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• 2023

Most cancer cells utilize glucose at a high rate to produce energy and precursors for the biosynthesis of macromolecules such as lipids, proteins, and nucleic acids. This phenomenon is called the Warburg effect or aerobic glycolysis- this distinct characteristic is an attractive target for developing anticancer drugs. Here, we found that Phosphofructokinase-1 (PFK-1) is a substrate of the Protein Phosphatase 4 catalytic subunit (PP4C)/PP4 regulatory subunit 1 (PP4R1) complex by using immunoprecipitation and in vitro assay. While manipulation of PP4C/PP4R1 does not have a critical impact on PFK-1 expression, the absence of the PP4C/PP4R1 complex increases PFK-1 activity. Although PP4C depletion or overexpression does not cause a dramatic change in the overall glycolytic rate, PP4R1 depletion induces a considerable increase in both basal and compensatory glycolytic rates, as well as the oxygen consumption rate, indicating oxidative phosphorylation. Collectively, the PP4C/PP4R1 complex regulates PFK-1 activity by reversing its phosphorylation and is a promising candidate for treating glycolytic disorders and cancers. Targeting PP4R1 could be a more efficient and safer strategy to avoid pleiotropic effects than targeting PP4C directly.

• Molecules and Cells
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• 제27권6호
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• pp.629-634
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• 2009

G protein-coupled receptors (GPCRs) are part of multi-protein networks called 'receptosomes'. These GPCR interacting proteins (GIPs) in the receptosomes control the targeting, trafficking and signaling of GPCRs. PDZ domain proteins constitute the largest protein family among the GIPs, and the predominant function of the PDZ domain proteins is to assemble signaling pathway components into close proximity by recognition of the last four C-terminal amino acids of GPCRs. We present here a machine learning based approach for the identification of GPCR-binding PDZ domain proteins. In order to characterize the network of interactions between amino acid residues that contribute to the stability of the PDZ domain-ligand complex and to encode the complex into a feature vector, amino acid contact matrices and physicochemical distance matrix were constructed and adopted. This novel machine learning based method displayed high performance for the identification of PDZ domain-ligand interactions and allowed the identification of novel GPCR-PDZ domain protein interactions.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.451-455
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• 1996

Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis (Btt) gene coding for insecticidal protein in recombinant Escherichia coli 537 were carried out to identify optimal culture condition. It was necessary to shift culture temperature from 30 to $42^{\circ}C$ to express the gene. Expression of the Btt toxin gene by recombinant E. coli 537 began within one hour after induction. Complex nitrogen sources increased production of the insecticidal protein. The total insecticidal protein was 0.5 g/I when using yeast extract as a complex nitrogen source. Soybean hydrolysate showed apparently the highest induction efficiency. After induction, the cellular content of the insecticidal protein was 5.4 times higher than it had been before induction. The optimal cultivation strategy was found to grow cells for 7hours at $30^{\circ}C$ and then 5-8 hours at $42^{\circ}C$. The optimal cultivation pH for the production of insecticidal protein was 6.5. The Btt toxin produced by the recombinant E. coli 537 was found to have the same level of potency against Colorado potato beetle as the original toxin.

• 대한방사성의약품학회지
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• 제5권2호
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• pp.83-88
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• 2019

In order to understand the in vivo biodistribution of repebody protein (RB), an efficient and simple radiolabeling method for the protein is needed. We demonstrate a detailed protocol for the radiosynthesis of an ¹¹¹In radiolabeled tetrazine prosthetic group and its application to the efficient radiolabeling of trans-cyclooctene-group conjugated repebody protein using inverse-electron-demand Diels-Alder reaction. First, 1,2,4,5-tetrazine (Tz) conjugated with a DOTA chelator, was used for preparing the radiolabeled DOTA complex with ¹¹¹In. Second, the trans-cyclooctene (TCO) functionalized repebody protein was synthesized which allows for the preparation of radiolabeled proteins by copper-free click chemistry. Following incubation with the ¹¹¹In-radiolabeled DOTA complex (¹¹¹In-Tz), the TCO-functionalized RB (TCO-RB) was radiolabeled successfully with ¹¹¹In, with a high radiochemical yield (69.5%) and radiochemical purity (>99%). The radiolabeling of repebody protein by copper-free click chemistry was accomplished within 20 min, with great efficiency in aqueous conditions. These results clearly indicate that the present radiolabeling method will be useful for the efficient and convenient radiolabeling of trans-cyclooctene-group containing biomolecules.