• Title/Summary/Keyword: homodimer

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Ligand and Dimerization Dependent Transactivation Capability of Aromatic Hydrocarbon Receptor

  • Park, Hyun-Sung
    • BMB Reports
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    • v.32 no.3
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    • pp.279-287
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    • 1999
  • The aromatic hydrocarbon receptor (AhR) is a cytosolic protein that binds the environmental pollutant, dioxin. The liganded AhR translocates into the nucleus where it heterimerizes with a constitutive nuclear protein, AhR nuclear translocator (Arnt). The N-terminal regions of both AhR and Arnt contain basic helix-loop-helix (bHLH) and Per-AhR-Arnt-Sim (PAS) motifs that are required for DNA binding, dimerization, and ligand binding whereas the C-terminal regions of both AhR and Arnt contain transactivation domains. Here, results from the mammalian two-hybrid system indicate that Arnt can make a homodimer but AhR cannot. In the presence of dioxin, the interaction between AhR and Arnt is stronger than that of the Arnt homodimer, suggesting that Arnt prefers to make a heterodimer with the liganded AhR rather than a homodimer. Transfection analyses using the GAL4-driven reporter system suggest that AhR's N-terminal region represses its own transactivation domain, as well as exogenous transactivation domains such as Sp 1 and VP16. Interestingly, the repressed transactivation domains of AhR are activated by ligand-dependent heterodimerization with Arnt. These observations suggest that heterodimerzation with Arnt is necessary not only for DNA binding but also for activation of the repressed transactivation capability of AhR.

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The treatment effect of novel hGHRH homodimer to male infertility hamster

  • Zhang, Xu-Dong;Guo, Xiao-Yuan;Tang, Jing-Xuan;Yue, Lin-Na;Zhang, Juan-Hui;Liu, Tao;Dong, Yu-Xia;Tang, Song-Shan
    • The Korean Journal of Physiology and Pharmacology
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    • v.22 no.6
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    • pp.637-647
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    • 2018
  • Extra-hypothalamic growth hormone-releasing hormone (GHRH) plays an important role in reproduction. To study the treatment effect of Grin (a novel hGHRH homodimer), the infertility models of 85 male Chinese hamsters were established by intraperitoneally injecting 20 mg/kg of cyclophosphamide once in a week for 5 weeks and the treatment with Grin or human menopausal gonadotropin (hMG) as positive control was evaluated by performing a 3-week mating experiment. 2-8 mg/kg of Grin and 200 U/kg of hMG showed similar effect and different pathological characteristics. Compared to the single cyclophosphamide group (0%), the pregnancy rates (H-, M-, L-Grin 26.7, 30.8, 31.3%, and hMG 31.3%) showed significant difference, but there was no difference between the hMG and Grin groups. The single cyclophosphamide group presented loose tubules with pathologic vacuoles and significant TUNEL positive cells. Grin induced less weight of body or testis, compactly aligned tubules with little intra-lumens, whereas hMG caused more weight of body or testis, enlarging tubules with annular clearance. Grin presented a dose-dependent manner or cell differentiation-dependentincrease in testicular GHRH receptor, and did not impact the levels of blood and testicular GH, testosterone. Grin promotes fertility by proliferating and differentiating primitive cells through up-regulating testicular GHRH receptor without triggering GH secretion, which might solve the etiology of oligoasthenozoospermia.

AtbZIP16 and AtbZIP68, two new members of GBFs, can interact with other G group bZIPs in Arabidopsis thaliana

  • Shen, Huaishun;Cao, Kaiming;Wang, Xiping
    • BMB Reports
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    • v.41 no.2
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    • pp.132-138
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    • 2008
  • AtbZIP16 and AtbZIP68 are two putative G group bZIP transcription factors in Arabidopsis thaliana, the other three members of G group bZIPs are GBF1-3 which can bind G-box. Members of G group have conservative protein structure: highly homological basic region and a proline-rich domain in the N-terminal region. Here, we report that AtbZIP16 and AtbZIP68 could bind cis elements with ACGT core, such as G-box, Hex, C-box and As-1, but with different binding affinities which from high to low were G-box > Hex > C-box > As-1; AtbZIP16 and AtbZIP68 could form homodimer and form heterodimer with other members of G group; N-terminal proline rich domain of AtbZIP16 had transactivation activity in yeast cells while that of AtbZIP68 did not; AtbZIP16 and AtbZIP68 GFP fusion protein localized in the nucleus of onion epidermal cells. These results indicated that AtbZIP16 and AtbZIP68 were two new members of GBFs. In Arabidopsis, AtbZIP16 and AtbZIP68 may also participate in light-responsive process in which GBF1-3 are involved.

Rice NAC proteins act as homodimers and heterodimers

  • Jeong, Jin Seo;Park, Yeong Taek;Jung, Harin;Park, Su-Hyun;Kim, Ju-Kon
    • Plant Biotechnology Reports
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    • v.3 no.2
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    • pp.127-134
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    • 2009
  • Members of the NAM-ATAF-CUC (NAC) protein family are plant-specific transcription factors that contain a highly conserved N-terminal NAC-domain and diverse C-terminal regions. They have been implicated in plant development and abiotic stress responses. To identify interacters of rice NAC-domain proteins (OsNACs), we performed yeast two-hybrid screening of rice cDNA library using OsNAC5 as a bait, and the results showed that OsNAC5 interacts with other OsNACs including itself. To delineate an interacting domain, a series of deletion constructs of four OsNACs were made and transformed into yeast in various combinations. The results revealed that the conserved NAC domain of OsNACs plays a primary role in homodimer and heterodimer formation, and a part of C-terminal sequence is also necessary for the interaction. In vitro pull-down assays using recombinant OsNAC proteins verified the dimer formations, together suggesting that OsNACs may act by forming homodimers and/or heterodimers in plants.

The Homodimerization of Thalictrum tuberosum O-Methyltransferases by Homology-based Modelling

  • Yang, Hee-Jung;Ahn, Joong-Hoon;Jeong, Karp-Joo;Lee, Sang-San;Lim, Yoong-Ho
    • Bulletin of the Korean Chemical Society
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    • v.24 no.9
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    • pp.1256-1260
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    • 2003
  • Two O-methyltransferases, OMTII-1 and OMTII-4 of meadow rue Thalictrum tuberosum showed a high sequence identity. Of 364 amino acids only one residue is not the same, which is Tyr21 or Cys21. Even if the 21st residues in these OMTs are not included in the binding sites of the enzymes, binding affinities of the enzyme homodimers over the same substrate are very different. While the binding affinity of one homodimer over caffeic acid is 100%, that of the other is 25%. Authors tried to predict the three-dimensional structures of Thalictrum tuberosum O-methyltransferases using homology-based modelling by a comparison with caffeic acid O-methyltransferase, and explain the reason of the phenomenon mentioned above based on their three dimensional structural studies. In the enzyme homodimer, the better binding affinity may be caused by the shorter distance between the 21st residue and the binding site of the other monomer.

Disulfide Bond Bridged Divalent Antibody-Toxin, $(Fab-PE38fl)_2$ with the Toxin PE38 Fused to the Light Chain

  • Won, Jae-Seon;Choe, Mu-Hyeon
    • Journal of Microbiology and Biotechnology
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    • v.18 no.8
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    • pp.1475-1481
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    • 2008
  • B3 antibody specifically binds the $Lewis^Y$-related carbohydrate antigen of many carcinomas, and it is used as a model antibody in this study. In a previous study, the Fab fragment of the antibody was fused to a 38 kDa truncated form of Pseudomonas exotoxin A, PE38, to make Fab-PE38, where PE38 is fused to the Fd fragment of the Fab domain. This parent monomer molecule, Fab-PE38, had no cysteine in the hinge region, and it could not make a disulfide bond to form a disulfide bond bridged homodimer. In this study, we constructed three different kinds of divalent Fab-toxin fusion homodimers where the toxin is fused to the light chain of Fab, $(Fab-PE38fl)_2$. In addition to the PE38 toxin fused to the light chain, these three molecules have different hinge sequences hi, h2, and h3 making Fabh1-, Fabh2-, and Fabh3-PE38fl monomers, respectively. These hinges contain only one cysteine on different positions of the hinge sequence. The disulfide bond between the hinge region of two monomers forms homodimers $(Fabh1-PE38fl)_2$, $(Fabh2-PE38fl)_2$, and $(Fabh3-PE38fl)_2$. The refolding yields of these dimers were 5-16-fold higher than a previously constructed dimer where the PE38 was fused to the Fd fragment $(Fabh2-PE38)_2$ [8]. Our data suggest that the steric repulsion between the two PE38s in $(Fabh1-PE38)_2$ during disulfide bridge formation is relieved by fusing it at the end of the light chain. The best cytotoxicity value of these dimers showed about 2.5-fold higher on an MCF7 cell line than that of the monovalent reference molecule in ng/ml scale, which is 15-fold higher in pM scale.

Expression of Human Serine Palmitoyltransferase Genes for Antibody Development (Antibody 제작을 위한 human serine palmitoyltransferase 유전자의 발현)

  • 김희숙
    • Journal of Life Science
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    • v.14 no.2
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    • pp.315-319
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    • 2004
  • For antibody development of human serine palmitoyltransferase (SPT, EC 2.3.1.50), SPTLC1 and SPTLC2 genes were subcloned in pRset vector and expressed in E. coli BL21 (DE3)pLys cells. Eucaryotic SPT is a membrane-bound heterodimer enzyme, while all other members are soluble homodimer enzymes. cDNA library were obtained from total RNA from human embryo kidney cell line, HEK293, using RT-PCR and PCR with specific primers was carried out for preparing SPTLC1 and SPTLC2 genes. pRset vector which can express hexahistidine-tag fusion protein was used and the DNA sequences of pRsetB/SPTLC1 and pRsetA/SPTLC2 were confirmed. Recombinant BL21 cells with SPTLC subunits were selected with LB plate containing ampicillin and chroramphenicol. SPTLC1 and SPTLC2 proteins were induced with 1 mM IPTG and seperated on 10% SDS-PAGE gel. Expressed proteins were confirmed by western blotting with His-tag antibody.

Characterization of CTLA-4 Antigen Expression: Identification of Molecules Composing Intracellular CTLA-4 Multiprotein Complex (CTLA-4 항원의 활성 T 세포내 발현의 특성: 세포질내 단백복합체 구성분자의 동정)

  • Rhim, Dae-Cheol;Chung, Yong-Hoon
    • IMMUNE NETWORK
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    • v.2 no.1
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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.

Purification and Biochemical Properties of Glutathione S-Transferase from Lactuca sativa

  • Park, Hee-Joong;Cho, Hyun-Young;Kong, Kwang-Hoon
    • BMB Reports
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    • v.38 no.2
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    • pp.232-237
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    • 2005
  • A glutathione S-transferase (GST) from Lactuca sativa was purified to electrophoretic homogeneity approximately 403-fold with a 9.6% activity yield by DEAE-Sephacel and glutathione (GSH)-Sepharose column chromatography. The molecular weight of the enzyme was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure. The activity of the enzyme was significantly inhibited by S-hexylGSH and S-(2,4-dinitrophenyl) glutathione. The enzyme displayed activity towards 1-chloro-2,4-dinitrobenzene, a general GST substrate and high activities towards ethacrynic acid. It also exhibited glutathione peroxidase activity toward cumene hydroperoxide.

Intercellular Adhesion Molecule-1 (ICAM-1)의 분자구조와 기능

  • 전창덕
    • The Zoological Society Korea : Newsletter
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    • v.18 no.2
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    • pp.33-37
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    • 2001
  • Intercellular adhesion molecule-1 (ICAM-1)은 T 세포의 활성이나 면역세포가 염증작용이 일어나고 있는 곳으로 옮겨갈 때 중요한 역할을 수행하는 당단백질 (glycoprotein)으로서 2-integrin 계열의 분자인 leukocyte function associated antigen-1(LFA-1)이나 Mac-1의 수용체이며 세포의 막에 homodimer인 상태로 발현되어진다. ICAM-1은 5개의 세포외 domain과 transmembrane domain(TM) 및 짧은 cytoplasmic domain을 갖고 있으며, dimerization motif는 domain 5 또는 TM인 것으로 알려져 있다. 최근의 crystal 구조분석 결과에 의하면 domain 1에도 dimerization motif가 있는 것으로 추정되어지지만 정확한 실험적인 뒷받침이 되어져 있지 않다. 본 연구에서는domain 1에 dimer를 형성할 수 있는 motif가 존재하는 지에 대한 실험적인 증명과 함께 ICAM-1이 세포의 막에서 어떠한 구조를 갖고 있는지에 대한 최근의 연구성과를 보고하고자 한다.

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