• Molecules and Cells
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• 제24권3호
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• pp.372-377
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• 2007

The orphan nuclear receptor, SF-1, plays a pivotal role in the development and differentiation of the endocrine and reproductive systems, and also regulates the transcription of a host of genes, including those encoding several steroidogenic enzymes and gonadotropins. We found that a previously unidentified repressor, EID-1, is an SF-1-interacting protein that inhibits the transactivation of SF-1. A transient transfection assay revealed that EID-1 inhibits SF-1, but not LRH-1, $ERR{\gamma}$, or mCAR. Using the yeast two hybrid and GST pull-down assays, we determined that EID-1 interacted strongly with SF-1. In addition, it colocalized with SF-1 in mammalian cells and interacted specifically with the AF-2 domain of SF-1, competing with SRC-1 to inhibit SF-1 transactivation. EID-1 is expressed in the mouse testis, and its expression decreases during testis development. The results of the present study suggest that EID-1 can act as a repressor, regulating the function of SF-1.

• IMMUNE NETWORK
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• 제2권3호
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• pp.150-157
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• 2002

Background: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. Methods: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. Results: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. Conclusion: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.

• Journal of Nutrition and Health
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• 제34권8호
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• pp.905-911
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• 2001

Phytoestrogens, especially soy-derived isoflavones, are receiving great scrutiny as a food supplement for preventing hormone dependent diseases such as cardiovascular diseases, cancer, and osteoporosis. These beneficial effects of phytoestrogens are caused by functioning as partial agonists or antagonists of estrogens. In contrast to the common usage of soy bean, Yak-kong(Rhynchosia Molubilis ; ) has been used as supplements of estrogen fir preventing postmenopausal osteoporosis in Oriental medicine. To investigate estrogenic effects of Yak-kong and soy bean on the proliferation of MG-63 osteoblastic cells, each bean was extracted with 70% methanol and dried by freeze-drying. Yak-kong treatment of MG-63 cells resulted in an increase of cell proliferation to a maximum of 76% compared to 68% of soy bean treatment. Treatment of MG-63 cells with Yak-kong extract also resulted in an increase of transactivation of an ERE(estrogen response element)-luciferase reporter plasmid and IGF-I expression selectively. Despite increased effects of both bean treatments on the expression of estrogen receptor $\alpha$(ER$\alpha$) and $\beta$(ER$\beta$), soy bean treatment decreased transactivation of an ERE-luciferase reporter plasmid and did not further enhance IGF-I expression. Together, our data demonstrates that the greater estrogenic response of Yak-kong extract for MG-63 cell proliferation is mediated by ER derived transactivation of ERE and selective induction of IGF-I expression.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2008년도 Proceedings of the Convention
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• pp.55-74
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• 2008

Previously, we have shown that hypoxia, through HIF-1, induces ligand-independent $ER{\alpha}$ activation and the physical interaction of HIF-1 and $ER{\alpha}$. However, the effect of hypoxia on the transactivation of $ER{\beta}$ is not yet known. In the present study, we found that hypoxia activated the $ER{\beta}$-mediated transcriptional response in the HEK 293 cell line, as determined by the transient expression of$ER{\beta}$ and ER-responsive reporter plasmids. The hypoxia-induced estrogen response element-mediated transcriptional response was dependent on $ER{\beta}$ expression and was inhibited by the ER antagonist ICI 182,780. Transactivation of $ER{\beta}$ was induced by the expression of HIF-$1{\alpha}$ under normoxic conditions, as determined by the expression of oxygen-independent stable GFP-HIF-$1{\alpha}$. HIF-$1{\alpha}$-induced $ER{\beta}$ transactivation was abolished by the inhibition of HIF-$1{\alpha}$ activation. This was determined by using chemical inhibitors for the MAPK pathway. In addition, HIF-$1{\alpha}$ interacted with $ER{\beta}$ in a mammalian-two hybrid assay. We conclude that hypoxia activates $ER{\beta}$ in a ligand-independent manner, possibly through the interaction of HIF-$1{\alpha}$ and $ER{\beta}$.

• BMB Reports
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• 제34권1호
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• pp.73-79
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• 2001

Transcriptional activation domains are known to be inherently "unstructured" with no tertiary structure. A recent NMR study, however, has shown that the transactivation domain in human p53 is populated with an amphipathic helix and two nascent turns. This suggests that the presence of such local secondary structures within the overall "unstructured" structural framework is a general feature of acidic transactivation domains. These pre-existing local structures in p53, formed selectively by positional conserved hydrophobic residues that are known to be critical for transcriptional activity, thus appear to constitute the specific structural motifs that regulate recognition of the p53 transactivation domain by target proteins. Here, we report the results of a NMR structural comparison between the native human p53 transactivation domain and an inactive mutant (22L,23W$\rightarrow$22R,23S). Results show that the mutant has an identical overall structural topology as the native protein, to the extent that the amphipathic helix formed by the residues 18T 26L within the native p53 transactivating domain is preserved in the double mutant. Therefore, the lack of transcriptional activity in the double mutant should be ascribed to the disruption of the essential hydrophobic contacts between the p53 transactivation domain and target proteins due to the (22L,23W$\rightarrow$22R,23S) mutation.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.265-265
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• 1994

본 연구는 간염 바이러스의 X 및 elongated X 유전자를 클로닝하여 E. coli에서 대량 발현시진 후, 그 기능을 여러 측면에서 연구하교 지금까지 알려진 oncogene products, tumor suppressor, 그리고 그 밖의 다른 암 유발인자와의 interaction에 대해 분석함으로써 간암 생성의 분자적 기작을 이해하고 더 나아가 간암의 예방 및 치료제의 개발을 목표로 하였다. 그 일차적 연구로서 이전에 플로닝된 mutant hepatitis Bvirus genome으로부터 X 및 elongated X 유전자를 클로닝하였으며, E. coli에서 대량 발현시키기 위하여 T7 bacteriophage promoter아래에 재 클로닝하였다. 이러한 X 및 ebngated X 유전자를 E. coli에서 대량 발현시킨 후, rabbit anti-X antibody를 이용하여 western blotting을 수행함으로서 이를 확인하였으며 DEAE-cellulose와 heparin-agarose chromategraphy를 이용하여 순수분리하였다. 순수분리된 X 및 etongated X 단백질을 highly differentiated hepatoma cell인 HepG$_2$ cell에 처리하여 transactivation activity를 측정하였다. 그 결과 순수분리된 단백질들이 SV4O promoter를 transactivation 함을 할 수 있었으며, 이로부터 클로닝된 유전자들이 모두 정상적인 기능을 가짐을 확인하였다. 그러고 X 유전자의 작용기작을 규명하기위하여 restriction endonuclease를 이용하여 5 개의 mutant X 유전자를 구성하였으며 현재 이를 HepG2 cell에 transfection 하여 그 기능을 연구하고 있다.

• BMB Reports
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• 제47권11호
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• pp.643-648
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• 2014

Stanniocalcin (STC), a glycoprotein hormone originally discovered in fish, has been implicated in calcium and phosphate homeostasis. While fishes and mammals possess two STC homologs (STC1 and STC2), the physiological roles of STC2 are largely unknown compared with those of STC1. In this study, we identified Ran-binding protein M (RanBPM) as a novel binding partner of STC2 using yeast two-hybrid screening. The interaction between STC2 and RanBPM was confirmed in mammalian cells by immunoprecipitation. STC2 enhanced the RanBPM-mediated transactivation of liganded androgen receptor (AR), but not thyroid receptor ${\beta}$, glucocorticoid receptor, or estrogen receptor ${\beta}$. We also found that AR interacted with RanBPM in both the absence and presence of testosterone (T). Furthermore, we discovered that STC2 recruits RanBPM/AR complex in T-dependent manner. Taken together, our findings suggest that STC2 is a novel RanBPM-interacting protein that promotes AR transactivation.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.85.3-86
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• 2003

Oltipraz, a cancer chemopreventive agent, induces CYP1A1 to a certain extent by transactivation of the gene via the Ah receptor (AhR)-xenobiotic response element (XRE) pathway. Previously, we showed that oltipraz promoted CCAAT/enhancer binding protein (C/EBP ) activation, which leads to the induction of glutathione S-transferase. Given that oltipraz activates C/EBP for gene transactivation and that the putative C/CBP binding site is located in CY)1A1 promoter region, this study investigated the effect of oltipraz on CYP1A1 induction by 3-methylcholanthrene (3-MC). (omitted)

• BMB Reports
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• 제49권9호
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• pp.497-501
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• 2016

Wide-line ¹H NMR intensity and differential scanning calorimetry measurements were carried out on the intrinsically disordered 73-residue full transactivation domain (TAD) of the p53 tumor suppressor protein and two peptides: one a wild type p53 TAD peptide with a helix pre-structuring property, and a mutant peptide with a disabled helix-forming propensity. Measurements were carried out in order to characterize their water and ion binding characteristics. By quantifying the number of hydrate water molecules, we provide a microscopic description for the interactions of water with a wild-type p53 TAD and two p53 TAD peptides. The results provide direct evidence that intrinsically disordered proteins (IDPs) and a less structured peptide not only have a higher hydration capacity than globular proteins, but are also able to bind a larger amount of charged solute ions.

• Molecules and Cells
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• 제20권2호
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• pp.263-270
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• 2005

Transactivation of EGF-receptor (EGFR) by G-protein coupled receptors (GPCRs) is emerging as an important pathway in cell proliferation, which plays a crucial role in the development of atherosclerotic lesion. Angiotensin II (Ang II) has been identified to have a major role in the formation of atherosclerotic lesions, although the underlying mechanisms remain largely unclear. We hypothesize that Ang II promotes the proliferation and migration of smooth muscle cells through the release of heparin-binding epidermal growth factor like growth factor (HB-EGF), transactivation of EGFR and activation of Akt and Erk 1/2, with matrix metalloproteases (MMPs) playing a dispensable role. Primary rat aortic smooth muscle cells were used in this study. Smooth muscle cells rendered quiescent by serum deprivation for 12 h were treated with Ang II (100 nM) in the presence of either GM6001 ($20{\mu}M$), a specific inhibitor of MMPs or AG1478 ($10{\mu}M$), an inhibitor of EGFR. The levels of phosphorylation of EGFR, Akt and Erk 1/2 were assessed in the cell lysates. Inhibition of MMPs by GM6001 significantly attenuated Ang II-stimulated phosphorylation of EGFR, suggesting that MMPs may be involved in the transactivation of EGFR by Ang II receptor. Furthermore Ang II-stimulated proliferation and migration of smooth muscle cells were significantly blunted by inhibiting MMPs and EGFR and applying HB-EGF neutralization antibody, indicating that MMPs, HB-EGF and EGFR activation is necessary for Ang-II stimulated migration and proliferation of smooth muscle cells. Our results suggest that inhibition of MMPs may represent one of the strategies to counter the mitogenic and motogenic effects of Ang II on smooth muscle cells and thereby prevent the formation and development of atherosclerotic lesions.