Structural studies of stress-responsive sensor protein kinases
| Project/Area Number |
15370046
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | NARA INSTITUTE OF SCIENCE AND TECHNOLOGY |
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Principal Investigator |
HAKOSHIMA Toshio Nara Institute of Science and Technology, Graduate School of Information Science, Professor, 情報科学研究科, 教授 (00164773)
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| Co-Investigator(Kenkyū-buntansha) |
KITANO Ken Nara Institute of Science and Technology, Graduate School of Information Science, Assistant Professor, 情報科学研究科, 助手 (40346309)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 2005: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2003: ¥8,700,000 (Direct Cost: ¥8,700,000)
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| Keywords | stress / protein / separation / purification / IRE / TRAF / IRF |
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| Research Abstract |
The accumulation of unfolded proteins in the lumen of the endoplasmic reticulum induces an unfolded protein response. The response alleviates stress by upregulating protein folding and degradation pathways in the endoplasmic reticulum and inhibiting protein synthesis. This response is mediated by a novel membrane-integrate protein serine/threonine kinase, IRE. IRE consists of a sensor domain in the lumen, a transmembrane helix region, and a cytoplasmic domain. The cytoplasmic domain has a linker at the juxtamembrane region, which is followed by protein kinase domain and nuclease domain. Interestingly, the nuclease activity was regulated by the kinase that is activated by the oligomerization of the protein through the sensor domain in the lumen. This nuclease domain resembles to that of RNase L, which is known to be a component of the interferon response pathway, and, again, the organization of the kinase-nuclease in one polypeptide chain is also common in these proteins. In this study, we tried to establish the protein preparation of IREs, human IREα,IREβ and yeast Ire1p, for the three-dimensional structural studies. We produced several kinds of constructs for protein expression in both E.coli cells and insect cells and tried to purify the expressed proteins. We found high protein expression and relatively efficient purification procedure of a protein using a construct of the cytoplasmic domain from Ire1p. We also found that constructs of the sensor domain of Ire1p provide proteins that can be efficiently purified. These advances in protein preparation enable us to prepare protein crystals for X-ray diffraction studies or to measure NMR spectra for structural analyses and determination.
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Report
(4 results)
Research Products
(34 results)
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[Journal Article] Crystal structure of the protein histidine phosphatase SixA in the multistep His-Asp phosphorelay.2005
Author(s)
Hamada, K., Kato, M., Shimizu, T., Ihara, K., Mizuno, T., Hakoshima, T.
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Journal Title
Genes to Cells 10(1)
Pages: 1-11
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Structural basis of recruitment of human flap endonuclease 1 to PCNA.2005
Author(s)
Sakurai, S., Kitano, K., Yamaguchi, H., Hamada, K., Okada, K., Fukuda, K., Uchida, M., Ohtsuka, E., Morioka, H., Hakoshima, T.
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Journal Title
EMBO J. 24(4)
Pages: 683-693
Description
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[Journal Article] Preparation and crystallization of human flap endonuclease FEN-1 in complex with proliferating-cell nuclear antigen, PCNA.2003
Author(s)
Sakurai, S., Kitano, K., Okada, K., Hamada, K., Morioka, H., Hakoshima, T.
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Journal Title
Acta Crystallogr. D59(5)
Pages: 933-935
Description
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[Journal Article] Leucine zippers.2003
Author(s)
Hakoshima, T.
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Journal Title
Nature Encyclopedia of the Human Genome (Nature Pub.Group) Vol.3
Pages: 679-683
Description
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[Journal Article] Parallel coiled-coil association of the RhoA-binding domain in Rho-kinase.2003
Author(s)
Shimizu, T., Ihara, K., Maesaki, R., Amano, M., Kaibuchi, K., Hakoshima, T.
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Journal Title
J.Biol.Chem. 278(46)
Pages: 46046-46051
Description
「研究成果報告書概要(欧文)」より
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