| Project/Area Number |
12558078
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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 | 展開研究 |
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| Research Field |
Structural biochemistry
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| Research Institution | OSAKA BIOSCIENCE INSTITUTE |
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Principal Investigator |
URADE Yoshihiro Osaka Bioscience Inst.2ND dep., HEAD, 第2研究部, 研究部長 (10201360)
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| Co-Investigator(Kenkyū-buntansha) |
INOUE Thutoshi Osaka University. dept. of Appl. Chem., Instructor, 大学院・工学研究科, 講師 (20263204)
EGUCHI Naomi Osaka Biosci. Inst., 2nd. dept., vice Head, 第2研究部, 副部長 (10250086)
HAYAISHI Osamu Osaka Biosci. Inst., 2nd. dept., Research Associate, 第2研究部, 研究員 (40025507)
KAI Yasushi Osaka University. dept. of Appl. Chem., Instructor, Professor, 大学院・工学研究科, 教授 (40029236)
望月 貴年 (財)大阪バイオサイエンス研究所, 第2研究部, 研究員 (40263933)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥13,700,000 (Direct Cost: ¥13,700,000)
Fiscal Year 2001: ¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 2000: ¥8,900,000 (Direct Cost: ¥8,900,000)
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| Keywords | prostaglandin D2 / transgenic mouse / eosinophil / Th2 cytokine / X-ray'crystallography / enzyme inhibitor / allergy / sleep / 肥満細胞 |
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| Research Abstract |
Prostaglandin (PG) D_2 is actively produced in the central nervous system and exhibits a variety of central actions such as sleep induction and pain modulation. POD2 is also produced by mast cells and Th2 cells and is released as an inflammatory and allergic mediator. There are two distinct types of POD synthase (PGDS) : one is lipocalin-type PODS (L-PGDS) localized in the central nervous system and the other is hematopoietic POD synthase (H-PODS) expressed in those allergy-related cells. These two enzymes catalyze the same reaction from POH_2 to POD2 but possess no homology at all in their amno acid sequences. Recombinant mouse L-PGDS and human H-PGDS were cocrystallized with several inhibitors selective to each PODS. We obtained the X-ray diffraction data of these crystals at KEK and Spring-8 synchrotron facilities. The 3-D coordinates of L-PODS complexed with its inhibitor AT-056 were determined at a resolution of 2.5 Å. We also determined the 3-D coordinates of H-PODS complexed wit
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h Mg^<2+> or Ca^<2+> and 4 different inhibitors, including an orally effective inhibitor HQL-79, at 1.2 to 1.8 Å resolution. As predicted, the tertiary structures of two enzymes are quite different from each other L-PGDS possesses β-barrell structure with hydrophobic pocket and H-PGDS, α+β structure with a catalytic creft containing the glutathione-binding site within 2 subdomains. Based on the crystallographic structures, we introduced the site-directed mutagenesis to identify the amino acid residues involved in the catalytic reactions by L-PODS and H-PODS. We then surveyed the functional abnormality of trarisgenic (TG) mice that over-expressed human L-PODS or H-PODS under the control of the β-actin promoter. We found that L-PGDS-TG mice showed a striking time-dependent increase in non-rapid eye movement sleep afler stimulation by tail clipping. The sleep induction in TO mice was positively correlated with the POD2 production in the brain. In an ovalbumin-induced asthma model, L-PODS- or H-PGDS-TG mice showed a pronounced infiltration of eosinophils and lymphocytes into the lung and upregulation of Th2 cytokines in the bronchoalveolar lavage fluid. These TO mice are useful to monitor the in vivo efficacy of the lead compounds to develop the PGDS inhibitors act as anti-doze drugs or anti-allergy drugs. Less
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