| Project/Area Number |
13142209
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Tokai University |
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Principal Investigator |
NAKAE Taiji Tokai University, Institute of Medical Science, Professor, 総合医学研究所, 教授 (50102851)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIHARA Eisaku Tokai University, Molecular Life Science, Associate Professor, 総合医学研究所, 助教授 (70167063)
NARITA Shin-ichiro Tokai university, molecular life science, Instructor, 総合医学研究所, 助手 (30338751)
TANI Ayako Tokai University, Molecular Life Science, Instructor, 総合医学研究所, 助手 (70284867)
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| Project Period (FY) |
2001 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥85,000,000 (Direct Cost: ¥85,000,000)
Fiscal Year 2005: ¥16,200,000 (Direct Cost: ¥16,200,000)
Fiscal Year 2004: ¥16,800,000 (Direct Cost: ¥16,800,000)
Fiscal Year 2003: ¥17,000,000 (Direct Cost: ¥17,000,000)
Fiscal Year 2002: ¥17,000,000 (Direct Cost: ¥17,000,000)
Fiscal Year 2001: ¥18,000,000 (Direct Cost: ¥18,000,000)
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| Keywords | drug resistance / efflux pump / transporter / xenobiotic antibiotic / bacteria / Psudomonas / membrane protein / cell membrane / 抗生物質 / 排出 / 結晶構造 / X線解析 / 異物 / 薬物 / ポンプ / 異物排出 / RND / エネルギー / 膜貫通領域 / MexB / 変異体 |
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| Research Abstract |
Pseudomonas aeruginosa expresses several tripartite xenobiotic-antibiotic exporters that confer the multidrug resistance to the cells. To elucidate molecular and atomic mechanism of this important transporter function, we did the following studies. (i) Crystal structure of MexAB-OprM pump subunits : Structure of MexA revealed a long sickle shape protein monomer that consisted of 4 main domains. The distal a-domain and proximal disordered domains were assigned to interact with OprM and MexB, respectively. Structure of MexA was registered as a totally new protein structure. Thus, the protein was designated as the molecular clump. The OprM structure revealed that the trimeric assembly of this subunit formed the transmembrane 13-barrel domain and the long a-helical periplasmic cavity domain. Thus, it was assumed that the antibiotics must be discharged through this OprM cavity. However, the size of the cavity end at periplasmic a-barrel was totally occluded and that of outer membrane 13-bar
… More
rel was very small that is difficult to accommodate the passage of antibiotics. It was assumed that external energy has to be exerted to open these OprM ends, probably through MexB and MexA. (ii) The substrate-recognizing domain of MexB transporteer was determined by constructing the MexB-MexY hybrid protein. The result revealed that the hybrid protein consisting of transmembrane domain of MexY and periplasmic domain of MexB showed the MexB type substrate selectivity. Therefore, it was concluded that the transporters recognize their substrate by the extramembrane periplasmic domain. This result established a new concept of xenobiotic export that the xenobiotic substrates were trapped at the periplasmic space before reaching to the cytoplasmic membrane or cytoplasm and efficiently discharged to the external medium. This mechanism of xenobiotic export sounds reasonable in terms for selfprotection. Mechanism of the transporter protein expression was studied by the experiment measuring the direct binding of the repressor protein and the operator-promoter DNA. MexR and MexZ repressor interacted with the respective operator-promoter DNA and the binding site was specified. This study revealed the mechanism of the xenobiotic transporter expression. (iii) Assembly mechanism of the MexAB-OprM transporter was studied by tagging in only one of the subunit and by the co-purification of untagged subunits. It was revealed that MexA subunit plays a central role in the assembly of the transporter complex. This conclusion sounds plausible because the MexA subunit seems to be the molecular damp as revealed by the xTay crystallographic study of MexA mentioned above. (iv)To yield large amount of transporter protein amenable for aystallization experiment, high expression vectors were constructed. The yield of MexB and MexY proteins became 50 to 100 times higher than that from the wild-type cells. Less
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