Elucidation of the role of claudin on the tight junction mechanism

• Project/Area Number: 25840028
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Shinoda Takehiro 国立研究開発法人理化学研究所, ライフサイエンス技術基盤研究センター, 研究員 (10597868)
• Project Period (FY): 2013-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2016: ¥520,000 (Direct Cost: ¥400,000、Indirect Cost: ¥120,000); Fiscal Year 2015: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2014: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2013: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 密着結合 / 大腸菌無細胞タンパク質合成技術 / 膜タンパク質 / X線結晶構造解析 / モノクローナル抗体 / 立体構造解析 / キメラタンパク質 / 単分散性

Outline of Final Research Achievements

In this study, we determined the crystal structure of the complex of human claudin-4 which is a main component of tight junction strand of the intestinal epithelial barriers with the C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE) at 3.5-angstrom resolution.
The complex structure of claudin-4 and C-CPE revealed the details about the interaction between them and the differences with the off-target claudin. Moreover, a comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced upon the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules.

Research Products

• [Journal Article] Structural basis for disruption of claudin assembly in tight junctions by enterotoxin. (2016)
  • Author(s): Shinoda, T., Shinya, N., Ito, K., Ohsawa, N., Terada, T., Hirata, K., Kawano, Y., Yamamoto, M., Kimura-Someya, T., Yokoyama, S., Shirouzu, M.
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 1-10
  • DOI: 10.1038/srep33632
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] A systematic cell-free methodology to produce mammalian membrane proteins for crystallography. (2016)
  • Author(s): Shinoda T, Shinya N, Ito K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Hirata K, Kawano Y, Yamamoto M, Tomita T, Ishibashi Y, Hirabayashi Y, Tomomi Kimura-Someya T, Shirouzu M, Yokoyama S
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 30442-30442
  • DOI: 10.1038/srep30442
  • Peer Reviewed / Open Access
• [Presentation] 密着結合タンパク質ヒトクローディン-4とウェルシュ菌毒素との結合様式の構造基盤およびヒトクローディン-5結合型C-CPE変異体の開発 (2015)
  • Author(s): 篠田雄大, 新屋直子, 伊東夏織, 大沢登, 寺田貴帆, 木村（染谷）友美, 横山茂之, 白水美香子
  • Organizer: 第38回日本分子生物学会年会 第88回日本生化学会大会 合同大会
  • Place of Presentation: 神戸ポートアイランド、兵庫県
  • Year and Date: 2015-12-01
• [Presentation] Preparation and Crystal Structure Determination of Eukaryotic Membrane Proteins (2015)
  • Author(s): H. Tanabe, T. Shinoda, S. Yokoyama, 他
  • Organizer: International Conference on Structural Genomics
  • Place of Presentation: Weizmann institute of science, Israel
  • Year and Date: 2015-06-07
  • Int'l Joint Research
• [Presentation] 無細胞タンパク質合成技術を利用した結晶構造解析用ヒト膜タンパク質生産の体系的手法 (2014)
  • Author(s): 篠田雄大, 新屋直子, 伊東夏織, 桂（石塚）芳子, 大沢登, 寺田貴帆, 平田邦生, 河野能顕, 山本雅貴, 富田泰輔, 石橋洋平, 平林義雄, 染谷友美, 白水美香子, 横山茂之
  • Organizer: 日本結晶学会平成26年度年会
  • Place of Presentation: 東京大学農学部
  • Year and Date: 2014-11-01
• [Presentation] ウェルシュ菌内毒素Clostridium perfringens enterotoxinによる、密着結合内クローディンアッセンブリーの破壊機構 (2014)
  • Author(s): 篠田雄大, 新屋直子, 伊東夏織, 大沢登, 寺田貴帆, 平田邦生, 河野能顕, 山本雅貴, 木村（染谷）友美, 横山茂之, 白水美香子
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 国立京都国際会館
  • Year and Date: 2014-10-17
• [Remarks] ウェルシュ菌毒素により引き起こされる密着結合破壊メカニズムの解明
  • URL: http://www.clst.riken.jp/ja/topics/research/161006news/
• [Remarks] 細胞を使わない膜タンパク質の合成技術 －ヒトの膜タンパク質などを標的とした新薬の創出が加速－
  • URL: http://www.riken.jp/pr/press/2016/20160801_1/
• [Patent(Industrial Property Rights)] 膜タンパク質の製造方法およびその利用 (2016)
  • Inventor(s): 横山茂之、篠田雄大、伊東夏織、白水美香子、染谷友美、その他5名
  • Industrial Property Rights Holder: 理化学研究所
  • Industrial Property Rights Type: 特許
  • Filing Date: 2016-07-29
  • Overseas