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ヒトカルシトニンのアミロイド線維形成および阻害の分子機構の解明

Publicly Offered Research

Project AreaDynamical ordering of biomolecular systems for creation of integrated functions
Project/Area Number 26102514
Research Category

Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Allocation TypeSingle-year Grants
Review Section Science and Engineering
Research InstitutionYokohama National University

Principal Investigator

内藤 晶  横浜国立大学, 工学(系)研究科(研究院), 名誉教授 (80172245)

Project Period (FY) 2014-04-01 – 2016-03-31
Project Status Completed (Fiscal Year 2015)
Budget Amount *help
¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000)
Fiscal Year 2015: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2014: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Keywordsカルシトニン / アミロイド線維 / 固体NMR / 電子顕微鏡 / 分子動力学 / ヒトカルシトニン / アミロイド線維形成 / REDOR / 球状中間体 / βーシート構造 / 2段階自己触媒反応機構 / 線維形成阻害機構
Outline of Annual Research Achievements

カルシトニンはカルシウム代謝に重要な役割を果たしているペプチドホルモンであり、骨粗鬆症の治療薬としても使用されているが、高濃度中性水溶液中で容易にアミロイド線維を形成して沈殿する性質が知られている。本研究ではヒトカルシトニン(hCT)のアミロイド線維形成過程を解明するため、固体NMR、TEMおよび分子動力学計算の手法を用いて線維形成反応機構の解析、線維の立体構造決定を行い、hCT線維形成の分子機構を明らかにすることを研究の目的とする。
固体NMR信号の測定により、ヒトカルシトニンは2段階自己触媒反応機構でアミロイド線維が形成すること、中央部が線維形成の過程でαーヘリックス構造からβーシート構造に転移することを明らかにした。また、電子顕微鏡でHEPES溶液中での線維形成を観測したところ、線維形成前駆体として球状中間体の存在が明らかになった。さらに、球状中間体から線維が成長する過程を直接観測することに成功した。
次に、13C,15Nで二重標識したヒトカルシトニン線維を用いて、固体NMRにより精密原子間距離測定を行い、これまで明らかになっていない酸性条件で生成するヒトカルシトニンアミロイド線維の精密立体構造決定を行った。この結果、逆平行βーシートと平行βーシートが混在した線維構造をとっていることを示唆する結果が得られた。次に線維形成阻害剤として知られているクルクミンの線維形成阻害機構について固体NMR測定から解析し、核形成段階が阻害されることが判明した。ヒトカルシトニンとクルクミンの系について分子動力学計算を行い、クルクミンとヒトカルシトニンが相互作用を持つことが判明した。この結果は、クルクミンが核形成の段階を阻害する機構であることを指示している。
本研究によりカルシトニンの線維形成機構およびクルクミンによる線維形成阻害の分子機構が明らかになった。

Research Progress Status

27年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

27年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2015 Annual Research Report
  • 2014 Annual Research Report
  • Research Products

    (36 results)

All 2016 2015 2014

All Journal Article (8 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 8 results,  Acknowledgement Compliant: 7 results) Presentation (25 results) (of which Int'l Joint Research: 12 results,  Invited: 6 results) Book (3 results)

  • [Journal Article] Polysilsesquioxanes with mixed self-assembled organic tethers: Alkyl chains and alkanoate-aminopropyl pairs2016

    • Author(s)
      H. Yoshitake, T. Kodate, T.Takagi, I. Kawamura, A. Naito
    • Journal Title

      Reactive and Functional Polymers

      Volume: 99 Pages: 9-16

    • DOI

      10.1016/j.reactfunctpolym.2015.12.002

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Mechanism for microwave heating of 1-(4'-cyanophenyl)-4-propylcyclohexane characterized by in situ microwave irradiation NMR spectroscopy.2015

    • Author(s)
      Y. Tasei, T. Yamakami, I. Kawamura, T. Fujito, K. Ushida, M. Sato, A. Naito
    • Journal Title

      J. Magn. Reson.

      Volume: 254 Pages: 27-34

    • DOI

      10.1016/j.jmr.2015.02.002

    • Related Report
      2015 Annual Research Report 2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] The microwave heating mechanism of N-(4-methoxybenzyliden)-4-butylaniline in liquid crystalline and isotropic phase as determined using in situ microwave irradiation NMR spectroscopy.2015

    • Author(s)
      Y. Tasei, F. Tanigawa, I. Kawamura, T. Fujito, M. Sato, A. Naito
    • Journal Title

      Phys. Chem. Chem. Phys.

      Volume: 17 Issue: 14 Pages: 9082-9089

    • DOI

      10.1039/c5cp00476d

    • Related Report
      2015 Annual Research Report 2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Characterization of photo-intermediates in the photo-reaction pathways of a bacteriorhodopsin Y185F mutant using in situ photo-irradiation solid-state NMR spectroscopy2015

    • Author(s)
      Kyosuke Oshima, Aris Shigeta, Yoshiteru Makino, Izuru Kawamura, Takashi Okitsu, Akimori Wada, Satoru Tuzi, Tetsuo Iwasa, Akira Naito
    • Journal Title

      Photochem. Photobiol. Sci.

      Volume: 14 Issue: 9 Pages: 2015-2015

    • DOI

      10.1039/c5pp00154d

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Recent solid-atate NMR studies of membrane-bound peptides and proteins2015

    • Author(s)
      Akira Naito, Izuru Kawamura, Namsrai Javkhlantugs,
    • Journal Title

      Annu. Rep. NMR Spectrosc.,

      Volume: 86 Pages: 333-411

    • DOI

      10.1016/bs.arnmr.2015.06.001

    • ISBN
      9780128021231
    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] Structure and orientation of antibiotic peptide alamethicin in phospholipid bilayers as revealed by chemical shift oscillation analysis of solid state nuclear magnetic resonance and molecular dynamics simulationfion2015

    • Author(s)
      Takashi Nagao, Daisuke Mishima, Namsrai Javlhalantugs, Jun Wang, Daisuke Ishioka, Kiyonori Yokota, Kazushi, Norisada, Izuru Kawamua, Kazuyoshi, Ueda, Akira Naito
    • Journal Title

      Biochim. Biophys. Acta

      Volume: 1848 Issue: 11 Pages: 2789-2798

    • DOI

      10.1016/j.bbamem.2015.07.019

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] Color Discriminating Retinal Configurations of Sensory Rhodopsin I by Photo-Irradiation Solid State NMR Spectroscopy.2014

    • Author(s)
      H. Yomonda, Y. Makino, Y. Tomonaga, T. Hidaka, I. Kawamura*, T. Okitsu, A. Wada, Y. Sudo*, A. Naito*.
    • Journal Title

      Angew. Chem. Int. Ed.

      Volume: 53 Issue: 27 Pages: 6960-6964

    • DOI

      10.1002/anie.201309258

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Interaction of extracellular loop II of kappa-opioid receptor (196-228) with opioid peptide dynorphin in membrane environments as revealed by solid state nuclear magnetic resonance, quartz crystal microbalance and molecular dynamics simulation.2014

    • Author(s)
      A. Kira, N. Javkhlantugs, T. Miyamori, Y. Sasaki, M. Eguchi, I. Kawamura, K. Ueda, A. Naito.
    • Journal Title

      J. Phys. Chem. B

      Volume: 118 Issue: 32 Pages: 9604-9612

    • DOI

      10.1021/jp505412j

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Presentation] Photoreaction pathways of photoreceptor membrane proteins revealed by in-situ photo-irradiation solid-state NMR2015

    • Author(s)
      Akira Naito
    • Organizer
      2015 International Chemical Congress of Pacific Basin Society (Pacifichem 2015)
    • Place of Presentation
      Honolulu, Hawaii
    • Year and Date
      2015-12-15
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Solid-state NMR studies for chromophore protein interaction changes in pharanis phoborhodopsin2015

    • Author(s)
      Izuru Kawamura, Ryota Nishikawa, Satoshi Nakatani, Takashi Okitsu, Akimori Wada, Naoki Kamo, Akira Naito
    • Organizer
      2015 International Chemical Congress of Pacific Basin Society (Pacifichem 2015)
    • Place of Presentation
      Honolulu, Hawaii
    • Year and Date
      2015-12-15
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Interaction of bombinin H4 containing a D-amino acid residue with lipid membranes as studied by solid-state NMR and MD simulations2015

    • Author(s)
      Yuki Kitahashi, Namsrai Javkhlantugs, Shiho Kaneda, Nyamsambuu Altannavch, Kazuyoshi Ueda, Akira Naito, Izuru Kawamura
    • Organizer
      2015 International Chemical Congress of Pacific Basin Society (Pacifichem 2015)
    • Place of Presentation
      Honolulu, Hawaii
    • Year and Date
      2015-12-15
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Hydrogen bond alteration of Tyr174 and Tyr199 of phoborhodopsin in the lipid environment as studied by solid-state 13C MAS NMR2015

    • Author(s)
      Ryota Nishikawa, Izuru Kawamura, Takashi Okitsu, Akimori Wada, Yuki Sudo, Naoki Kamo, Akira Naito
    • Organizer
      2015 International Chemical Congress of Pacific Basin Society (Pacifichem 2015)
    • Place of Presentation
      Honolulu, Hawaii
    • Year and Date
      2015-12-15
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Structural changes in the photo-reaction pathways of retinal in bacteriorhodopsin studied by in-situ photo-irradiation solid-state NMR2015

    • Author(s)
      Aris Shigeta, Kyosuke Oshima, Ryota Miyasa, Miyako Horigome, Izuru Kawamura, Takashi Okitsu, Akimori Wada, Satoru Tuzi, Akira Naito
    • Organizer
      2015 International Chemical Congress of Pacific Basin Society (Pacifichem 2015)
    • Place of Presentation
      Honolulu Hawaii
    • Year and Date
      2015-12-15
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Structural diversity of amyloid fibril and its intermediate in human calcitonin as studied by solid-state NMR and MD simulation2015

    • Author(s)
      Shuhei Toyoda, Lkhamsuren Ganchimeg, Namsrai Javkhlantugs, Hikari-Itoh-Watanabe, Izuru Kawamura, Kazuyoshi Ueda, Akira Naito
    • Organizer
      2015 International Chemical Congress of Pacific Basin Society (Pacifichem 2015)
    • Place of Presentation
      Honolulu, Hawaii
    • Year and Date
      2015-12-15
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Photo reaction pathways of sensory rhodopsin I as revealed by in-situ photo-irradiation solid-state NMR2015

    • Author(s)
      Yoshiteru Makino, Hiroki Yomoda, Yuya Tomonaga, Teturou Hidaka, Izuru Kawamura, Takashi Okitsu, Akimori Wada, Yuki Sudo, Akira Naito
    • Organizer
      2015 International Chemical Congress of Pacific Basin Society (Pacifichem 2015)
    • Place of Presentation
      Honolulu, Hawaii
    • Year and Date
      2015-12-15
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] In situ光照射固体NMRによるD96N-bR変異体の光反応サイクルにおけるM-中間体の解析2015

    • Author(s)
      大谷優人、重田安里寿、槙野義輝、宮佐亮太、川村 出、沖津貴志、和田昭盛、辻暁、内藤 晶
    • Organizer
      第54回NMR討論会
    • Place of Presentation
      千葉工業大学
    • Year and Date
      2015-11-06
    • Related Report
      2015 Annual Research Report
  • [Presentation] 13-cis, 15-syn型バクテリオロドプシンの光中間体のin situ光照射固体NMRによる検出2015

    • Author(s)
      重田安里寿、大島恭介、川村 出、沖津貴志、和田昭盛、辻暁、内藤 晶
    • Organizer
      第54回NMR討論会
    • Place of Presentation
      千葉工業大学
    • Year and Date
      2015-11-06
    • Related Report
      2015 Annual Research Report
  • [Presentation] In situ光照射固体NMRによる光受容膜タンパク質ppR/pHtrIIの光活性中間体の解析2015

    • Author(s)
      槙野義輝、友永雄也、日高哲郎、川村 出、沖津貴志、和田昭盛、須藤雄気、加茂直樹、内藤 晶
    • Organizer
      第54回NMR討論会
    • Place of Presentation
      千葉工業大学
    • Year and Date
      2015-11-06
    • Related Report
      2015 Annual Research Report
  • [Presentation] 固体NMRによるヒトカルシトニンのアミロイド線維中間体から線維への構造変化の解析2015

    • Author(s)
      豊田修平、Lkhamsuren Ganchimeg, Namsrai Javkhlantugs, 渡邉(伊藤)ひかり、川村 出、上田一義、内藤 晶
    • Organizer
      第54回NMR討論会
    • Place of Presentation
      千葉工業大学
    • Year and Date
      2015-11-06
    • Related Report
      2015 Annual Research Report
  • [Presentation] 31P MNRを用いたペプチドホルモングルカゴンによるDMPC脂質二分子膜の形態変化と線維形成過程の解析2015

    • Author(s)
      羽矢和未、菊池章江、川村 出、内藤 晶
    • Organizer
      第54回NMR討論会
    • Place of Presentation
      千葉工業大学
    • Year and Date
      2015-11-06
    • Related Report
      2015 Annual Research Report
  • [Presentation] In situ光照射固体NMRによるバクテリオロドプシンY185F変異体の光反応経路の解明2015

    • Author(s)
      大島恭介、重田安里寿、槙野義輝、川村 出、沖津貴志、和田昭盛、辻暁、岩佐達郎、内藤 晶
    • Organizer
      第53回日本生物物理学会年会
    • Place of Presentation
      金沢大学
    • Year and Date
      2015-09-13
    • Related Report
      2015 Annual Research Report
  • [Presentation] The microwave heating mechanism of MBBA as studied by microwave irradiation NMR spectroscopy2015

    • Author(s)
      Akira Naito, Yugo Tasei, Teruaki Fujito, Izuru Kawamura
    • Organizer
      The 19th International Society of Magnetic Resonance Conference
    • Place of Presentation
      Shanhai, China
    • Year and Date
      2015-08-16
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 13C solid-state NMR study of hydrogen bonding alteration of Tyr174 and Tyr199 on phoborhodopsin in the lipid environment2015

    • Author(s)
      Izuru Kawamura, Ryota Nishikawa, Takashi Okitsu, Akimori Wada, Yuki Sudo, Naoki Kamo, Akira Naito
    • Organizer
      19th International Society of Magnetic Resonance Conference
    • Place of Presentation
      Shanghai, China
    • Year and Date
      2015-08-16
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Color-discriminating retinal configuration change of sensory rhodopsin as revealed by in situ photo-irradiation solid-state NMR2015

    • Author(s)
      Yoshiteru Makino, Hiroki Yomoda, Yuya Tomonaga, Tetsurou Hidaka, Izuru Kawamura, Takashi Okitsu, Akimori Wada, Yuki Sudo, Akira Naito
    • Organizer
      19th International Society of Magnetic Resonance Conference
    • Place of Presentation
      Shanghai, China
    • Year and Date
      2015-08-16
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Structural changes in the photo-excited process of bacteriorhodopsion by photo-irradiation solid-state NMR2015

    • Author(s)
      Aris Shigeta, Kyosuke Oshima, Takashi Okitsu, Akimori Wada, Satoru Tuzi, Akira Naito
    • Organizer
      19th International Society of Magnetic Resonance Conference
    • Place of Presentation
      Shanghai, China
    • Year and Date
      2015-08-16
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Determination of photo-intermediates and photo-reaction pathways of photoreceptor membrane proteins by in-situ photo-irradiation solid-state NMR2015

    • Author(s)
      Akira Naito, Yoshiteru Makino, Hiroki Yomoda, Yuya Tomonaga, Tetsurou Hidaka, Izuru Kawamura, Yuki Sudo, Akimori, Wada, Takashi Okitsu, Naoki Kamo
    • Organizer
      The 6th Asia-Pacific NMR Symposium
    • Place of Presentation
      Hong Kong, China
    • Year and Date
      2015-08-13
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 固体NMRによる抗菌ペプチドの生体膜結合構造と膜相互作用解析2015

    • Author(s)
      内藤 晶
    • Organizer
      第15回 日本蛋白質科学会年会
    • Place of Presentation
      あわぎんホール、徳島市
    • Year and Date
      2015-06-24
    • Related Report
      2015 Annual Research Report
    • Invited
  • [Presentation] 固体NMRとTEMによるヒトカルシトニンにおけるアミロイド線維形成と阻害機構の解明2014

    • Author(s)
      内藤 晶、伊藤ー渡邉 ひかり、上平ー石島 美弥、N. Javkhlantugs、近藤正志、中越雅道、川村 出、辻 暁、斉藤 肇、上田一義
    • Organizer
      第53回NMR討論会(2014)
    • Place of Presentation
      大阪大学コンベンションセンター
    • Year and Date
      2014-11-04 – 2014-11-06
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] 光照射固体NMRによる光受容膜タンパク質の光反応経路の解明2014

    • Author(s)
      内藤 晶、槙野義輝、重田安里寿、四方田博紀、川村 出、沖津貴志、和田昭盛、須藤雄気、加茂直樹
    • 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
      2014-10-15 – 2014-10-18
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Detection of photo-intermediates of microbial sensory rhodopsin by in situ photo-irradiation solid-state NMR2014

    • Author(s)
      A. Naito, Y. Makino, H. Yomoda, Y. Tomonaga, T. Hidaka, I. Kawamura, Y. Sudo, A. Wada, T. Okitsu, N. Kamo,
    • Organizer
      16th ICRP
    • Place of Presentation
      Nagahama, Japan
    • Year and Date
      2014-10-05 – 2014-10-10
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] NMR, QCM, MDシミュレーションによるk-オピオイド受容体細胞外第2ループとダイノルフィンの細胞膜中での相互作用解析2014

    • Author(s)
      A. Naito, A. Kira, N. Javkhlantugs, T. Miyamori, Y. Sasaki, M. Eguchi, I. Kawamura, K. Ueda,
    • Organizer
      第52回日本生物物理学会年会
    • Place of Presentation
      札幌コンベンションセンター
    • Year and Date
      2014-09-25 – 2014-09-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] Detection of light activated intermediates of photoreceptor membrane proteins by in-situ photo-irradiatied solid-state NMR2014

    • Author(s)
      A. Naito, Y. Makino, Y. Tomonaga, T. Hidaka, I. Kawamura, Y. Sudo, A. Wada, T. Okitsu, N. Kamo, A. Ramamoorthy
    • Organizer
      16th ICMRBS
    • Place of Presentation
      Dallas, Texas, USA
    • Year and Date
      2014-08-24 – 2014-08-29
    • Related Report
      2014 Annual Research Report
  • [Presentation] 固体NMR,QCM,MDシミュレーションによるk-オピオイド受容体細胞膜外第2ループとダイノルフィンとの相互作用解析2014

    • Author(s)
      内藤 晶、N. Javkhlantugs, 宮森丈敬、佐々木慶幸、江口政幸、川村 出、上田一義、吉良敦史
    • Organizer
      第14回蛋白質科学会年会
    • Place of Presentation
      ワークピア横浜
    • Year and Date
      2014-06-25 – 2014-06-27
    • Related Report
      2014 Annual Research Report
  • [Book] 光と生命の事典 第5章 光による生命現象の計測 第176節 NMR分光法2016

    • Author(s)
      内藤 晶
    • Total Pages
      4
    • Publisher
      朝倉書店
    • Related Report
      2015 Annual Research Report
  • [Book] Photoactivated structural changes in ohotoreceptor membrane proteins as revealed by in situ solid-state NMR spectroscopy. (Advances in Biological Solid-State NMR)2014

    • Author(s)
      A. Naito, I. Kawamura, (Editors F. Separovic, A. Naito)
    • Publisher
      Royal Society of Chemistry
    • Related Report
      2014 Annual Research Report
  • [Book] 電磁場相互作用のNMRによる研究 (最新マイクロ波エネルギーと応用技術)2014

    • Author(s)
      内藤 晶 (編集委員長 吉川 昇)
    • Publisher
      産業技術サービスセンター
    • Related Report
      2014 Annual Research Report

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Published: 2014-04-04   Modified: 2018-03-28  

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